Population Genetic Tools Research Articles

<p class="Body"><strong>Development and characterization of six novel microsatellite markers for honey bee parasitic mite <em>Varroa destructor</em> (Mesostigmata: Varroidae) </strong></p>

The Varroa destructor is an ectoparasitic mite and the most serious biotic threat to honey bee and apiculture worldwide. Genetic types of V. destructor determine its ability of transmission and pathogenicity. Population genetics tools could supply useful information for comprehensive management of the mite. In this study, transcriptome information of V. destructor was analyzed for mining more polymorphic microsatellite markers for the population genetics investigation with further experimental verified. A total of 83,711 unigenes were assembled with the N50 length of 1,826 bp and the GC content of 40.03%. A total of 27,775 potential microsatellite loci were identified in 18,563 unigenes. The di-nucleotide and mono-nucleotide were most abundant repeat motifs and the most dominant di-nucleotides and mono-nucleotide repeat motifs were A/T and AT/TA. Forty-two of sixty randomly selected microsatellite loci were successfully amplified. Six of them were confirmed to be polymorphic in five V. destructor geographical populations. Our result showed transcriptomic data provide valuable resources for molecular marker development, and these novel microsatellite loci would be valuable in facilitating population genetic and evolutionary of V. destructor and related species.

Island invasion and reinvasion: Informing invasive species management with genetic measures of connectivity

Abstract Invasive species are the major threat to island biodiversity world‐wide. Genetic analyses assist in identifying invasion routes as well as revealing population connectivity, which both represent crucial information for conservation management strategies and risk evaluation. Such information is critical to safeguarding vulnerable species on nearshore islands, which often serve as wildlife sanctuaries. The house mouse Mus musculus has invaded islands around the world and is a model species for showcasing how genetic tools can be used to inform biosecurity management. The genetic population structure of 316 mice from 12 locations in the upper South Island of New Zealand was determined, using novel GenePlots and traditional population genetic tools based on 10 microsatellite loci, to identify gene flow and reinvasion pathways among mainland and island populations over a decade. On the mainland, populations remained genetically homogeneous over landscape scales of many tens of kilometres. In contrast, historically established island populations only kilometres offshore had low genetic diversity from prolonged isolation. Two islands were potentially colonized from both the mainland and each other in a hybrid swarm. Islands that had recently been invaded or reinvaded in the past decade had genetic profiles consistent with the adjacent mainland, suggesting failure of biosecurity procedures to prevent reinvasion, rather than eradication survivors. Although two islands were invaded by only a few individuals, on a third island many invaders simultaneously arrived. Synthesis and applications. Assessing the genetic structure and connectivity of mainland and island populations of an invasive species, using a combination of traditional and novel visualization tools, has uncovered a spectrum of invasion mechanisms and pathways. These results have informed ongoing biosecurity measures by revealing the locations and intensities of biosecurity threats, allowing targeted management actions to reduce the likelihood of island reinvasion.

Development and Validation of an Open Access SNP Array for Nile Tilapia (Oreochromis niloticus).

Tilapia are among the most important farmed fish species worldwide, and are fundamental for the food security of many developing countries. Several genetically improved Nile tilapia (Oreochromis niloticus) strains exist, such as the iconic Genetically Improved Farmed Tilapia (GIFT), and breeding programs typically follow classical pedigree-based selection. The use of genome-wide single-nucleotide polymorphism (SNP) data can enable an understanding of the genetic architecture of economically important traits and the acceleration of genetic gain via genomic selection. Due to the global importance and diversity of Nile tilapia, an open access SNP array would be beneficial for aquaculture research and production. In the current study, a ∼65K SNP array was designed based on SNPs discovered from whole-genome sequence data from a GIFT breeding nucleus population and the overlap with SNP datasets from wild fish populations and several other farmed Nile tilapia strains. The SNP array was applied to clearly distinguish between different tilapia populations across Asia and Africa, with at least ∼30,000 SNPs segregating in each of the diverse population samples tested. It is anticipated that this SNP array will be an enabling tool for population genetics and tilapia breeding research, facilitating consistency and comparison of results across studies.

Multilocus Sequence Typing of Enterocytozoon bieneusi Isolates From Various Mammal and Bird Species and Assessment of Population Structure and Substructure.

