Edited by: Sun Hee Woo, Chungbuk National University, South Korea
Reviewed by: Md. Shafikur Rahman, Patuakhali Science and Technology University, Bangladesh; Abu Hena Mostafa Kamal, Korea Research Institute of Bioscience and Biotechnology, South Korea
*Correspondence: Nnadozie Oraguzie, Irrigated Agriculture Research and Extension Centre, Washington State University, 24106 North Bunn Road, Pullman, WA 99350, USA. e-mail:
This article was submitted to Frontiers in Crop Science and Horticulture, a specialty of Frontiers in Plant Science.
This is an open-access article distributed under the terms of the
Most previous studies on genetic fingerprinting and cultivar relatedness in sweet cherry were based on isoenzyme, RAPD, and simple sequence repeat (SSR) markers. This study was carried out to assess the utility of single nucleotide polymorphism (SNP) markers generated from 3′ untranslated regions (UTR) for genetic fingerprinting in sweet cherry. A total of 114 sweet cherry germplasm representing advanced selections, commercial cultivars, and old cultivars imported from different parts of the world were screened with seven SSR markers developed from other
Sweet cherry (
The Washington State University (WSU) sweet cherry breeding program came to a stand-still about 25 years ago following the retirement of the then breeder, Dr. Tom Toyama. However, active evaluation of the germplasm continued (although many breeding records were lost) which led to the release of several new sweet cherry cultivars (Oraguzie et al.,
Most published genetic studies in cherry have typically been based on microsatellite or simple sequence repeat (SSR) markers (Dirlewanger et al.,
Previously, SNP analysis was carried out only in plants with large genome sequence databases including
New methods and technologies are continuously being developed and utilized with the aim to improve the identification and genetic characterization of plant species. For example, high resolution melting (HRM) is a recent advance for the detection of SNPs. This technique measures temperature induced strand separation of short PCR amplicons and is also able to detect variation as small as one base pair difference between samples (Hoffmann et al.,
Our aim in this study is to establish genetic identity, verify parentage, and also determine the relatedness of sweet cherry advanced selections, commercial cultivars, and old cultivars obtained from diverse geographical regions, as well as newly released cultivars from the WSU sweet cherry breeding programme, using SNP markers. We will validate the results by comparison with data generated by screening similar germplasm with SSR markers.
A total of 110 and 103 sweet cherry cultivars respectively, were used for the SNP experiment and the SSR study (Table
Cultivar/Selection | Presumed parentage | Origin | Self (in) compatibility status |
---|---|---|---|
99FI131RJ (0900A) | – | Turkey | – |
99FI132R4 | – | Turkey | – |
99FI150RJ (0900) | – | Italy | – |
99FI154R4 | – | Italy | – |
Ambrunes | Unknown | Spain | |
Angela | Unknown | – | |
Attika/Kordia | Unknown | Czechoslovakia | |
Balaton | Tart cherry, |
Hungary | – |
Benton | Stella × Beauliue | WSU | |
Bing | Black Republican op | USA | |
Black Republican | Unknown | – | |
Black Gold | Unknown | – | – |
Black Tartarian | Unknown | – | |
Chelan | Stella × Beaulieu | WSU | |
Columbia | Benton synonym? | WSU | |
Compact Stella | Unknown | BC, Canada | |
