Abstract
The U.S. Department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS), Plant Genetic Resources Unit apple (Malus) collection in Geneva, NY, conserves over 2500 trees as grafted clones. We have compared the genotypes of 1131 diploid Malus ×domestica cultivars with a total of 1910 wild and domesticated samples representing 41 taxonomic designations in the NPGS collection to identify those that are genetically identical based on nine simple sequence repeat (SSR) loci. We calculated the probability of identity for samples in the data set based on allelic diversity and, where possible, use fruit images to qualitatively confirm similarities. A total of 237 alleles were amplified and the nine SSRs were deemed adequate to assess duplication within the collection with the caveat that “sport families” likely would not be differentiated. A total of 238 M. ×domestica and 10 samples of other taxonomic groups shared a genotype with at least one other M. ×domestica individual. In several cases, genotypes for cultivars matched genotypes of known rootstocks and indicated that these accessions may not accurately represent the indicated named clones. Sets of individuals with identical genotypes and similar cultivar names were assigned to sport families. These 23 sport families, comprised of 104 individuals, may have mutational differences that were not identified using the nine SSR loci. Five of the selected markers (CH01h01, CH02d08, CH01f02, G12, GD147) overlap with sets of markers that have been used to fingerprint European apple collections, thus making it possible to compare and coordinate collection inventories on a worldwide scale.
The global production of apples is threatened by disease, pest susceptibility, suboptimal cold-hardiness and heat tolerance, minimal resistance to drought and wet soils, undesirable storage and transport characteristics, and expensive production methods. The U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS) National Plant Germplasm System, Plant Genetic Resources Unit (PGRU) apple collection in Geneva, NY, conserves key genetic resources useful for breeding and research programs that address threats to apple crop production. The Malus collection is maintained as grafted, clonally propagated trees and own-rooted seedling trees. The grafted orchards include 33 species of Malus, of which only one species, Malus ×domestica, is cultivated for fruit production, as well as some hybrid materials. These species are represented by between one and 1372 unique accessions each. The taxonomic and ecological breadth of this collection makes it a vital genetic resource for both the United States and the world.
The clonally propagated apple field collection is grafted on ‘East Malling 7’ (EMLA 7) rootstock (Wertheim, 1998), which was selected for its dwarfing and increased resistance to fireblight infection (Aldwinckle et al., 2004; Forsline et al., 2010). Currently, the grafted collection has ≈2500 clones acquired primarily from gene banks, breeders, and wild collection trips (Table 1). The field collection also includes several thousand Malus species seedling (non-grafted) trees, derived primarily from seeds collected during plant exploration trips. Field trees are expensive to maintain; the most recent estimated cost was more than $50 per tree each year (Hokanson et al., 1998), and this figure has almost certainly been exceeded in recent years. Thus, it is critical to understand the genetic constitution of the collection materials to ensure continued maintenance of high-priority genetic resources using limited financial resources.
Taxonomic designation and number of individual inventories of Malus trees in the Geneva, NY, Plant Genetic Resources Unit (PGRU) grafted orchard, the number of those grafted trees that are known to be diploid, the number of individuals included in the nine simple sequence repeat (SSR) data set, and the number of seedling trees in the nine SSR data set.
Microsatellite markers have been used successfully to assess the diversity of wild and clonally propagated fruit collections (Gökirmak et al., 2009; Koehmstedt et al., 2011; Laucou et al., 2011; Njuguna et al., 2011; Urrestarazu et al., 2012; van Treuren et al., 2010). As a result of the high levels of allelic diversity within most Malus species, relatively few markers are required to differentiate among unique M. ×domestica cultivars (Hokanson et al., 1998) or among half-sib individuals from wild populations of M. sieversii and M. orientalis (Richards et al., 2009a, 2009b; Volk et al., 2005, 2008). Conversely, the very low genomic coverage of microsatellite data sets limits our ability to differentiate among individuals in “sport” families, because these individuals may exhibit distinct phenotypic traits that are based on one or a few mutations (Venturi et al., 2006; Wünsch and Hormaza, 2002).
In this work, we determined whether a set of nine SSR loci is sufficiently variable to differentiate among siblings and identify identical accessions in the USDA-ARS Malus field collection based on the probability of identity (ProbI) for the data set. We identified genetically identical M. ×domestica cultivars maintained as clones in the field collection and, where possible, used fruit images to qualitatively confirm similarities.
Materials and Methods
Plant material.
