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

Abstract

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 & Pear Forecast”). In addition, commercial apples have been shown to be closely related to each other genetically (Migicovsky et al., 2021; Noiton & Alspach, 1996). Reliance on a limited number of closely related cultivars means that consumers experience an extremely limited fraction of the apple's genetic diversity. However, the extent of phenotypic diversity captured by the top commercial cultivars remains to be quantified. Fruit firmness is strongly associated with consumers “likeness” of cultivars and is, therefore, a key target for breeders (Szczesniak 2002; 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 & Volk, 2019). Extensive germplasm resources are already available for major crops such as maize and rice which can be stored as seeds, but resources for perennial fruit crops are lacking as they are expensive to establish and maintain (Flint-Garcia et al., 2005; Jackson, 1997; Migicovsky & Myles, 2017). Despite the high cost, living germplasm collections are essential for woody perennials which are difficult to conserve in seed banks or tissue culture. There is increasing interest in using germplasm collections to not only preserve biodiversity and exploit valuable phenotypes but also to perform genetic mapping (Migicovsky et al., 2019). The high genetic diversity and sample sizes present in many germplasm collections can result in well-powered genetic mapping studies that identify genetic markers useful for genomics-assisted breeding (Zhu et al., 2008). In addition, germplasm collections often contain wild relatives with novel traits that can be introgressed into elite cultivars (Migicovsky & Myles, 2017). There are numerous apple germplasm collections that have been established worldwide (e.g., USA (Gross et al., 2013), China (Gao et al., 2015), New Zealand (Kumar et al., 2010), and Europe (Jung et al., 2020)). Within Canada, there are apple collections located across the country, for example, in Ontario and British Columbia (Hampson et al., 2009; 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 & Silsby, 1992). The combination of these indicators was used to determine whether an accession was ready to be harvested. Flowering date was measured as the date when the youngest wood displayed >80% of flowers at the king bloom stage (McClure, 2017). Flowering date was recorded in 2016, with trees being assessed every 3 days during this period. Time to ripen was calculated as the time (in days) between flowering date and harvest date. Precocity was recorded as the year in which a tree first bloomed after establishment in the orchard and converted into a score: a score of 1 corresponded to 2014, a score of 2 corresponded to 2015, a score of 3 corresponded to 2016, and a score of 4 indicated that the tree had not yet bloomed as of 2016. The fruit quality traits measured included weight (g), firmness (kg/cm2), acidity (g/L malic acid), soluble content and of fruit In measurements were taken from 5 a sample of apples was used in 2016 possible and a for each tree was were not recorded for trees with apples available for In 2016, an phenotyping was used to fruit quality In measurements were collected as the between 2016 and we assessed the between years for each weight of the apples in a sample were measured and an weight apple was calculated by the recorded weight by the number of apples in the measurements for apples were recorded using a Fruit a a small section of skin the of the apple was removed to the to the apple apple was on the that the the of the apple the skin had been There was for the of the apple from which the was The firmness measurements were to a value for the tree. acidity in a sample of a of each apple was using a The from the apples belonging to a tree was to make a acidity was measured using the This 1 of the sample with of content was measured on the sample using the The was calculated by measurements by acidity apples that were not were in cold storage 3 months in these apples were removed and at until fruit quality be measured on the using the same methods apples from each accession were assessed for fruit quality both at harvest and after 3 months of storage. measurements were taken from a of 3 and a of apples During the 2016 fruit were collected and into a using and stored at were in duplicate for the of using in an solution in 2 A for was performed to the of by the of the color were as & et al., 2008). The of apple were in of weight of an The the content present in plant and was as & 2009; 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 & Pear that are in the ABC to the phenotypes of the accessions in the The of the top cultivars that are present in the ABC are as and The and measurements were calculated for the and for the other ABC were performed to determine whether measurements of the from the measurements of the other ABC The proportion of the in measurements by the cultivars was calculated by the in measurements of the by the of the were also performed to determine whether phenotypic traits Malus geographic and was calculated using from the in The was to for Canada's Apple Biodiversity Collection (ABC) to capture of the diversity of apples and contains 1,119 accessions from with a on apples from Canada and the USA The measurements from over fruit from 1,000 unique apple accessions across 2 years are in Data S1. phenotypes were measured in both 2016 and the of flowering and other phenotypes in the main of the are from 2017 due to the larger sample in that The between years for phenotypes from to and of of the phenotypes had significant The were for the phenotypic changes during storage. For example, the was for change in weight during storage The between years was for harvest date 1 The of phenology and fruit quality traits are shown in The of these traits for the year 2016 are in The for each of the phenotypes are in Data Flowering date days with a M. sieversii flowering first on May and a M. domestica flowering on Harvest as as The harvested accession was on August and the harvested accession was on Time to ripen days. had the time to ripen days) and had the time to ripen We a in apple weight from to The accession was and the was by the accession was and the most was 3 a M. sylvestris There was a in firmness accessions at the accession was with a of the accession was at The accession was at and the was as at content by of from to The accessions with the content were and the advanced breeding the apple had the of the phenotypes of the cultivars from the of the accessions in the ABC after for The measurements of the