Coral | Unknown | – | |
Corum | Unknown | Turkey | |
Cristalina | Star × Van | BC, Canada | |
Dame Roma | Black Douglas × Stella | Australia | |
Early Robin | Unknown | BC, Canada | |
Ebony | Unknown | – | – |
Emperor Francis | Unknown | – | |
Gil Peck | Napoleon × Giant | Cornell University, USA | |
Hedelfingen | Unknown | Europe | |
Kiona | Glacier × Cashmere | WSU | |
Kootenay Lambert | Unknown | – | – |
Kreiger | Unknown | – | – |
Kristen | Emperor Francis × Gil Peck | Cornell University, USA | |
Lala Star | Compact Lambert × Lapins | Italy | |
Lambert 685 | Napoleon × Blackheart | Western US | |
Lamida | Lambert op | Idaho, USA | |
Lapins | Van × Stella | BC, Canada | |
Larian | Unknown | CA, USA | |
Lyons | Unknown | – | |
Margit | Germersdorfer op | Hungary | |
Merton Biggareau | Unknown | – | |
Merton Heart | Unknown | England | |
Moby Dick | Unknown | Australia | – |
Moreau | Unknown | France | |
Napoleon | Unknown | France | |
NY 54 | Unknown | USA | |
Olympus | Lambert × Van | Cornell University, USA | |
13/20 | Moldevian Black × H236 | Hungary | – |
6/240 | Unknown | Hungary | – |
PC7144-11 | Stella × Early Burlat? | WSU | – |
PC7146-16 | Stella × Beaulieu | WSU | – |
PC7146-17 | Stella × Beaulieu | WSU | |
PC7147-1 | Stella op | WSU | – |
PC7147-9 | Stella op | WSU | |
PC7214-3 | – | WSU | – |
PC7217-2 | Bing × Stella | WSU | – |
PC7336-1 | – | – | – |
PC7309-4 | Stella × Bing | WSU | – |
PC7614-2 | – | WSU | – |
PC7635-4 | – | WSU | – |
PC7903-2 | – | WSU | |
PC7636-1 | – | WSU | – |
PC8005-1 | – | WSU | – |
PC8008-10 | – | WSU | – |
PC8008-5 | – | WSU | – |
PC8008-8 | – | WSU | – |
PC8011-10 | PC7147-4 × PC66138-2 | WSU | – |
PC8011-2 | PC7147-4 × PC66138-2 | WSU | – |
PC8011-3 | PC7147-4 × PC66138-2 | WSU | – |
PC8011-4 | PC7147-4 × PC66138-2 | WSU | – |
PC8011-5A | PC7147-4 × PC66138-2 | WSU | – |
PC8011-5B | PC7147-4 × PC66138-2 | WSU | – |
PC8011-6 | PC7147-4 × PC66138-2 | WSU | – |
PC8012-1 | – | WSU | – |
PC8012-5 | – | WSU | – |
PC8012-9 | – | WSU | – |
PC8014-1 | – | WSU | – |
PC9805-104 (HH) | PC7214-3 op | WSU | – |
PC9816-144 (EE) | PMR-1 × Rainier | WSU | |
PC9816-31 (FF) | PMR-1 × Rainier | WSU | – |
PC9816-67 (AA) | PMR-1 × Rainier | WSU | – |
PC9816-104 (DD) | PMR-1 × Rainier | WSU | – |
PC9816-96 (JJ) | PMR-1 × Rainier | WSU | |
PC9817-97(GG) | Rainier × PMR-1 | WSU | |
PMR-1 | Unknown | WSU | |
Rainier | Bing × Van | WSU | |
Regina | Schneiders × Rube | Germany | |
Salmo | Lambert × Van | BC, Canada | |
Sam | Windsor op? | BC, Canada | |
Sandra Rose | 2C-61-18 × Sunburst | BC, Canada | |
Santina | Stella × Summit | BC, Canada | |
Saylor 153 | Unknown | – | – |
Saylor Ocis | Unknown | – | – |
Schnieders Spate | Unknown | Germany | |
Selah | P8–9 | Stella | |
Seneca | Unknown | – | |
Sir Tom | Black Douglas × Stella | Australia | |
Skeena | (Bing × Stella) × (Van × Stella) | BC, Canada | |
Sparkle | Unknown | – | |
Spalding | Unknown | USA | |
Star | Unknown | BC, Canada | |
Stella | Lambert × J12420 | BC, Canada | |
Sunburst | – | BC, Canada | |
Sunset Bing | Unknown | USA | – |
Sweet Anne | Unknown | – | – |
Sweet September | Unknown | – | – |
Sweetheart | Van × Newstar | BC, Canada | |
Tieton | Stella × Early Burlat | WSU | |
Valera | Unknown | Vineland, Canada | |
Van | Empress Eugenie op | Summerland, Canada | |
Vega | Bing × Victor | Vineland, Canada | |
Velvet | Unknown | – | |
Venus | Hedelfingen × Windsor | – | |
Vic | Unknown | Vineland, Canada | |
