Simple Sequence Repeat Fingerprinting Research Articles

Assessment of Genetic Diversity and Relatedness of Local Banana (Musa spp.) Cultivars in Malaysia Using Simple Sequence Repeat (SSR) Markers

Banana (Musa spp.) is an economically important crop widely consumed all over the world. Understanding the genetic diversity and relatedness between various banana cultivars is important to continuously improve its fruit characteristics, nutritional value, and disease resistance. This study aimed to develop a DNA fingerprinting panel using polymorphic simple sequence repeat (SSR) markers for the genetic characterisation of 11 banana cultivars in Malaysia. A total of 10 polymorphic SSR markers were developed into a multiplex PCR reaction and capillary electrophoresis to uniquely profile our collection of banana cultivars. The developed fingerprinting panel successfully amplified a total of 95 alleles, with 6 to 13 alleles per SSR marker. The average SSR marker polymorphic information content (PIC) value is 0.812, indicating the informativeness of the panel. Analysis of molecular variance (AMOVA) shows that 97% of the molecular variance from our banana collection is due to inter-variety genetic diversity, while the remaining 3% is due to intra-variety genetic diversity. A population structure analysis groups our collection of banana varieties according to the presence of at least one M. balbisiana (B) genome in their genetic makeup. The Cavendish variety, however, showed a distinct structure compared to the other cultivars. This SSR fingerprinting panel provides valuable insights into the genetic diversity and relatedness between banana cultivars in Malaysia. It has the potential to assist future banana breeding initiatives and serve as an effective quality control measure for verifying varieties in a tissue culture facility involved in banana planting materials production.

Assessment of Genetic Diversity and Fingerprinting of Sugarcane Varieties Using Simple Sequence Repeat (SSR) Markers

Simple sequence repeat (SSR) fingerprinting was chosen because of its high polymorphism, which enables precise analysis of genetic diversity, for the assessment of genetic variety among several sugarcane varieties. This method focuses on certain areas of the sugarcane genome and offers important insights into the distinctive genetic patterns and relationships between the many sugarcane types under study. To increase sugarcane production and resilience, crop development plans, breeding programmes, and conservation activities must be guided by the data obtained by SSR fingerprinting. This study aimed to assess the genetic diversity and fingerprinting of eight sugarcane varieties (Isd 16, Isd 20, Isd 21, Isd 24, Isd 28, Isd 29, Isd 30, and Isd 31) using four SSR primers. A simple and efficient method for DNA isolation was employed, and the primers successfully amplified a total of 59 bands from the eight varieties. The results showed that the marker SMC687CS exhibited the highest number of alleles and genotypes per locus, followed by SMC336BS and SMC334BS, while SMC119CG showed the lowest. The highest PIC value was obtained from the marker SMC687CS, indicating its high level of polymorphism. The four SSR markers were able to distinguish 56.25% of all the cultivars evaluated, with the highest linkage distance recorded between the varieties Isd 24 and Isd 28. This study identified and classified the eight sugarcane varieties, indicating genetic differences among them that coincide with their field performances.

Construction of Longan (Dimocarpus longan Lour.) Simple Sequence Repeat Fingerprints and Application in Distinctness, Uniformity and Stability Testing

Simple sequence repeat (SSR) markers are highly polymorphism, good reproducibility, abundant number and co-dominant inheritance, and were considered to be one of the preferred molecular markers for DNA fingerprints in distinctness, uniformity and stability (DUS) testing. In this study, 10 representative Dimocarpus longan Lour (longan) varieties with significant differences were selected from 63 longan varieties according to the morphological characteristics. Based on PCR amplifications of the 10 selected varieties, 24 SSR primers pairs were screened from total 300 SSR primers pairs, to establish SSR fingerprints for all 63 longan varieties. The results showed that a total of 127 alleles were detected in 63 longan varieties, with an average of 5.29 alleles for each pair of primers. The Shannon’s index of the 24 pairs of SSR markers ranged from 0.64 to 1.58, with an average of 1.20. The polymorphism information content of each locus ranged from 0.32 to 0.72, with an average of 0.58. Clustering analysis indicated that most of the varieties with close genetic relationships tended to fall in the same cluster, and only a few in different clusters or sub-clusters. The 63 longan varieties were completely identified by the optimal combination of 6 pairs of SSR primers (LY161, LY252, LY137, LY130, LY25 and LY34). Overall, DNA fingerprints of the 63 longan varieties were constructed based on these 24 pairs of SSR primers. This study may provide a technical support for variety identification and similar variety screening in DUS detection in longan.

