Introduction

The cultivated potato, Solanum tuberosum L., is considered the most important tuber crop in the world and among the four most valuable crops world-wide (Ross 1986). Germplasm with adaptation to different climatic and cultural conditions has been essential to the development of new potato cultivars (Tarn et al. 1992). Canadian potato cultivars are unique in their geographic and climatic ranges of adaptation (Anstey 1986; Tarn et al. 1992). Potato breeding in Canada began in 1888 with the goal to improve potato yield of early introductions (Anstey 1986; Turner and Molyneaux 2004). Since then, breeding efforts have gone through several major stages from early selection and adaptation, selection for disease resistance, obtaining disease-free stock, to selection for processing quality (Anstey 1986). Great improvements have been made in traits associated with production, utilization, disease control, in vitro culture, and processing quality (Tarn et al. 1992). Hundreds of potato cultivars have been developed and released, many of which have had significant impacts on Canadian agriculture (Anstey 1986; Guenthner 2001; Turner and Molyneaux 2004). There are still concerns about the narrowing of the North American Solanum gene pool as a result of the small number of parental lines used since potato breeding began (Love 1999; Simko et al. 2004). However, no assessment has been made on the genetic diversity of the Canadian Solanum germplasm.

To enhance potato breeding programs and conserve the Solanum germplasm released from the Canadian potato breeding programs, the Potato Gene Resources Repository was established in 1992 at the Potato Research Centre of Agriculture and Agri-Food Canada, Fredericton, New Brunswick. The Repository is a clonal collection of potato cultivars with an emphasis on heirloom varieties, Canadian-bred cultivars, and elite breeding lines from the Canadian breeding programs. Currently, the Repository contains 132 clones originating from eight countries, of which 119 are maintained in vitro and 13 as tubers. Recently some effort was made to recruit 37 more domestic and exotic accessions for genetic research and possible addition to widen the genetic diversity of the collection. However, little is known about the genetic diversity and genetic structure of this Canadian dominant collection.

Characterization of plant germplasm using molecular techniques has played an increasingly important role in the management and utilization of plant genetic resources (Karp 2002). It can also enhance plant breeding in selection of diverse parents to widen the breeding gene pool (Fu 2006). Efforts have been made to characterize potato germplasm using allozyme (Ortiz and Huaman 2001), restriction fragment polymorphism (RFLP) (Powell et al. 1991), random amplified polymorphic DNA (RAPD) (Hosaka et al. 1994; Demeke et al. 1996; del Rio et al. 1997), amplified fragment length polymorphism (AFLP) (Milbourne et al. 1997; Kim et al. 1998), inter simple sequence repeat (ISSR) (Bornet et al. 2002), and simple sequence repeat (SSR) markers (Ghislain et al. 2004, 2006; Braun and Wenzel 2005; Feingold et al. 2005; Ispizúa et al. 2007). These characterizations have not only provided useful information for understanding the genetic diversity and structure of various potato gene pools established in different geographic regions for an effective management of potato germplasm, but have also facilitated some potato breeding programs by identifying released cultivars and determining their genetic relationships.

The objective of this study was to assess the genetic diversity, structure, association, and distinctiveness of 169 Canadian and exotic potato accessions using SSR markers. It is our hope that this molecular assessment generates some baseline information not only for managing our potato germplasm collection, but also for identifying diverse germplasm for potato breeding.

Materials and Methods

Plant Materials

The potato germplasm used in this study consisted of 132 potato accessions currently held at the Potato Gene Resources Repository, Fredericton, New Brunswick, and 37 recently recruited clones (Table 1). The Repository maintains 68 Canadian cultivars released since the start of modern breeding in the 1930s, 56 heirloom varieties mostly of North American origin, and eight accessions introduced from Great Britain before 1900. The recruited accessions, which are not yet included in the Repository, consisted of 24 modern cultivars, mostly Canadian, and 13 long-day adapted selections of diploid and tetraploid cultivated potato species from the Andes. These Andean accessions represented a set of late blight differentials (the NRBK clones) that were derived from S. demissum Lindl. and some breeding lines. Ten accessions are known to be diploid and all other accessions in the study are believed to be tetraploid. To assess the diversity changes over time, the 68 Canadian cultivars released from 1919 to 2002 were classified into four groups based on date of cross (period 1 from 1919 to 1959; period 2 from 1960 to 1970; period 3 from 1971 to 1980; period 4 from 1981 to 2002). To assess the genetic structure, 169 accessions were grouped into three germplasm types: heirloom variety, modern cultivar, and elite line.

