Novel Microsatellite Markers Acquired from Rubus coreanus Miq. and Cross-Amplification in Other Rubus Species

The Rubus genus consists of more than 600 species that are distributed globally. Only a few Rubus species, including raspberries and blueberries, have been domesticated. Genetic diversity within and between Rubus species is an important resource for breeding programs. We developed genomic microsatellite markers using an SSR-enriched R. coreanus library to study the diversity of the Rubus species. Microsatellite motifs were discovered in 546 of 646 unique clones, and a dinucleotide repeat was the most frequent (75.3%) type of repeat. From 97 microsatellite loci with reproducible amplicons, we acquired 29 polymorphic microsatellite markers in the Rubus coreanus collection. The transferability values ranged from 59.8% to 84% across six Rubus species, and Rubus parvifolius had the highest transferability value (84%). The average number of alleles and the polymorphism information content were 5.7 and 0.541, respectively, in the R. coreanus collection. The diversity index of R. coreanus was similar to the values reported for other Rubus species. A phylogenetic dendrogram based on SSR profiles revealed that seven Rubus species could be allocated to three groups, and that R. coreanus was genetically close to Rubus crataegifolius (mountain berry). These new microsatellite markers might prove useful in studies of the genetic diversity, population structure, and evolutionary relationships among Rubus species.


Introduction
The Rubus genus in the Rosaceae family consists of more than 600 species grouped in 12 subgenera. A few of these species, including raspberries, blackberries, dewberries, arctic fruits, and flowering raspberries, have been domesticated and are the focus of breeding programs [1]. Both cultivated and wild Rubus species have the potential to interact with other species belonging to different Rubus subgenera. Cross-fertilization within the Rubus genus implies that wild populations could be useful resources to improve domesticated species [2][3][4]. For this reason, Rubus coreanus, which is distributed throughout Southeast Asia, could be a valuable resource for breeding programs and the biotech industry [5,6].
More reliable molecular markers are needed to enhance genetic analyses of Rubus species. The objectives of this study were to develop novel genomic SSR markers using a microsatellite-enriched library of R. coreanus and to test their transferability across six other Rubus species. New microsatellite markers with polymorphisms and transferability across species might be valuable tools to evaluate genetic variability and identify genes controlling agronomic traits in the Rubus genus.

Results and Discussion
In this study, 684 positive colonies were randomly selected from an SSR-enriched library and sequenced. A total of 358 kb sequences were acquired in 684 clones, and 646 singleton sequences were used to search for SSR motifs. A total of 38 (5.6%) of the 684 clones were duplicates. Microsatellite motifs were discovered in 546 clones. The enrichment efficiency of microsatellites (84.5%) was higher than for groundnut at 68% [17], Japanese apricot at 57.0% [18], lychee at 52.0% [19], and Actinidia arguta at 74.2% [20].
Most of the microsatellite motifs (75.3%) were dinucleotide repeats, followed by trinucleotide repeats (20.7%) and repeat motifs that were tetranucleotide or greater (4.0%). Among the SSR motifs, AG/CT and CTT/AAG represented the majority of di-and trinucleotide motifs at 57.1% and 6.0%, respectively. This result corresponds to the general SSR motif distribution in dinucleotide motifs [21,22]. The most common repeat in plants is the AT repeat; however, it was rare (1.1%) in this study. The AT motif is not suitable for hybridization, due to its autocomplementarity [23].
Among the 546 clones containing microsatellite motifs, 263 primer pairs were designed based on flanking SSR regions. The SSR motifs biased over the acquired sequences might affect primer variation. The divergent distribution of DNA fragments in the SSR-enriched library could introduce variation among crops [24]. In total, 97 (46.2%) of the microsatellite markers covered by the 263 primer pairs were successfully amplified and had reproducible amplicons (Supplementary Table S1); the remaining markers had no amplicons or multi-bands between two R. coreanus accessions, and were thus excluded from further analysis.
We applied the 97 microsatellite markers to analyze their transferability to other Rubus species ( Table 1). The results for six other Rubus species indicated that, except for eleven loci with no amplified products, 86 (88.7%) of the microsatellite markers produced at least one amplicon in other Rubus species. A total of 26 (26.8%) of the microsatellite markers had PCR products in all tested samples ( * Accessions used to check for amplicon production using the designed primer pairs. Among the transferable microsatellite markers, we identified 29 polymorphic markers in selected R. coreanus accessions, while the remaining markers were monomorphic. These 29 markers were used to create DNA profiles of 48 Rubus accessions (R. coreanus, 32 accessions; other Rubus species, 16 accessions). Polymorphism scores for 29 microsatellite markers were calculated for the R. coreanus collection and for other Rubus species (Table 3). The number of alleles (NA) varied among loci, and ranged from two to 13 (mean = 5.7 alleles) alleles in R. coreanus and from four to 15 (mean = 9.6 alleles) alleles in other Rubus species. Within the R. coreanus collection, the mean expected heterozygosity and observed heterozygosity were 0.582 and 0.558, respectively, and the mean polymorphism information content (PIC) was 0.541 (range, 0.147-0.863). GB-RC-245 had the highest PIC value of 0.863 (NA = 13), followed by 0.826 for GB-RC-091 and GB-RC-247 (NA = 8 and 10, respectively). Castillo et al. [25] reported 12 microsatellite markers from red raspberry (R. idaeus) and blackberry (Rubus L. hybrids), with an average PIC value of 0.55 in the red raspberry collection. Michael et al. [26] reported an average PIC value of 0.49 (mean NA = 8.6) using 21 polymorphic Rubus SSR primers in cultivated and wild black raspberry (R. occidentalis L.) collections. Our results revealed a similar diversity index in the R. coreanus collection compared to previously reported microsatellite markers in other Rubus species. This implies that the microsatellite markers developed in this study might be useful for the genetic assessment of Rubus species with high transferability.
Microsatellite analyses of R. coreanus and the genetic relatedness among seven Rubus species based on SSR profiles were not previously reported, whereas several studies have focused on cultivated Rubus species such as red raspberry [10,25]. In this study, we constructed phylogenetic trees based on the DNA profiles of 48 Rubus accessions using 29 microsatellite loci. The Rubus species were divided into three groups (Figure 1). Rubus coreanus was genetically close to mountain berry (R. crataegifolius) in the third group (G-3). The alleles of R. crataegifolius for the 29 microsatellite loci were similar to those of R. coreanus, although R. crataegifolius had low transferability (59.8%). The first group (G-1) consisted of other mountain berries (R. crataegifolius var. subcuneatus and R. parvifolius), while the second group (G-2) included blackberry (Rubus fruticosus and R. ursinus) and red raspberry. These microsatellite markers will be useful for studies of the genetic diversity, population structure, and evolutionary relationships among Rubus species.