Vista | Hedelfingen × Victor | Vineland, Canada | |
Viva | Unknown | Vineland, Canada | |
Vogue | Unknown | ||
Windsor | Unknown | – |
The methodology for SNP discovery and primer design is described in Koepke et al. (
SNP | Forward primer | Reverse primer |
---|---|---|
SNP2 | GCCATGAACCAGCTTGTAGC | AGCTGAGCTCGCAAAACC |
SNP4 | TTATCAAGACGCTTGCCTGGT | GAAGGAAACCCCCAAAATGT |
SNP5 | TTGGTTTGAAGTGCTGAGGA | TCCTGGAGAAATAACCATTCAAA |
SNP8 | TTTCTGCAAGTAGCAAACTCCA | TTGTGGCTCAAACTTTTTGCT |
SNP16 | GCCAATTATCGTGATTTCCA | CACTTGGGCTACAAACCACA |
SNP18 | GGCTTACCATTTTCCTCAAGC | TTCGGTAAATTGCCAAAACA |
SNP23 | TTGATCTGTTTGGATTTTGGTC | GCACCCCTTTTCCATTCATA |
SNP24 | TTTTAAGTGCATCCATGTTGTG | CCCCTGAAAGCAATCTTCAC |
SNP32 | TGGTGAGTTTCTCCCCATGT | AAAAGTCTGAGCCAATGGGATA |
SNP34 | TTGCATTTGGTGACTTCAGG | CCAAATAAATTAGAAATCCAAGTCG |
SNP36 | TCTGTGGAACATAATTCAAAATGTA | TGTTACACAGGTCGAATGCAA |
SNP44 | TGTTTGGTTTATGGGCAACA | ATGACGTTTGCACTGTGAGC |
SNP52 | TGTTTTATGTCTGTTTTATGTTGTACG | CACATTCATGGTGGCCAAA |
SNP54 | TCTTGTCGGCTACATTCTCG | ACCAATCACAGTAGCAAACTGA |
SNP55 | TCGCTGCTGTCTTGGTTATG | CCACCAACTCATGCATTTACA |
SNP59 | TTTACGGATTTGTTTTGCTGT | TTTCATGGTAAAGATCAAGAATCA |
SNP65 | TGCTGCTCTGGAGAAGGATT | CTCCTTCATCGCCATCATCT |
SNP69 | GGCGTCCTATTCTATTTCTTCAA | GAAAGAATACCTTTTTCCAACGAA |
SNP82 | CATTCTCACTTTCTTCAGCATTTT | TCTTCTTGCTTCCTTGTCGAG |
SNP91 | TCATCCCCATCTTCAAGGTC | CCTGAATAACAAATATACCCGACA |
SNP100 | GAGGAAAACGGTATTCTGATGC | GGGACTTCCTTTCCAACCTC |
SNP101 | AAGCCTCGGCAGATGAATTA | GCAAAACTCCGACTCCAAAG |
SNP110 | ACATGGCATGGTGGAAGTTT | TGGTTGAAGAAAGGCTGTTCA |
SNP120 | AGCAGAAATTGACTCCATTGAA | TTGAGGATTTTTCAGCTTTTGA |
SNP122 | AGCGAAGCAGATCCAGAAGA | TTCCAAAACCAGAACCTTCAA |
SNP128 | GAACGACAACATTTCGTATTGC | CAACAAGAACGAACGCTCAA |
SNP138 | AGCACTTTAGGATGGCAAGG | GCTTCAAATTAGCACCGATGA |
SNP144 | GAAAGAGACAATCTACCAGTGATGAA | TTGCATTCAAAAGCAATCCA |
SNP161 | TCAAAGCCCTTGGATCATTC | TTTCCCACCCTAACCATGAC |
SNP172 | GGATGATGAAGGGGAGGATT | AGGCATCACCACATGACAGA |
SNP173 | ACTACCACGCCACAGGTGAT | GGAAAAATGAAAGCCACACC |
SNP174 | TGTGTTAGGGAATATGGAAAAATC | AAATTGACATTTGCTCGCTTC |
SNP181 | AAGTGACCTGCCTCTGCAAT | TATCAACCCCATCCATCTGC |
SNP189 | GGATCCTGGGGGATGTATTC | CTCGTTGCCATAGTCGAACA |
SNP195 | AAGCAGACAGTGGATCATTCC | CAATGATAGAGATCAGTAAGTGGGAT |
SNP197 | TACCCTCGTCAGGGATCTTG | TTAAGCACACCACGCATTTT |
SNP202 | GCCATGTGGTTGTAGCAGAA | TGGAATACTCCAACCCTAAGC |
SNP204 | AGTTGGTGTGCAAAAATAGCA | GGCGTTCATTTCCATCATTT |
SNP212 | TCTCGTGCTTCTTGCCTTCT | TCCTAACAACTTTTCACAATCACC |
SNP221 | TCACATTCATATCAGTGTCCTGTC | CCTACGAGCTTTTGCCACAT |
PCR amplifications were performed on a LightCycler®480 Real-Time PCR System (Roche, EEUU). A total of 100 SNP primers were used initially to screen a panel of 30 sweet cherry cultivars of which 40 that showed polymorphism were chosen for screening all germplasm (including the initial 30 cultivars and the remaining 80 accessions). Cultivar Dame Roma was also extracted and run twice to check for marker consistency along with the 30 initial sweet cherry cultivars. HRM PCR amplifications were performed in a total volume of 12.5 μl, using the LightCycler®480 HRM Master 1× with 2.5 mM MgCl2, 0.2 μM of each primer, and 2.5 ng of genomic DNA. All the SNP primers were amplified using the same PCR conditions including 10 min of pre-incubation at 95°C; 45 cycles of amplification at 95°C for 10 s, 58°C for 20 s, and 72°C for 30 s; one cycle of HRM at 95°C for 1 min, 40°C for 1 min, 65°C for 1 s, and finally, one cycle of cooling at 40°C for 10 s. Polymorphism was scored based on the melting temperature (