Table 1 Summary of potato accessions studied and SSR variation with respect to country of origin
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DNA Extraction

All clones were grown in the greenhouse at the Potato Research Centre. Young leaves were harvested, placed into plastic bags with paper towels moistened with sterile RO water, placed into coolers with ice packs and shipped to Plant Gene Resources of Canada (PGRC), Saskatoon, Saskatchewan. The leaves were freeze-dried in a Labconco Freeze Dry System for 5 days to 7 days, and stored at −20°C. From each sample, dry leaves were ground to a fine powder in a 2 ml Eppendorf tube with three 3 mm glass beads in a multidirectional shaker. Genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacture’s directions. Extracted DNA was quantified by fluorimetry using Hoechst 33258 stain (Sigma Chemical Co., St. Louis, USA), followed by the dilution to 10 ng µL−1 for SSR analysis.

SSR Analysis

Based on reported polymorphism and genome coverage (Milbourne et al. 1998; Ghislain et al. 2004; Feingold et al. 2005), 14 genomic and 22 EST derived SSR primer pairs were selected for this assessment. All polymerase chain reactions (PCRs) were performed in an MJ Research DYAD thermocycler (BioRad, Mississauga, ON, Canada). PCRs for the STM series of primers (Ghislain et al. 2004) were preformed in a 25 µl total volume using 25 ng genomic DNA, 1× PCR buffer (Promega, ON, Canada), 2.5 mM MgCl2 (Promega, ON, Canada), 0.2 mM each dNTPs, 0.5 µM each forward and reverse primer, and 0.5 U/reaction Taq polymerase (New England Biolabs, ON, Canada). Cycling conditions were 95°C 10 s, Tm for 30 s, 72°C 40 s for 35 cycles. Those annealing temperatures indicated by a range were preformed using a touchdown PCR decreasing by 0.5°C per cycle until the lower temperature was reached. Twenty-five additional cycles were preformed at the lower temperature. PCRs for the StI series of primers (Feingold et al. 2005) were preformed in 25 µl total volume containing 50 ng genomic DNA, 1× ThermoPol buffer (New England Biolabs, ON, Canada) containing a 2 mM MgSO4, 0.2 mM each dNTPs, 0.2 µM each forward and reverse primer, and 0.5 U/reaction Taq polymerase (New England Biolabs, ON, Canada). Cycling conditions were 95°C 10 s, Tm for 30 s, 72°C 40 s for 30 cycles. Those annealing temperatures indicated by a range were preformed using a touchdown PCR decreasing by 1°C per cycle until the lower temperature was reached. Thirty additional cycles were preformed at the lower temperature. The PCR products were separated on a 1.5 mm thick 6% (w v−1) non-denaturing acrylamide:bis-acrylamide (19:1) gel in 1× TBE buffer with 0.5 mg L−1 ethidium bromide for 2 h to 2.5 h (Wang et al. 2003) and recorded on a digital gel documentation system.

Data Analysis

To generate a dataset of SSR allele counts for each accession, DNA fragments amplified by SSR primer pairs were identified based on their sizes in base pairs measured with a 10 bp DNA ladder (Invitrogen, Carlsbad, CA, USA) and compared with the fragment sizes reported in the literature (Ghislain et al. 2004; Feingold et al. 2005). Frequencies of the scored alleles were calculated with respect to primer, country origin, and germplasm type. The number of likely loci detected for each primer pair were determined by the observed allelic pattern and the possible number of alleles per single tetraploid plant. The polymorphic information content (PIC) was calculated for each primer pair, as described in Roussel et al. (2004) with adjustment to the number of likely loci, to assess the informativeness of each marker.

An analysis of molecular variance (AMOVA; Excoffier et al. 1992) that was based on the dissimilarity matrix of pairwise accessions was also performed using Arlequin version 3.1 (Excoffier et al. 2005) to assess the genetic structure of the potato germplasm. This analysis not only allowed the partition of the total SSR variation into within- and among- group variation components, but also provided measures of inter-group genetic distance as the proportion of the total SSR variation residing between any two groups (Phi statistic; Excoffier et al. 1992). Six models of genetic structuring were examined as (1) existing vs new germplasm, (2) germplasm of various country origin, (3) Canadian vs exotic germplasm, (4) Canadian germplasm of four breeding periods, (5) diploid vs tetraploid germplasm, and (6) germplasm type. Significance of resulting variance components and inter-group genetic distances was tested with 10,100 random permutations.