Most of the trees in the grafted PGRU orchards were sampled for potential inclusion in the genetic analyses, and, in some cases, seedling trees were included as additional representatives of wild species. For both grafted and seedling trees, only diploid trees had microsatellite signatures that could be scored in a comparative manner, and only these were included in the final data set. The final data set included 1910 individuals, or accessions, each identified with a unique Plant Introduction number [PI# (for grafted trees)] or Geneva Malus number (for seedlings in most cases) identification numbers (Table 1). For the present analyses, we focus on 1131 diploid M. ×domestica cultivars in the PGRU orchards. To identify duplicates, each M. ×domestica genotype has been compared with all the other domesticated genotypes as well as 779 wild genotypes in the 1910 individual data set.
Microsatellite markers.
Genomic DNA was extracted from leaf tissue using DNeasy 96 plant kits (Qiagen, Valencia, CA). Nine previously published SSRs [GD12, GD15, GD96, GD142, GD147, GD162, CH01h01, CH01f02, and CH02d08 (Hokanson et al., 1998; Liebhard et al., 2002)] chosen based on their ease of scoring, variation, and overlap with SSRs used to evaluate Malus collections in other countries were amplified in all samples. Primer sequences, amplicon size ranges, and annealing temperatures are listed in Gross et al. (2012). Forward primers, labeled with either IRD 700 or IRD 800, were obtained from MWG-Biotech (High Point, NC). Unlabeled reverse primers were purchased from IDT (Coralville, IA). All polymerase chain reactions (PCRs) were carried out in 15 μL total volume using previously published methods (Volk et al., 2005). PCR products were visualized and scored in one of two ways. Some were visualized on a slab sequencer (LI-COR 4200; LI-COR, Lincoln, NE); digital images were collected from the sequencer using LI-COR Saga Generation 2 software and were manually interpreted and scored using the Saga software. Others were visualized on a capillary sequencer (ABI 3730; Applied Biosystems, Foster City, CA); chromatograms were scored using the GeneMarker software (SoftGenetics, State College, PA). Peaks were scored automatically based on allelic bins created for each SSR locus and then corrected manually for cases of errors or nonsensical automatic scoring. When a single SSR was scored using both the LI-COR and ABI systems, a minimum of 10 individuals were run on both systems to control for allele length differences resulting from instrumentation.
Probability measurements and identification of “duplicates.”
Basic diversity statistics, along with polymorphism information content and discriminatory power for each locus in the nine SSR data set, were calculated using GenoDive and CERVUS (Botstein et al., 1980; Kalinowski et al., 2007; Meirmans and Van Tienderen, 2004; Tessier et al., 1999). Note that expected heterozygosity (He) and observed heterozygosity (Ho) were calculated for reference but are not comparable to those calculated for a randomly mating population. To evaluate our ability to correctly identify duplicated genotypes using the nine SSR data set, we calculated ProbI for each locus and across multilocus genotypes. ProbI is an estimate of the probability that two individuals will have the same multilocus genotype by chance alone (Peakall and Smouse, 2006; Taberlet and Luikart, 1999). To account for relatedness among samples, we also calculated the more conservative ProbIsibs, which allows for the possibility of identity resulting from sibling relationships. Both ProbI and ProbIsibs were calculated using GenAlEx (Peakall and Smouse, 2006). The nine SSR data set, with data missing at a maximum of one locus for any individual, was checked for duplicate genotypes using GenoDive (Meirmans and Van Tienderen, 2004). Genotypes were considered to be duplicated if they matched at all alleles across all nine SSRs with the exception of missing data, which were ignored (i.e., counted neither for nor against identity).
Phenotypes and fruit images.
Fruit images for each accession identified as a duplicate were downloaded from the Germplasm Resources Information Network database (USDA, 2012). Images were qualitatively compared for fruit size, fruit ground color, fruit over color, shape, and russet appearance.
Results
This research focuses on the M. ×domestica cultivars in the grafted collection but does include some seedling materials for comparison purposes. The M. ×domestica collection has 1372 individuals, of which 1240 (90%) are diploid, and thus produced amplification products that could be accurately scored. Individuals with missing data at more than one locus were not included in the data set, leaving 1131 M. ×domestica accessions that were compared with each other as well as the additional 779 diploid individuals from other species in the data set.
Nine SSRs were amplified in the 1910 individuals, and a total of 237 alleles (average of 26.3 alleles per locus) were present across the nine SSRs. Levels of heterozygosity were high; Ho was greater than 0.50 and He was greater than 0.85 across all species for all loci except for GD15 (Table 2). In M. ×domestica, Ho was greater than 0.70 for all loci except for GD15 (data not shown). ProbI and ProbIsibs were calculated for the data set to determine whether any empirically determined duplicated genotypes were likely the result of chance alone or whether they were the result of true identity at the genetic level. Although there is not a firm cutoff for when to interpret a level of ProbI as sufficient, one suggested standard (Peakall et al., 2006) is the reciprocal of the sample size (1/1910 = 0.00052). Thus, a value for ProbI or ProbIsibs < 0.00052 would indicate that when samples do share identical genotypes, it is the result of true identity between the individuals. The values of ProbI and ProbIsibs are plotted in Figure 1, and the required level of ProbI is achieved with two SSRs for ProbI and with seven SSRs for the more conservative ProbIsibs. ProbI values for each SSR locus, representing a measure of the discriminatory power of a given marker, are given in Table 2.