Enterocytozoon bieneusi is one of the most common intestinal pathogens in humans and animals. E. bieneusi has been confirmed to be complex microsporidian species. Approximately 500 ITS genotypes of E. bieneusi have been defined. With the establishment and application of multilocus sequencing typing and population genetic tools in E. bieneusi, the studies on these aspects have been carried out worldwide, but little information is available. To understand genetic variation of mini-/micro-satellites and the population structure and substructure of E. bieneusi in northeastern China, 305 E. bieneusi DNA specimens composed of 28 ITS genotypes were from 13 mammal species and five bird species in the investigated areas. They were characterized by nested-PCR amplification and sequencing at four mini-/micro-satellite loci (MS1, MS3, MS4, and MS7). At the MS1, MS3, MS4, and MS7 loci, 153 (50.16%), 131 (42.95%), 133 (43.61%), and 128 (41.97%) DNA specimens were amplified and sequenced successfully with 44, 17, 26, and 24 genotypes being identified, respectively. Multilocus genotypes (MLGs) showed a higher genetic diversity than ITS genotypes. 48 MLGs were produced out of 90 ITS-positive DNA specimens based on concatenated sequences of all the five genetic loci including ITS. Linkage disequilibrium (LD) and limited genetic recombination were observed by measuring LD using both multilocus sequences and allelic profile data, indicating an overall clonal population structure of E. bieneusi in the investigated areas. These data will aid in the longitudinal tracking of the attribution of source of infection/contamination and in elucidating transmission dynamics, and will provide valuable information for making efficient control strategies to intervene with and prevent occurrence of microsporidiosis caused by E. bieneusi among animals and transmission of E. bieneusi from animals to humans in the investigated areas. Phylogenetic and network analyses identified three different subgroups, revealing the presence of host-shaped segregation and the absence of geographical segregation in E. bieneusi population. Meanwhile, the MLGs from zoonotic ITS genotypes were observed to be basically separated from the MLGs from host-adapted ones. Assessment of substructure will have a reference effect on understanding of zoonotic or interspecies transmission of E. bieneusi and evolution direction from zoonotic genotypes to host-adapted genotypes.

Joint Genetic Analyses of Mitochondrial and Y-Chromosome Molecular Markers for a Population from Northwest China.

The genetic markers on mitochondria DNA (mtDNA) and Y-chromosome can be applied as a powerful tool in population genetics. We present a study to reveal the genetic background of Kyrgyz group, a Chinese ethnic group living in northwest China, and genetic polymorphisms of 60 loci on maternal inherited mtDNA and 24 loci on paternal inherited Y-chromosome short tandem repeats (Y-STRs) were investigated. The relationship between the two systems was tested, and the result indicated that they were statistically independent from each other. The genetic distances between Kyrgyz group and 11 reference populations for mtDNA, and 13 reference populations for Y-STRs were also calculated, respectively. The present results demonstrated that the Kyrgyz group was genetically closer to East Asian populations than European populations based on the mtDNA loci but the other way around for the Y-STRs. The genetic analyses could largely strengthen the understanding for the genetic background of the Kyrgyz group.

Development of 19 novel microsatellite markers of lily-of-the-valley (Convallaria, Asparagaceae) from transcriptome sequencing.

The lily-of-the-valley Convallaria (Asparagaceae) consists of three herbaceous perennial species. The plants are commonly found in northern hemisphere, and are best-known for their ornamental and pharmaceutical value. In order to assess the genetic structure, diversity and demographic history of Convallaria species, 19 novel microsatellite markers were developed based on transcriptome data of C. keiskei. Polymorphism and cross-amplification of the markers were tested in three populations of C. keiskei and one population each of C. majalis and C. montana. The transferability rate in two species was both 89.5%. The average number of alleles detected per locus was 7.7, 3.3 and 2.7 in C. keiskei, C. majalis and C. montana, respectively, and the polymorphism information content correspondingly varied from 0.067 to 0.730, from 0.071 to 0.637 and from 0.195 to 0.680 at the population level. The observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.833, respectively. Seven of the 19 loci showed significant deviation from Hardy-Weinberg equilibrium. The availability of these markers will provide a useful molecular tool for further population genetics, phylogeographic and breeding studies of Convallaria species.

Gridlock and beltways: the genetic context of urban invasions.