To assess the genetic associations of the potato accessions, two approaches were applied. First, a principal component analysis of 169 accessions was conducted using NTSYS-PC 2.01 (Rohlf 1997) based on the similarity matrix of 232 SSR alleles, and plots of the first three resulting principal components were made to assess the accession associations and to identify genetically distinct accessions. Second, a neighbor-joining analysis of 169 accessions was also made using PAUP* (Swofford 1998) and a circulating tree was displayed using MEGA 3.01 (Kumar et al. 2004) to confirm the genetic association of individual accessions and to identify any genetic clustering without restriction to country of origin or germplasm type.

To assess the genetic distinctiveness of the potato accessions, the similarities of each accession with the remaining accessions assayed were calculated using the simple matching coefficient (Sokal and Michener 1958) as: S ij =(a+d)/(a+b+c+d), where S ij is the SSR similarity between the accession i (i = 1 to n) and the other accession j [j = 1 to (n−1)], a is the number of alleles (from all SSR loci) shared in both i and j, b is the number of alleles present in i but not shared in j, c is the number of alleles present in j but not shared in i, and d is the number of alleles absent from both i and j. The SSR dissimilarity for each pair of accessions can be defined as 1- S ij . The average SSR dissimilarity for the accession i can be obtained by averaging all of the n−1 SSR dissimilarities that the accession was associated with. This average dissimilarity measures the overall genetic difference between the accession (i) of interest and the remaining accessions assayed. A higher average dissimilarity obtained from unlinked markers means that the accession has a genetic background more distinct from the other accessions (Fu 2006). This assessment was done using a specific SAS program written in SAS IML (SAS Institute Inc. 2004). The specific SAS program is available upon request.

Results and Discussion

SSR Variation

The 36 SSR primer pairs detected a total of 64 likely loci on all 12 homologous linkage groups of potato (Table 2; Milbourne et al. 1998; Ghislain et al. 2004; Feingold et al. 2005). Twenty-two likely loci detected by EST-derived SSR markers should represent transcribed chromosomal regions, while the other 42 likely loci detected by genomic SSR markers may largely sample non-genic chromosomal segments. A total of 232 SSR alleles were detected in this study, but they could include some null alleles, because it was difficult to separate non-amplification due to experimental errors from null alleles. The number of detected alleles per primer pair ranged from two for four primer pairs (StI062, StI064, STM1053, STM1017) to 17 for StI049 with an average of 6.4 alleles per primer pair. Values of each marker polymorphic information content (PIC) ranged from 0.01 to 0.49 with an average of 0.25. Note that these PIC values could be biased by the determination of likely loci, as this variation was not significantly (P = 0.08) associated with the number of alleles detected at each primer pair. The three most informative loci were StI062 on linkage group I, StI028 on linkage group XI, and STM0030 on linkage group XII. EST-derived SSR markers appear to be more informative (average PIC of 0.28) than genomic SSR markers (average PIC of 0.19). Thus, the SSR markers revealed a relatively large amount of variation in the sampled genome.

Table 2 Details of 36 SSR markers applied in this study
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The observed occurrence frequencies of the 232 alleles ranged from 0.01 to 0.98 with an average of 0.35 (Table 1). There were 12 alleles with an occurrence frequency of 0.95 or larger in the assayed accessions and 51 with frequencies greater than 0.60, while 55 alleles were detected with frequencies less than 0.05, and 24 alleles with frequencies less than 0.02. Some of the rare alleles may be useful as diagnostic markers for some of the assayed potato cultivars.

Genetic Structure of Potato Accessions

Six models of genetic structuring were examined in this assessment. The proportion of the total SSR variation which resided between existing and newly recruited potato accessions was 1.8% (Table 3), but slightly more diversity was observed within newly recruited accessions. The proportion of the total SSR variation explained by country origin was 2.1% (Table 3). Eight Dutch accessions displayed more within-country variation (Table 1), probably due to the introgression of S. demissum genetic background. The Canadian germplasm (although with 114 accessions) harbored 0.6% less SSR variation than the other 55 potato accessions from seven countries (Table 1), largely due to the mixture of genetic backgrounds of diverse origin. Considering only the 68 Canadian accessions released from 1919 to 2002, it was found that 2.7% of the total SSR variation resided among accessions released over the four major breeding periods. The period 1 accessions (i.e., released before 1960) were the most diverse, followed by the accessions for period 3 (from 1971–1980), period 4 (after 1981), and period 2 (from 1960 to 1970). The proportion of the total SSR variation explained by ploidy levels was 3.7% (Table 3), but diploid accessions unexpectedly displayed more variation than tetraploid accessions. The highest proportion of the total SSR variation (4%) was found to reside among three germplasm types. The modern cultivars were the most diverse, followed by the heirloom varieties and elite lines.