Reliable Propagation of Persian Walnut Varieties Using SSR Marker-based True-to-type Validation

Simple sequence repeat (SSR) markers were used to authenticate ramets of 11 Persian walnut (Juglans regia L.) varieties. All varieties and 28 of their ramets (n = 39) were genotyped with 17 SSR markers. The genetic profiles revealed two off-types: the ramets Serr 4 (S4) and Vina 1 (V1). SSR fingerprints individuating 11 walnut varieties were possible using 13 polymorphic SSRs that could be used in the future to identify clones of these varieties. Except for ‘Chandler’, each cultivar could be distinguished using a combination of two SSR loci. This result emphasizes the efficacy of the SSR markers in true-to-type validation of walnut orchards.

Simple Sequence Repeat and S-Locus Genotyping to Assist the Genetic Characterization and Breeding of Polyploid Prunus Species, P. spinosa and P. domestica subsp. insititia

Polyploid Prunus spinosa (2n = 4 ×) and P. domestica subsp. insititia (2n = 6 ×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programs. In Hungary, 16 cultivar candidates and a recognized cultivar ‘Zempléni’ were selected from wild-growing populations including ten P. spinosa, four P. domestica subsp. insititia and three P. spinosa × P. domestica hybrids (2n = 5 ×) were also created. Genotyping in eleven simple sequence repeat (SSR) loci and the multiallelic S-locus was used to characterize genetic variability and achieve a reliable identification of tested accessions. Nine SSR loci proved to be polymorphic and eight of those were highly informative (PIC values ˃ 0.7). A total of 129 SSR alleles were identified, which means 14.3 average allele number per locus and all accessions but two clones could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified and the complete and partial S-genotype was determined for 10 and 7 accessions, respectively. The DNA sequence was determined for a total of 17 fragments representing 11 S-RNase alleles. ‘Zempléni’ was confirmed to be self-compatible carrying at least one non-functional S-RNase allele (SJ). Our results indicate that the S-allele pools of wild-growing P. spinosa and P. domestica subsp. insititia are overlapping in Hungary. Phylogenetic and principal component analyses confirmed the high level of diversity and genetic differentiation present within the analysed accessions and indicated putative ancestor–descendant relationships. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species but non-related accessions sharing common S-alleles may distort phylogenetic inferences.

Autochthonous Austrian Varieties of Prunus avium L. Represent a Regional Gene Pool, Assessed Using SSR and AFLP Markers

Sweet cherry production faces new challenges that necessitate the exploitation of genetic resources such as varietal collections and landraces in breeding programs. A harmonized approach to characterization is key for an optimal utilization of germplasm in breeding. This study reports the genotyping of 63 sweet cherry accessions using a harmonized set of 11 simple sequence repeat (SSR) markers optimized in two multiplexed PCR reactions. Thirty-eight distinct allelic profiles were identified. The set of SSR markers chosen proved highly informative in these germplasm; an average of 6.3 alleles per locus, a PIC value of 0.59 and above-average expected and observed heterozygosity levels were detected. Additionally, 223 amplified fragment length polymorphism (AFLP) markers derived from eight selective primer combinations were employed to further differentiate 17 closely related accessions, confirming the SSR analysis. Genetic relationships between internationally known old cultivars were revealed: SSR fingerprints of “Schneiders Späte Knorpelkirsche” and “Germersdorfer” were found to be identical to those of the standard cultivar “Noire de Meched”, among others, whereas four accessions known as “Hedelfinger Riesenkirsche” and four known as “Große Schwarze Knorpelkirsche” showed allelic differences at various loci. The genetic diversity of locally-grown cultivars worldwide might be currently underestimated. Several autochthonous Austrian sweet cherry germplasm accessions were genotyped for the first time and their genetic relationships analyzed and discussed. Interestingly, seven Austrian sweet cherry landraces were shown to be clearly genetically separated from international and modern varieties, indicating that Austrian germplasm could include valuable genetic resources for future breeding efforts.