Genetic parameters for each of the nine simple sequence repeat (SSR) loci used to determine duplicate genotypes in the Malus collection, including number of alleles (A), observed heterozygosity (Ho), expected heterozygosity (He), the probability of identity (ProbI), the discriminating power (D), and the polymorphic information content (PIC).
Probabilities of identity (ProbI) for increasing combination of the nine simple sequence repeat (SSR) loci arranged in decreasing order of ProbI for each locus. Dashed lines show the standard ProbI, and solid lines show the more conservative ProbIsibs.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.333
Given that the number and variability at the loci used in this study are likely sufficient to discriminate between individuals and matches that represent true identity, another way to interpret the data are to consider the probability (however low) that two individuals would match by chance alone. For the standard value of ProbI, assuming no relatedness, the probability is 4.8 × 10−15, or one in 21 trillion. For the more conservative value of ProbIsibs, it is 7.7 × 10−5, or one in 13,000. The real value is likely somewhere in between these two extremes, because some of the individuals are undoubtedly related, but many are not, given the number of different species present in the data set.
The explicit possibility that full- or half-sibs might have the same genotype based on their close relatedness also must be considered, especially in the context of the sport families. The probability that two full-sibs will have the same genotype is mainly dependent on the level of homozygosity of the parents and ranges from 0.25 if the parents are heterozygous with four different alleles across a single locus to 1.0 if the parents are homozygous. There are a variety of ways that these probabilities can be combined across nine SSRs. However, even if one or the other parent is homozygous at each of the nine SSRs (although the other is heterozygous), the probability that the siblings would have identical genotypes would be 0.00195, or less than one in 500. Given the high levels of observed heterozygosity for most loci in the data set (Ho greater than 0.70 for eight of the nine loci in M. ×domestica), it is unlikely that two parents would have this many homozygous loci. Thus, any matching genotypes are much more likely to represent duplicates than full-sibs.
Overall, based on the allelic diversity, levels of heterozygosity, and ProbI and ProbIsibs values for the data set, the nine SSRs were deemed adequate to assess duplication within the collection with the caveat that “sport families” likely would not be differentiated. A total of 238 M. ×domestica and 10 samples of other taxonomic groups (eight Malus hybrid, one M. pumila, one Malus species) shared a genotype with at least one other M. ×domestica individual (Fig. 2). When available, fruit image data generally supported the genetically similar assessments based on the nine SSR loci (Table 3; Fig. 3). Fifty-two cultivars with unique names shared 24 genotypes and their similarities could be confirmed visually with image data for those individuals. Seven pairs of cultivars with very similar names also shared image and genotypic identities. Seventy individuals shared 32 genotypes, but image data were not available (Table 3).
Plant Introduction (PI) or Geneva Malus (GMAL) number, cultivar name (followed by species name if not Malus ×domestica), and country of origin for matching genotypes or sets of genotypes, arranged horizontally.z
Chromatograms of locus CH02d08 for two sets of duplicated Malus ×domestica genotypes. The first two are PI #437036 and PI #613830 (cultivars Aromat de Vara and Inducoa No. 11) with alleles of length 212 and 230 bps. The second two are PI #129820 and PI #249925 (cultivars Cole and King Cole) with alleles of length 214 and 230 bps.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.333
Representative sets of Malus ×domestica fruit identified sharing identical genotypes based on nine simple sequence repeat (SSR) loci. PI number and cultivar names are provided on the figure for pairs of accessions with matching genotypes and different names (A–D), cultivars matching rootstock accessions (E–F), and a portion of the ‘Golden Delicious’ sport family (G). Fruit are arranged on 1-cm2 grids.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 137, 5; 10.21273/JASHS.137.5.333
The data set included two rootstock accessions named ‘Malling XIII’ (M.13) with identical genotypes. The genotype of PI #589710 ‘Merton 793’ matched that of PI #263633 Malus sp. ‘Rootstock’, and the genotype of PI #589923 ‘Red Dijmanszoet’ and PI #613944 ‘Coop 44’ (sp.) matched PI #588812 ‘Malling Merton 111’ (MM.111), another known rootstock. Based on the identical genetic signatures between these “cultivars” and rootstocks, it appears as if these specific accessions may not represent accurately the indicated named clones (Table 3). Instead, these individuals may have been received as materials believed to be clones, although they were actually rootstocks at the original source planting. When sets of individuals were identified as being genetically identical, had related cultivar names, and mostly similar fruit appearances, they were assigned to “sport” families. There were 23 sport families containing between two and 15 individuals each for a total of 105 individuals in all (Table 4). These sport families may have mutational differences that could not be identified using the nine SSR loci.