Seven SSR markers previously developed in peach, almond, and sweet cherry (Table
SSR locus | Sequence | Ta | LG | Reference |
---|---|---|---|---|
CPPCT021F | CGGATCCCAGTTGTATTAAATG | 60 | G6 | Aranzana et al. ( |
CPPCT021R | GAGGAACTGGTTATCACCTTGG | |||
PMS02F | CACTGTCTCCCAGGTTAAACT | 55 | G2 | Cantini et al. ( |
PMS02R | CCTGAGCTTTTGACACATGC | |||
PMS40F | TCACTTTCGTCCATTTTCCC | 55 | G1 | Cantini et al. ( |
PMS40R | TCATTTTGGTCTTTGAGCTCG | |||
PS12e2F | GCCACCAATGGTTCTTCC | 56 | G7 | Joobeur et al. ( |
PS12e2R | AGCACCAGATGCACCTGA | |||
CPDCT025F | GACCTCATCAGCATCACCAA | 62 | G3 | Mnejja et al. ( |
CPDCT025R | TTCCCTAACGTCCCTGACAC | |||
EPPCU9168F | TCCCTTCTCCATGTTTTCCA | 60 | G4 | Howad et al. ( |
EPPCU9168R | GGAATCGGCATAAGCAAAA | |||
EPDCU5100F | CTCTTCTCGCCTCCCAATTT | 57 | G1 | Howad et al. ( |
EPDCU5100R | TGCTTAGCCCTGGGTACAAG |
Both SNP and SSR data were scored on the basis of presence or absence of marker alleles and this was used to generate a binary matrix. This data was used to estimate genetic similarity between individuals based on Nei and Li (
Initial screening of a panel of 30 sweet cherry cultivars with 100 SNP markers resulted in the pre-selection of 40 highly polymorphic primers for further amplification of the rest of the germplasm. The allele call was based on the
SNP locus | Ho | He | PIC | |
---|---|---|---|---|
SNP2 | 0.50 | 0.53 | 6.00 | 0.52 |
SNP4 | 0.47 | 0.52 | 6.00 | 0.51 |
SNP8 | 0.53 | 0.54 | 6.00 | 0.53 |
SNP16 | 0.40 | 0.59 | 5.00 | 0.59 |
SNP18 | 0.48 | 0.52 | 6.00 | 0.52 |
SNP23 | 0.40 | 0.58 | 5.00 | 0.58 |
SNP24 | 0.29 | 0.42 | 9.00 | 0.41 |
SNP32 | 0.24 | 0.72 | 7.00 | 0.72 |
SNP34 | 0.37 | 0.58 | 5.00 | 0.58 |
SNP36 | 0.15 | 0.43 | 4.00 | 0.42 |
SNP44 | 0.49 | 0.52 | 6.00 | 0.52 |
SNP52 | 0.40 | 0.58 | 5.00 | 0.58 |
SNP54 | 0.42 | 0.58 | 5.00 | 0.58 |
SNP55 | 0.49 | 0.53 | 6.00 | 0.52 |
SNP59 | 0.14 | 0.63 | 5.00 | 0.63 |
SNP65 | 0.45 | 0.59 | 5.00 | 0.59 |
SNP69 | 0.51 | 0.52 | 6.00 | 0.52 |
SNP82 | 0.25 | 0.71 | 7.00 | 0.71 |
SNP91 | 0.14 | 0.66 | 5.00 | 0.66 |
SNP100 | 0.51 | 0.53 | 6.00 | 0.53 |
SNP101 | 0.66 | 0.66 | 7.00 | 0.66 |
SNP110 | 0.46 | 0.51 | 6.00 | 0.51 |
SNP120 | 0.25 | 0.40 | 9.00 | 0.39 |
SNP122 | 0.27 | 0.73 | 7.00 | 0.73 |
SNP128 | 0.44 | 0.51 | 6.00 | 0.50 |
SNP138 | 0.52 | 0.51 | 5.00 | 0.50 |
SNP144 | 0.55 | 0.54 | 6.00 | 0.54 |
SNP161 | 0.41 | 0.59 | 5.00 | 0.59 |
SNP172 | 0.38 | 0.57 | 5.00 | 0.57 |
SNP173 | 0.64 | 0.67 | 7.00 | 0.66 |
SNP174 | 0.22 | 0.38 | 9.00 | 0.37 |
SNP181 | 0.45 | 0.59 | 5.00 | 0.59 |
SNP189 | 0.25 | 0.40 | 9.00 | 0.39 |
SNP195 | 0.49 | 0.52 | 6.00 | 0.52 |
SNP197 | 0.46 | 0.51 | 6.00 | 0.51 |
SNP202 | 0.42 | 0.58 | 5.00 | 0.58 |
SNP204 | 0.52 | 0.53 | 6.00 | 0.53 |
SNP212 | 0.40 | 0.60 | 5.00 | 0.59 |
SNP221 | 0.27 | 0.40 | 9.00 | 0.40 |
Mean | 0.403 | 0.5519 | 6.00 | 0.55 |
SD | 0.129 | 0.0871 |
All 7 SSR loci from the different
SSR locus | Ho | He | PIC | |
---|---|---|---|---|
CPDCT025 | 0.32 | 0.49 | 4.00 | 0.48 |
CPPCT021 | 0.30 | 0.56 | 4.00 | 0.55 |
EPDCU5100 | 0.45 | 0.65 | 5.00 | 0.65 |
EPPCU9168 | 0.59 | 0.79 | 10.00 | 0.79 |
PMS02 | 0.44 | 0.80 | 8.00 | 0.80 |
PMS04 | 0.65 | 0.66 | 8.00 | 0.65 |
Ps12e2 | 0.60 | 0.78 | 11.00 | 0.78 |
Mean | 0.44 | 0.68 | 7.14 | 0.67 |
St. Dev | 0.21 | 0.12 |