A new SSR fingerprinting set and its comparison to existing SSR- and SNP-based genotyping platforms to manage Pyrus germplasm resources

Ensuring the clonal identity of accessions within a germplasm repository, such as the US National Pyrus collection, is critical. Additionally, pedigree confirmation and understanding population structure is an important part of breeding and managing genetic resources. The ability to validate pedigree and identity is challenging and inconclusive through morphology alone. DNA information can be used to confirm parentage and identity by descent. A Pyrus fingerprinting set was previously developed by the European Cooperative Program for Plant Genetic Resources (ECPGR) and consists of 12 dinucleotide-containing simple sequence repeats (SSRs) markers that are amplified in two PCR reactions. The ECPGR set is difficult to use because dinucleotide-containing SSRs often exhibit a number of amplification artifacts such as stutters, split peaks, and binning errors. High-core repeat (3–6 bp motifs) SSRs do not exhibit many of the artifacts displayed by dinucleotide-containing SSRs. Therefore, an easy-to-use 10-SSR fingerprint set containing high-core repeat SSRs that can be evaluated in a single reaction was developed. This fingerprinting set, known as the US Pyrus Genetic Resources (USPGR) set, was compared with the ECPGR set and to single nucleotide polymorphism (SNP) markers found on the new 70K pear Axiom™ array for its ability to assess diversity, population structure, pedigree, and identity. The USPGR set performed similarly to both genotyping platforms while being easier to use. Additionally, the present study demonstrates the usefulness of SSRs in the age of high-throughput genotyping and sequencing platforms.

Microsatellite DNA fingerprinting of Coffea sp. germplasm conserved in Costa Rica through singleplex and multiplex PCR

Abstract A large collection of coffee genetic resources is conserved in Costa Rica. In this study, microsatellite DNA fingerprinting of coffee through singleplex and multiplex PCR approaches coupled with capillary electrophoresis are described. To validate both methods, germplasm of Coffea spp. (Arabica and non-Arabica) and intraspecific F1 hybrids were analyzed using fourteen microsatellite markers. It was observed that through both PCR methods the fingerprinting profile of a subset of samples was identical. The genetic analyses revealed that non-Arabica coffee displayed greater genetic variation than Arabica coffee did. In addition, microsatellite analyses allowed the separation of C. arabica from other species using the principal coordinate analysis (PCoA) approach. The neighbor-joining tree clustering analysis revealed either a grouping of wild genotypes separated from cultivars of C. arabica, or a relation of intraspecific F1 hybrids with parental lines. The utility of our methodology for the characterization of F1 hybrids not previously analyzed through SSR (Simple Sequence Repeats) fingerprinting is demonstrated.

Severe outbreaks of Phytophthora infestans on potato in Turkey caused by recent changes in the pathogen population structure

Potato late blight, caused by Phytophthora infestans, has been the most damaging disease in Turkey since 2010. In this study, 127 isolates of P. infestans were obtained from the main growing areas of Turkey between 2015 and 2017. Their phenotypic and genotypic features were revealed and presented with those of reference isolates. These isolates were categorized by their mating type, in vitro mefenoxam sensitivity, mtDNA haplotype, RG57 DNA fingerprinting patterns, simple sequence repeat (SSR) markers, and aggressiveness on a set of potato differential lines. All isolates were of the A2 mating type and mtDNA haplotype Ia, were resistant to mefenoxam, and had RG57 and SSR fingerprints similar to the 13_A2 clonal lineage reported in Europe. This is the first report of 13_A2 in Turkey. Virulence abilities against potato resistance (R) genes R1, R2, R3, R4, R6, R7, R10, and R11 were observed in most of the isolates. The mating type ratios and SSR marker analysis indicate that in Turkey, the sexual reproduction of P. infestans is limited. These results underline that the movement of asexual individuals and the generation of sub-clonal difference are the factors driving the population structure of P. infestans in Turkey.