Plant Materials and Genomic DNA Extraction
DNA was extracted from 48 Rubus accessions (32 R. coreanus accessions and 16 accessions of other Rubus species) that were conserved at the Bokbunja substation of the Gochang Agricultural Center (Table 1). Total genomic DNA was extracted from pulverized leaf samples using Plant DNAzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the supplier's protocol. The DNA concentration was determined using an ultraviolet-visible spectrophotometer (ND-1000; NanoDrop, Wilmington, DE, USA). The final concentration of each DNA sample was adjusted to 20 ng/µL in TE buffer before implementing the PCR procedure.

Construction of an SSR-Enriched Library and Primer Design
A modified biotin-streptavidin capture method was used to construct an SSR-enriched library of R. coreanus [27]. Genomic DNA of R. coreanus was digested with restriction enzymes (AluI, DraI, HaeIII, RsaI, EcoRV, and NruI), and the fragmented DNA was eluted on a 1.5% agarose gel; the fragments ranged in size from 300 to 1500 bp. After adaptor ligation and pre-amplification, the DNA mixture was hybridized with the following biotin-  15. The hybridized DNA fragments were recovered with streptavidin-coated magnetic beads (Promega, Madison, WI, USA). The fragments were cloned by TOPO TA cloning (Invitrogen). Recombinant colonies, identified as white colonies on an LB plate containing ampicillin, were sequenced using an ABI3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). SSR Manager [28] was used to identify the SSR motif and to design primers flanking these regions.

PCR Amplification and Genotyping
The M13-tailed primer method, in which the M13 sequence is attached to the 5'-end of the forward primer, was used to determine the sizes of the amplified products, as described by Schuelke [29]. PCR amplification was performed in a total volume of 20 µL, which consisted of 50 ng of genomic DNA, 2 pmol of a specific primer, 4 pmol of the fluorescently labeled M13 universal primer

Analysis of Transferability, Genetic Variability, and Evolutionary Relationships
The 97 microsatellite loci with a reproducible amplicon in R. coreanus were cross-amplified in other Rubus species, and their transferability was analyzed. PCR and resolving the amplified products were performed as described above. Genotypes of the 48 Rubus accessions were based on the 29 polymorphic microsatellite markers, and these DNA profiles were used to analyze genetic variability and construct a phylogenetic tree. PowerMarker (ver. 3.25) [30] was used to measure the genetic variability at SSR loci among Rubus species. A genetic distance matrix was created as a function of shared alleles [31]. The construction of a phylogenetic tree and bootstrapping were performed using PowerMarker (ver. 3.25) according to the un-weighted pair-group method with arithmetic averages. A consensus tree was created using the PHYLIP software package (ver. 3.695) [32].

Conclusions
We created 29 new polymorphic microsatellite markers based on the 97 transferable microsatellite markers in the Rubus species. The genetic diversity index and clustering results show that these markers are informative for genetic assessments of R. coreanus and other Rubus species. These new microsatellite markers can be used for genetic map construction, molecular breeding, and germplasm management of Rubus species.

Acknowledgments
Research supported by the Research Program for Agricultural Science and Technology Development (Code no. PJ010883) from National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.

Author Contributions
Gi-An Lee and Jae Young Song performed the experiments and wrote the paper and; Heh-Ran Choi contributed materials; Jong-Wook Chung, Young-Ah Jeon and Jung-Ro Lee analyzed the data; Kyung-Ho Ma and Myung-Chul Lee conceived and designed the experiments. All authors read and approved the final manuscript.

Conflicts of Interest
The authors declare no conflict of interest.