Although some sweet cherry cultivars have previously been characterized using molecular markers, most selections in this study were genotyped for the first time using SNP and SSR markers. The dendrograms from UPGMA cluster analysis were constructed using 110 and 103 accessions, respectively, for both the SNP and SSR studies. There was no clear pattern of differentiation of the germplasm based on country of origin. However, both “Balaton” (tart cherry) and “NY 54” (wild cherry) used as an out-group grouped together in cluster 1 (SNP dendrogram) and in cluster 3 (SSR dendrogram) were clearly distinguished from sweet cherry (Figures
Both SNPs dendrogram and SSR dendrogram showed three main clusters, with Cluster 2 further subdivided into two sub-clusters which include most of the selections and cultivars analyzed (75%). However, in the SSR dendrogram some of the relationships are not consistent with presumed pedigree relationships of the accessions (Figure
The close relationship of “Dame Roma,” “Tieton,” “Benton,” and “Sweetheart” in the upper part of Cluster 2 is clearly supported with “Stella” being one of the parents of “Dame Roma,” “Tieton,” and “Benton,” and the grand parent of “Sweetheart.” Both “7217-2” (“Bing” × “Stella”) and “7214-3” (with unknown parentage) in the lower part of Cluster 2 are related through both “Bing” and “Stella” as parents based on their inheritance of alleles from both cultivars. Moreover, both came from crosses made in 1972 in the WSU breeding programme. Also, the close affinity of “7309-4” (“Stella” × “Bing”) with “Kiona” (“Glacier” × “Stella” × “Early Burlat”) × “Cashmere” (“Stella” × “Early Burlat”) in the lower part of Cluster 2 confirms their relatedness through “Stella.” Finally, “Lala Star” presumed to be an offspring of “Lambert” and “Lapins” grouped with “8011-6,” “8011-5B,” and “Angela” in the upper part of Cluster 2. Although the pedigree information on these accessions is not available, the close affinity with “Lala Star” is likely to be through “Lapin’s” grandparent, “Stella.” The heavy use of “Stella” in breeding programmes to impart self fertility following introduction in 1968 (Lapins,
Cluster 3 on the other hand, shows some groupings that seem to contradict the pedigree relationships of some accessions. For example, the close grouping of “Black Gold” (with unknown parentage) with “Regina” (“Schneiders” × “Rube”) and “Selah” (“P8-9” × “Stella”). “Selah” is self-fertile (
Molecular markers have been successfully used for the study of genetic relationship in sweet cherry. These studies have been carried out using isoenzyme markers (Ducci and Santi,
Simple single nucleotides loci are generally bi-allelic and it is supposed to have occurred only once in evolutionary time. As a consequence SNPs are generally polymorphic only for a particular species or genetic material. Thus, contrary to SSRs, these markers are being created to evaluate variation within a specific species, which is in this study
The Ho obtained with SNPs and SSRs were similar; 0.40 vs. 0.44. Furthermore, the Ho is very close to those reported in other sweet cherries studies such as Ohta et al. (
Although sweet cherry is characterized by GSI, the level of heterozygosity observed here is much lower than in other
Assessment of genetic diversity of cultivated sweet cherries may aid in crop improvement strategies. Molecular markers such as SNPs may be applied to assess diversity at the DNA level and thus provide an effective tool for decision making regarding the choice of parental genotypes for use in crosses and for germplasm conservation.