Keragaman Genetik Kultivar Pisang Diploid (AA) Koleksi Cibinong Science Center Berdasarkan Marka RAPD dan ISSR

The banana (Musa acuminata Colla) is considered as an important crop plant due to its high economic value which also has good dietary source. Here, the genetic variation of 20 diploid (AA) banana cultivars from Cibinong Science Center collection were analyzed. Random amplified polymorphic DNAs (RAPDs) and Inter Simple Sequence Repeats fingerprinting of these banana cultivars were carried out by four primers of RPDSs and two primers of inter simple sequence repeats (ISSRs) led to DNA amplification. The amplification products of RPADs and ISSRs were polymorphic, 97.83% and 95%, respectively. Size of the bands was varied from 350bp to 2.0 kbp. The range of genetic distance was from 0.06 to 0.07. The molecular data showed that these banana varieties were diverse collection.

An overview of apricot breeding in China

Over the past 8 years, apricot (Prunus armeniaca L.) breeding and related studies in China have made significant progress. Based on phenotypic and molecular data, a core collection of apricot germplasm was constructed that well represents the genetic diversities of the entire collection of germplasm resources recorded in the China Fruit Tree Monographs, Apricot Flora. DNA fingerprinting of major apricot cultivars was conducted using polymorphic alleles from simple sequence repeat (SSR) loci. Efficiency of identification of apricot cultivars using SSR fingerprinting was high. Guidelines for conducting tests of distinctness, uniformity, and stability of apricot were issued in 2013 as a national standard, which contributes significantly to protecting the intellectual property of apricot breeders. More than 40 apricot cultivars have been released. The breeding objectives for fresh marketing apricot cultivars have changed from increasing yield to improving fruit quality, such as taste, attractiveness, firmness, disease resistance, and early ripening. Some molecular markers linked to traits of interest have been found, and marker-assisted selection and molecular breeding have started to become practical ways of accelerating new cultivar development.

Enhanced genetic diversity of weedy rice populations associated with latitude decreases revealed by simple sequence repeat fingerprints

AbstractWeedy rice (WR, Oryza sativa L. f. spontanea) is a noxious agricultural weed, infesting rice fields worldwide and causing tremendous yield losses of cultivated rice. However, little is known about the relationship between genetic diversity and distribution of WR populations across a wide latitudinal gradient, in addition to its reasons for genetic differentiation. To determine the distribution of genetic diversity and differentiation, we analyzed 20 WR populations collected from wide geographic ranges of rice‐planting regions across Northeast, Jiangsu, and Guangdong provinces of China, and Sri Lanka, based on 20 simple sequence repeat loci. Our results indicated a significant negative correlation (R = 0.84, P < 0.01) between genetic diversity and latitudinal locations of WR populations. The Mantel test (R2 = 0.49, P < 0.01) showed distinct groupings of WR populations from different rice‐planting regions, fitting an isolation‐by‐distance pattern. In addition, the STRUCTURE analysis and principal coordinates (PCoA) analysis indicated considerable genetic differentiation of WR from different rice‐planting regions, which was associated with the types of co‐occurring rice cultivars. We conclude, based on the above results, that WR genetic diversity is affected by the latitudes where WR populations are located. The genetic differentiation of WR populations is determined by their spatial distances and co‐occurring rice cultivars. Such a pattern of genetic diversity and differentiation across different regions might facilitate the design of effective WR control, in addition to understanding the adaptive evolution of this weed.

SSR fingerprinting of 203 sweetpotato (Ipomoea batatas (L.) Lam.) varieties

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F1 individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.

Impact of differential levels of sodium alginate, calcium chloride and basal media on germination frequency of genetically true artificial seeds of Rauvolfia serpentina (L.) Benth. ex Kurz.