cultivars capture between and of the variation in the ABC across For the cultivars are to the of the ABC and capture only of the variation in In of the variation in soluble is captured by the and for the to be above the of the Precocity from 1 year to over 4 years across but of the cultivars in the first 2 years after establishment in the orchard. were between measurements taken and after storage for fruit quality traits On apples of their acidity, of their firmness, and of their but in soluble by during 3 months of cold storage The change in acidity, firmness, soluble and weight for the cultivars not from the of the However, the cultivars to experience a of firmness and acidity to the of the We calculated the fruit and storage traits Data for the 2016 are in and Data the were significant after for significant were and 4 were Harvest date was with weight 1 change in 1 and change in acidity and weight at harvest were content and weight at harvest were and flowering date were at harvest was with phenology including flowering date 5 harvest date 1 and time to ripen 1 in firmness during storage was associated with flowering date harvest date and time to ripen For of the we significant between apples to geographic between M. domestica accessions 3 days 1 days were harvested days were 1 and had content M. sieversii accessions New apples days were and had content apples. apples days were were and had content apples content was the only of the 39 that a significant change over time after for the year of of named cultivars was with content Apple cultivars after had a content of which is cultivars food and the long-term of on the and characterization of germplasm collections that the genetic diversity for future crop improvement. of the phenotypes within these germplasm collections is to breeders to and improved plant this comprehensive apple evaluation phenotyping of Canada's Apple Biodiversity Collection which contains over apple The degree of phenotypic variation in the ABC was for some traits For example, apples can by in in acidity, and in such as harvest also across the days. In flowering within a are in with from diverse apples planted Europe harvest was also the flowering (Jung et al., that are to development under are at of from and flowering therefore, be a breeding target & 2013). Time to ripen is also a for apple and we a in the time to ripen between the with the days) and days) to There is a for fruit that the fruit on the tree to potentially or and the the of fruit and there is interest in breeding flowering and ripening cultivars to the by & & 2003). provide not only an of the levels of variation across apple phenotypes but also phenotypic variation that is for breeding improved apple cultivars. the apple breeding to as as wild relatives such as M. can to the development of elite cultivars with improved and fruit The cultivars in the USA not from the collection as a for that the most apples in the USA are not in of their phenotypes to the variation within the However, there is for improvement through breeding to for numerous For there are apples within the ABC with acidity ripening and flowering dates that the within the cultivars. the of soluble was the accession in the of the cultivars had for fruit quality and phenology In addition, the of measurements for the a of of the variation displayed by the collection. had the a value which had the content the cultivars. In addition, the only captured of the in flowering date across the and the cultivars a of days to ripen the apple in the orchard. This that the most apples in the USA capture only a fraction of the phenotypic diversity in the and that most phenotypic variation remains to consumers and In addition, genetic of the USDA apple collection that over half the collection is related through a of due to the of the most cultivars in breeding (Migicovsky et al., 2021). A of the apple genetic resources the germplasm resources the apple industry remains due to the limited number of cultivars in commercial production et al., 2015). apples not experience a domestication many breeding to make of only a small fraction of apple diversity, it to in genetic and phenotypic diversity in the future (Gross et al., Noiton & Alspach, 1996). the diversity of M. domestica and M. sieversii is not only useful to the genetic but it is also for novel traits into new cultivars that can meet apple and the of is a important target for breeders consumers apples that have been stored for within the ABC that performed during storage with of firmness, acidity, and weight may serve as useful breeding material to develop cultivars with how acidity, soluble firmness, and weight over 3 months of we that the of change in each was not across cultivars. For example, firmness is expected during a small number of cultivars to firmness during storage can be by due to the of the apples from a accession measured at harvest were the apples measured after storage from that accession were not the same apples as measured at harvest. Despite this the of phenotypic change during storage was with firmness, acidity, and weight during et al., For example, apples a of their acidity during which is with (Kouassi et al., 2009; et al., 2019). Apple is strongly by acidity, most malic and the of acidity during storage to fruit quality et al., et al., of the other key of fruit quality both at harvest and after months of storage is fruit firmness et al., et al., Despite the apple's to retain its firmness during storage to many other there is tremendous to apples by breeding novel cultivars that during of storage. 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In addition, harvested cultivars levels of which to their texture after storage (Watkins, 2003). at harvest is also a of firmness after storage 1 that the texture of an apple after storage is by how it was when The time it an apple to ripen is the of time between its flowering date and its harvest date. ripening may appear they result in fruit that they also result in to and In addition, flowering cultivars are they are to & may cultivars with ripening selection for ripening will result in apples with texture that make to Apple cultivars with ripening and be for by breeders they had It is possible that a of the cultivars we that ripen but also firmness (e.g., and may the key to ripening time from that enable the between ripening time and firmness to be novel apple cultivars in the future that ripen are harvested and are both at harvest and after storage. between domesticated crops and their wild can reveal which traits were during domestication and improvement et al., 2012). 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