In our study, a dendrogram using SSR markers was also generated in order to be compared with the one generated using SNPs. We observed in both cases, that the cultivars were grouped into three main clusters, with the second cluster subdivided into additional subgroups. As expected, both dendrograms are not identical, but share at least 75% of the clusters. The cultivar “Compact Stella,” which is a mutation of “Stella,” could not be differentiated from “Stella” using SSR markers but this was possible with SNPs. This is probably due to the intrinsic nature of SSRs and its inability to differentiate mutants that differ from the original genotype in one gene. It also suggests that SNPs may be able to distinguish somatic mutants from the mother plants.
The history of the pedigree relationships of sweet cherry is fraught with challenges including complications with selfing, outcrossing, and presence of synonyms. Of a total of 243 seedlings from 22 populations screened with PCR-based
Although the 40 SNPs used in this study were able to identify duplicates, confirm most parentages and also determine relatedness of the accessions, more SNPs may be necessary for a better resolution of the relationships.
Although contrary to conventional approaches, we analyzed melting peaks and scored
The study has demonstrated the utility of SNPs markers for genetic fingerprinting in sweet cherry. Although SSRs had a higher mean number of alleles per locus as well as higher heterozygosity and PIC values than SNPs, both markers showed similar groupings for the sweet cherry accessions as shown in the dendrogram. In fact, SNPs were able to distinguish between mutants and their wild type germplasm thus making SNPs a more valuable tool for cultivar fingerprinting and identification than SSRs. Further, SNPs confirmed the parentage of some accessions and determined relationships consistent with pedigree relationships. We recommend the use of SNPs for genetic fingerprinting, parentage verification, gene mapping, and study of genetic diversity in sweet cherry and believe it will prove useful in most other related species within Amygdaloidea as well.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Sarah O’Neill, a High School intern in Dr. Oraguzie’s lab, for carrying out the initial DNA extraction and HRM analysis. Nnadozie Oraguzie, Amit Dhingra, Tyson Koepke, Angel Fernandez i Marti, and BlessingAthanson designed the study. Amit Dhingra and Tyson Koepke performed transcriptomic sequencing and identified SNPs while Nnadozie Oraguzie and Amit Dhingra designed the primers. BlessingAthanson and Angel Fernandez i Marti collected leaf samples and performed melting curve analysis on the LightCycler 480. Angel Fernandez i Marti performed SSR analyses and along with CF carried out data analyses while Nnadozie Oraguzie supervised the research and guided data interpretation. Angel Fernandez i Marti and Nnadozie Oraguzie wrote the paper. All authors read and approvedthe final manuscript. Angel Fernandez i Marti was supported by a post-doctoral fellowship (CA-1-10) from the CAI/government of Aragon/(Spain). This work was funded in part with Royalty funds from the Sweet cherry breeding program, Washington State University, Irrigated Agriculture and Extension Centre, Prosser, WA, USA. We thank Drs. Chuck Brown and Kate Evans for their discussion and critical review of the manuscript.