The present research work demonstrates the influence of encapsulating agent, matrix and substrate levels on single bead alginate-encapsulation of in vitro shoot tips (STs) of Rauvolfia serpentina (L.) Benth. ex Kurz., their germination and retention of genetic fidelity. Use of 3% (w/v) sodium alginate (SA) with 75mM calcium chloride (CaCl2·2H2O) (CC) developed isodiametric artificial seeds, encapsulating the STs (3–4mm long), with 84% germination frequency. Concentrations of SA lower or higher than 3% were unsuitable ensuing artificial seeds either of indefinite shape, too soft to handle with weak gelling ability or darker, too viscous and harder hindering emergence of shoot. An increase in concentrations of CC from 75mM to 100mM complexed with 3% SA resulted in declined germination frequency from 84% to 52%. The ameliorated (88%) germination of artificial seeds was achieved on half-strength liquid Murashige and Skoog medium preferred over its other fractions and semisolid state. Randomly amplified polymorphic DNA fingerprinting and inter simple sequence repeats fingerprinting approach, utilized on indiscriminately chosen shootlets from germinated artificial seeds, approved the post-germination genetic constancy within them and with their in vitro source.

Development and mapping of microsatellite markers from transcriptome sequences of European hazelnut (Corylus avellana L.) and use for germplasm characterization

Microsatellite markers, also known as simple sequence repeat (SSR) markers, are useful in genetics and plant breeding for marker-assisted selection, cultivar fingerprinting, and kinship studies. They were previously developed for European hazelnut (Corylus avellana L.) from enriched libraries, inter-simple sequence repeat (ISSR) fragments, and bacterial artificial chromosome (BAC) sequences. This study utilized the ‘Jefferson’ transcriptome sequence to develop microsatellite markers for hazelnut. Microsatellites were mined from the transcriptome sequence and aligned with the corresponding genomic sequence using a BLASTN search, and primers were designed from the genomic sequence. From a total of 1432 identified microsatellites, 382 primer pairs were designed for repeats with motifs of three base pairs or longer and at least five repeats, and a few di-repeat sequences. The primers were initially screened for polymorphism with a set of 24 C. avellana accessions, and those that were polymorphic were characterized using a set of 63 accessions. One hundred nine polymorphic microsatellite markers that generated one or two fragments per accession were characterized, with AAG, AGC, and ACT being the most common motifs. Two additional SSR primer pairs generated three PCR products in a few accessions. Of the 109 loci, 53 were placed on the linkage map of the mapping population OSU 252.146 × OSU 414.062, and 22 were assigned to linkage groups using alternate segregating populations. A dendrogram constructed from the SSR fingerprints at the 109 new marker loci mostly showed clustering of accessions from similar geographic origins and confirmed the tremendous amount of genetic diversity present within C. avellana. These markers will be useful for fingerprinting, marker-assisted selection, and genetic studies in hazelnut.

Molecular identification of variety purity in a cotton hybrid with unknown parentage using DNA-SSR markers.

Molecular identification of hybrid purity is difficult in regional trials of cotton varieties and hybrid trials. In particular, the molecular detection of hybrid purity has not yet been reported in the case of unknown parentage. In this study, we screened 5000 pairs of primers and chose 17 pairs of core simple sequence repeat (SSR) primers to determine the F1 purity of Han6402. The results showed that the purity based on SSR markers reached 100%. Twelve of the 17 pairs of primers exhibited co-dominant banding patterns, and 5 showed non-co-dominant banding patterns. Moreover, we constructed an F1 SSR fingerprinting profile that enabled the identification of the authenticity of Han 6402. Using these primers, we subsequently detected 44 individual F2 seedlings, and the results exhibited different extents of separation, in which the majority of genotypes were heterozygous with co-dominance at most of the loci that differed from each other. The results validated the underlying heterozygous status of the F2 population at the molecular level. Therefore, we conclude that the set of core SSR primers can be used for the laboratory identification of the authenticity and purity of cotton hybrids, not only for distinguishing Fl hybrids or segregating F2 populations, but also for detecting volunteer seeds as fake F1 hybrids in the cotton hybrid industry, based on the hybrid fingerprinting.

The molecular characterization by SSRs reveals a new South Italian kinship and the origin of the cultivar Uva di Troia.