Sport family name, PI number, cultivar name, and country of origin for matching genotypes or sets of genotypes, arranged horizontally.
Discussion
The USDA seeks to maintain a diverse collection of Malus genetic resources. Given the maintenance cost per tree and the limited space available, it is critical that new materials strategically expand the collection by capturing novel genetic diversity that has potential value to the user community. Effectively maintaining the genetic diversity of clonal crop collections such as the NPGS Malus orchard is particularly important in the context of modern production, in which the occasional infusions of genetic diversity through cross-pollination with wild relatives, once a major source of new variation, are rarely incorporated into crop germplasm (McKey et al., 2010). Indeed, the U.S. apple industry is based primarily on 11 apple cultivars (Dennis, 2008), and these modern commercial cultivars are derived from progeny of only four seedling parents: ‘Cox’s Orange Pippin’, ‘Golden Delicious’, ‘Jonathan’, and ‘McIntosh’ (Brown, 2012; Luby, 2003; Noiton and Alspach, 1996). Diverse, well-characterized apple collections of both M. ×domestica as well as wild Malus species provide breeding programs with novel alleles that have the potential to increase yield, value, and quality as well as decrease susceptibility to biotic and abiotic stresses. Essentially, these collections serve as a resource to maintain a high overall genetic diversity for a crop despite very low diversity in the field.
Genetic assessments of the USDA-ARS-NCGRP apple collection allow for a more in-depth understanding of diversity represented by the greater than 1000 diploid M. ×domestica trees currently maintained as part of the orchard. This research focuses on identifying and classifying the “duplicate” materials within the collection. Accurately identifying genetically identical samples in a data set of any sort (crop collections, mark-recapture studies, forensics) requires that the selected loci be sufficiently variable and that the calculated probability of two genotypes matching take into account the reality of population substructure and relatedness among individuals to avoid matches that are the result of relatedness alone (Peakall et al., 2006; Waits et al., 2001). We have dealt with these issues by calculating both ProbI and ProbIsibs. The latter measure serves as a “conservative upper bound for the probability of observing identical multilocus genotypes between two individuals sampled from a population” (Waits et al., 2001). The large number of alleles amplified at the SSR loci used in this study (with the exception of GD15), combined with high levels of heterozygosity in wild and domesticated apple, yielded levels of ProbI and ProbIsibs that were low enough to be considered sufficient for both the size of the data set (0.00052) and the recommended levels of ProbI [0.01–0.0001 (Waits et al., 2001)]. Thus, the “duplicates” identified by this genetic analysis (and confirmed, in some cases, by fruit images) are likely to be very closely related, if not actually identical.
With “duplicates” identified in studies like this one, curators can assess if likely misidentified materials (such as the cultivars that match rootstocks) should be maintained or removed to accommodate new samples. In addition, materials identified as identical based on the nine SSRs can be characterized further at the phenotypic level to determine if the cultivars possess unique traits or if they might be similar enough to be considered synonyms of genotypes in the collection. This approach can also be taken with the sets of sport families, which can be characterized for phenotypic or physiological traits of interest. Sport family members may have unique, desirable traits that are based on simple mutations (potentially as a result of retrotransposon activity) compared with other family members (Kobayashi et al., 2004; Sun et al., 2010; Venturi et al., 2006). The cultivar Wijcik McIntosh is an excellent example of such a sport; it exhibits a unique, heritable columnar growth habit. Sports like ‘Wijcik McIntosh’ with desirable traits could be selected for further genetic analyses to identify new loci controlling traits of interest or new alleles at known loci that could be useful for future breeding efforts.
This research presented data from nine SSR loci that are sufficient to determine potential duplicates among 1910 accessions in the USDA Malus collection and likely from other large collections of Malus as well. These markers overlap those previously used to assess diversity within wild species (Richards et al., 2009a, 2009b; Volk et al., 2005, 2008, 2009) and among M. ×domestica, M. orientalis, M. sieversii, and M. sylvestris in the USDA-ARS-NPGS collection (Gross et al., 2012). Five of the markers (CH01h01, CH02d08, CH01f02, G12, GD147) overlap with the markers that have been selected within the European community for collection comparison purposes (F. Laurens, personal communication; M. Ordidge, personal communication), so it will be possible to extend this type of analysis across collections in the future. Ultimately, studies of this type can be used to lay the groundwork for genomic analyses of apple diversity and functional loci.
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