Table 3 Results for analyses of molecular variance (AMOVA) for 169 potato accessions with six models of genetic structuring
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These results indicate that the Solanum germplasm held at the Potato Gene Resources Repository is genetically narrow. Canadian germplasm displayed less SSR variation than accessions from the other countries. Existing diversity is not much improved if newly recruited germplasm is added to the collection. Interestingly, the heirloom varieties harbored slightly less diversity than the modern cultivars. These findings were well illustrated in the genetic associations of 169 potato accessions shown in Fig. 1. The two components accounted for only 8.8% and 7.9% of the total SSR variation. Also, newly recruited accessions had a better spread over the plot than existing accessions (Fig. 1a), as reflected in the lower genetic relatedness among these recruited accessions. The Canadian germplasm was well overlapped with the exotic germplasm (Fig. 1b). The modern cultivars were widely spread over the plot (Fig. 1c).

Fig. 1
figure 1

Plot of the first two principal component scores based on the Euclidean distances converted from the simple matching coefficient matrix of 232 SSR alleles for 169 potato accessions. These two components accounted for 8.8% and 7.9% of the total SSR variance, respectively. a Individual accessions are separately labeled for existing and newly recruited germplasm. b Canadian accessions are identified against those from other countries. c accessions are labeled for germplasm type. Note that a, b and c are the same plot with different labeling for clear illustration

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Limited genetic diversity observed in these potato accessions and little differentiation among potato groups are not surprising (Simko et al. 2006). First, only a small number of introduction events formed the basis of modern potato germplasm (Hawkes 1978; Ames and Spooner 2008). Second, the USA cultivar Katahdin is responsible for almost a quarter of the germplasm that makes up prominent North American cultivars (Love 1999). Third, the Canadian potato breeding has long been limited to a narrow genetic base with fewer introgressions of new exotic germplasm (Anstey 1986; Tarn et al. 1992; Turner and Molyneaux 2004). Recent efforts to incorporate germplasm from Andean cultivated species into some Canadian cultivars (de Jong et al. 2001; Lynch et al. 2004) yielded an improvement only in the level of genetic relatedness, but not in the magnitude of genetic diversity.

Genetic Association of Potato Accessions

Clustering of 169 potato accessions by neighbor-joining analysis revealed several variation patterns (Fig. 2). First, about ten clusters were found, but they were not well separated due to low SSR variation. Second, nine of the ten diploid accessions were clustered together in one group. Third, accessions of three germplasm types were well mixed in various clusters and no single clusters were found separately for heirloom or modern cultivars. Fourth, exotic accessions were widely spread into various clusters, reflecting their different genetic backgrounds. Fifth, five Canadian accessions (Mrs. Moehrle’s Yellow Fleshed, Siberian, LRC 373-5, LRC 4373-5b and 13594-070) and one Germany accession (NRBK-05) were genetically distinct from the other accessions, as reflected in the longer branch lengths. Sixth, some highly related (or genetically duplicated) accessions could be identified. The obvious candidates were the modern cultivar pairs of Canso vs Canus and Rose Gold vs Red Gold and the elite line pair of LRC 373-5 vs LRC 4373-5b, which is expected from the known pedigrees (Lynch et al. 2004). This study confirms the results of two groupings identified by X.-Q. Li (personal communication) through fingerprinting at the Potato Research Centre. One group consists of the blue-fleshed clones Sharon’s Blue, River John Blue, Nova Scotia Blue, MacIntosh Black, Congo, and British Columbia Blue, to which this study added McIntyre Blue. The other is a group of fingerlings: Banana, Kifli, Corne de Mouton, Jogeva Yellow Estonian, and Fingerling.