Vitis vinifera L. varieties were spread through cuttings following historic migrations of people, trades, or after biological crises due to pests outbreaks. Some today’s varieties could be more than a 1000 years old and, although over the centuries these varieties generated most of the remaining cultivars, their origin could be impossible to track back. The Italian grapevine biodiversity is one of most important, most likely due to its strategic position in the middle of the Mediterranean sea. Unravelling of its structure is challenging because of its complexity and the lack of historical documentation. In this paper molecular data are compared with historical documentations. Simple Sequence Repeats fingerprinting are molecular markers best suited to investigate genetic relationships and identify pedigrees. South-Italian germplasm was studied with 54 nuclear microsatellites. A family was identified, consisting of two parents and three siblings and further genetically characterized with six nuclear and five chloroplast microsatellites and described with ampelographic and phylometric analysis. Although these latter were not informative for the kinship identification. The common Bombino bianco was the female parent and the previously unknown Uva rosa antica was the male parent. Bombino nero, Impigno and the popular Uva di Troia, all typical of the south-east Italy, were the offspring. Further research showed that the Uva rosa antica was a synonym of Quagliano and Bouteillan noir, both minor varieties. Quagliano was considered to be autochthonous of some alpine valleys in the north-west of Italy and Bouteillan noir is a neglected variety of Vancluse in France. This finding uncovers the intricate nature of Italian grape cultivars, considered peculiar of an area, but possibly being the remains of ancient latin founding varieties. Consequently, intriguing new hypotheses are discussed and some conclusions are drawn, based on the peculiar geographical origin of the parents, on the distribution of the offspring, on the chance of a single, and perhaps intentional, crossing event.

佛坪国家级自然保护区秦岭箭竹克隆结构的SSR分析

佛坪国家级自然保护区秦岭箭竹克隆结构的SSR分析

SSR fingerprinting of a German Rubus collection and pedigree based evaluation on trueness-to-type

Eighty-two genotypes of Rubus available in germplasm collections, nurseries and home gardens were collected and evaluated using a set of 16 simple sequence repeat (SSR) markers to estimate the level of genetic diversity and relatedness of the germplasm and for testing them on trueness-to-type. Each of the 16 SSRs was successful in amplifying alleles from most genotypes. Fifteen of the markers produced polymorphic bands, whereas marker RhM023 was monomorphic. The polymorphic information content among genotypes varied from 0.056 to 0.83 with an average of 0.348. A neighbor-joining analysis allocated the genotypes to four major clusters containing 11, 24, 39 and eight genotypes, respectively. Cluster I consists of floricane-fruiting cultivars originating from the Scottish and/or British breeding programs or cultivars which have those cultivars in their pedigree. Cluster II included cultivars that have ‘Autumn Bliss’ or ‘Tulameen’ in their pedigree. Cluster III consists of summer-bearing raspberry cultivars, some primocane-fruiting cultivars, and a few intermediate summer-fall-bearing types. Cluster IV consists of the blackberry ‘Navaho’ (R. fruticosus L.), the interspecific hybrid ‘Dorman Red’ and a few other raspberry varieties. A number of yellow fruited varieties was dispersed on three different clusters suggesting a convergent evolution of this trait. The pedigree of several genotypes could be confirmed using a Pedimap based approach, whereas other cultivars were found to be genetically identical. The results disclose the alarming narrow genetic base of Rubus resources in Germany. Broadening of this base is urgently needed.

Development of genomewide simple sequence repeat fingerprints and highly polymorphic markers in cucumbers based on next‐generation sequence data

AbstractCucumbers are one of the most important vegetable crops worldwide. The molecular markers have substantial potential to improve the efficiency of conventional cucumber breeding. In this study, a total of 101 157 simple sequence repeats (SSRs) were identified across the cucumber 9930 genome. Most (69 526 of 76 103 or 91.36%) SSRs with unique flanking sequences (unique SSRs) had polymorphisms among 103 cucumber genomes; the number of alleles ranged from 2 to 34 with an average of 4.70. Next, we selected 7486 unique SSR loci with polymorphism information content (PIC) ≥0.5 and major allele differences (the size difference between the alleles with the highest and the second highest frequency) ≥3 bp to design primers. The SSR marker polymorphisms were experimentally validated; 50 highly polymorphic SSR markers had polymorphisms with an average PIC of 0.44. PCR analysis also indicated that these SSR markers could be effectively used as molecular markers. The SSR fingerprints and polymorphic SSR markers with large major allele differences could enhance the efficiency of genetic research and marker‐assisted breeding in cucumbers.