Fig. 2
figure 2

Clustering of 169 potato accessions obtained from the neighbor-joining analysis of 232 SSR alleles. Accession label includes the accession name, followed by ISO two-letter code for its country origin and one letter code for its germplasm type (H heirloom variety; M modern cultivar; E elite line). Diploid accessions are marked with black diamonds; examples of highly related (or genetically duplicated) accessions with black circles, except the genetically distinct pair of LRC 373-5 and LRC 4373-5b; and examples of congruent or incongruent accessions with respect to known pedigree with black triangles

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Further assessments on the clustering with respect to known pedigree revealed congruency of some accessions. For example, OAC Temagami and OAC Ruby Gold shared the same parents of Rhinered and Red Gold and were grouped together. Similarly, Mirton Pearl and Anson shared the same parents of Mira and F53018 and were clustered together. There were also many inconsistencies between grouping and known pedigree. For example, Acadia Russet and Shepody shared the same female parent of Bake King, but were located in separate clusters. Similarly, Aquilon and Mouraska shared the same female parent of Hudson, but were grouped in different clusters. Inconsistencies between cultivar clustering and known pedigrees are largely expected for these potato accessions with such a narrow genetic base (Loiselle et al. 1989b; Kim et al. 1998; Love 1999). Also, the limited sampling of the potato genome revealed by only 36 SSR loci may contribute to such inconsistencies. Applications of more mapped markers across the genome would improve the resolution to the genetic relationships of these potato cultivars. However, the estimated genetic relationships should still offer a useful guide for potato breeding, as they are more informative than parental selection (Hosaka et al. 1994; Demeke et al. 1996; Kim et al. 1998) and traditional pedigree analysis (Mendoza and Haynes 1974; Loiselle et al. 1989a, b).

Genetic Distinctiveness of Potato Accessions

The genetic distinctiveness of a potato accession was measured by the average dissimilarity (AD) of the accession against the remaining accessions assayed. The higher the AD, the greater is the distinctiveness of the genetic background. The AD of the accessions ranged from 0.241 for Pink Pearl (a Canadian modern cultivar) to 0.353 for NRBK-05 (the germplasm recruited from Germany) with a mean of 0.277 (Table 4). Ranking these AD values generated the genetic distinctiveness for all the accessions assayed. Table 4 lists 31 accessions with the highest AD values and 31 accessions with the lowest AD values. The 31 genetically most distinctive accessions listed in Table 4 contains a diverse group of four diploid accessions, 16 elite lines, ten heirloom varieties, and five modern cultivars. In contrast, among the 31 genetically least distinctive accessions, there were no diploid accessions, one elite line, eight heirloom varieties, and 22 modern cultivars. This contrast demonstrates again the low level of genetic distinctiveness in modern cultivars.

Table 4 Sixty-two potato accessions with the highest (left) or lowest (right) values of average dissimilarity (AD)
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The ADs shown in Table 4 are limited to only those accessions assayed. The AD values would change if more potato accessions were assessed. This method can recognize the distinctiveness, but not necessarily the relatedness, of accessions (Fu 2006). For example, two closely related cultivars that were quite distinct from the remaining cultivars could have similar higher levels of AD than the others and both cultivars would have been identified as genetically distinct. In spite of these limitations, the relative measure of genetic distinctiveness reported here could provide a guide for selecting specific germplasm with distinct genetic background for potato breeding programs (Demeke et al. 1996; Braun and Wenzel 2005).

Implications for Potato Germplasm Management and Breeding

This SSR analysis revealed a narrow genetic base for Canadian potato cultivars generally and specifically for the germplasm held in the Canadian Potato Gene Resources Repository. Although the mandate of the Repository focuses more on the Canadian potato germplasm, more efforts to expand the diversity range of the potato collection are warranted with targeted acquisitions of potato germplasm from countries with temperate climates similar to that of Canada. The characterization data presented here are useful for managing the existing potato germplasm in germplasm addition, accession identification, duplication verification, and germplasm structuring. Also, the analysis showed that Canadian potato germplasm harbored less SSR variation than those accessions from the other seven countries. This narrow genetic base persists in spite of the use in breeding of Andean cultivated potatoes of groups Phureja, Andigena and Tuberosum and as many as 14 wild species (Plaisted and Hoopes 1989; Spooner and Salas 2006). Canadian breeders have worked with germplasm from the Phureja and Andigena Groups (de Jong et al. 1981; Tarn and Tai 1983) and have released disease resistant germplasm (de Jong et al. 2001; Lynch et al. 2004). However, continuing efforts are still needed to diversify the Canadian potato gene pool to ensure sustainable breeding programs in the future. The findings reported here on genetic association and distinctiveness are helpful for parental selection of diverse plants for potato breeding. Moreover, the molecular characterization of potato germplasm generated not only essential information for managing the potato collection, but also provided a useful guide for selecting specific germplasm with distinct genetic background for diversifying potato breeding program.