The rapid expansion of urban land across the globe presents new and numerous opportunities for invasive species to spread and flourish. Ecologists historically rejected urban ecosystems as important environments for ecology and evolution research but are beginning to recognize the importance of these systems in shaping the biology of invasion. Urbanization can aid the introduction, establishment, and spread of invaders, and these processes have substantial consequences on native species and ecosystems. Therefore, it is valuable to understand how urban areas influence populations at all stages in the invasion process. Population genetic tools are essential to explore the driving forces of invasive species dispersal, connectivity, and adaptation within cities. In this review, we synthesize current research about the influence of urban landscapes on invasion genetics dynamics. We conclude that urban areas are not only points of entry for many invasive species, they also facilitate population establishment, are pools for genetic diversity, and provide corridors for further spread both within and out of cities. We recommend the continued use of genetic studies to inform invasive species management and to understand the underlying ecological and evolutionary processes governing successful invasion.

Development of genome-wide polymorphic microsatellite markers for Trichinella spiralis

BackgroundTrichinella nematodes are globally distributed food-borne pathogens, in which Trichinella spiralis is the most common species in China. Microsatellites are a powerful tool in population genetics and phylogeographic analysis. However, only a few microsatellite markers were reported in T. spiralis. Thus, there is a need to develop and validate genome-wide microsatellite markers for T. spiralis.MethodsMicrosatellites were selected from shotgun genomic sequences using MIcroSAtellite identification tool (MISA). The identified markers were validated in 12 isolates of T. spiralis in China.ResultsA total of 93,140 microsatellites were identified by MISA from 9267 contigs in T. spiralis genome sequences, in which 16 polymorphic loci were selected for validation by PCR with single larvae from 12 isolates of T. spiralis in China. There were 7–19 alleles per locus (average 11.25 alleles per locus). The observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.325 to 0.750 and 0.737 to 0.918, respectively. The polymorphism information content (PIC) ranged from 0.719 to 0.978 (average 0.826). Among the 16 loci, markers for 10 loci could be amplified from all 12 international standard strains of Trichinella spp.ConclusionsSixteen highly polymorphic markers were selected and validated for T. spiralis. Primary phylogenetic analysis showed that these markers might serve as a useful tool for genetic studies of Trichinella parasites.

Coalescent Simulation with msprime.

Coalescent simulation is a fundamental tool in modern population genetics. The msprime library provides unprecedented scalability in terms of both the simulations that can be performed and the efficiency with which the results can be processed. We show how coalescent models for population structure and demography can be constructed using a simple Python API, as well as how we can process the results of such simulations to efficiently calculate statistics of interest. We illustrate msprime's flexibility by implementing a simple (but functional) approximate Bayesian computation inference method in just a few tens of lines of code.

Microsatellite analysis reveals connectivity among geographically distant transmission zones of Plasmodium vivax in the Peruvian Amazon: A critical barrier to regional malaria elimination.

Despite efforts made over decades by the Peruvian government to eliminate malaria, Plasmodium vivax remains a challenge for public health decision-makers in the country. The uneven distribution of its incidence, plus its complex pattern of dispersion, has made ineffective control measures based on global information that lack the necessary detail to understand transmission fully. In this sense, population genetic tools can complement current surveillance. This study describes the genetic diversity and population structure from September 2012 to March 2015 in three geographically distant settlements, Cahuide (CAH), Lupuna (LUP) and Santa Emilia (STE), located in the Peruvian Amazon. A total 777 P. vivax mono-infections, out of 3264, were genotyped. Among study areas, LUP showed 19.7% of polyclonal infections, and its genetic diversity (Hexp) was 0.544. Temporal analysis showed a significant increment of polyclonal infections and Hexp, and the introduction and persistence of a new parasite population since March 2013. In STE, 40.1% of infections were polyclonal, with Hexp = 0.596. The presence of four genetic clusters without signals of clonal expansion and infections with lower parasite densities compared against the other two areas were also found. At least four parasite populations were present in CAH in 2012, where, after June 2014, malaria cases decreased from 213 to 61, concomitant with a decrease in polyclonal infections (from 0.286 to 0.18), and expectedly variable Hexp. Strong signals of gene flow were present in the study areas and wide geographic distribution of highly diverse parasite populations were found. This study suggests that movement of malaria parasites by human reservoirs connects geographically distant malaria transmission areas in the Peruvian Amazon. The maintenance of high levels of parasite genetic diversity through human mobility is a critical barrier to malaria elimination in this region.

New approach to simultaneously identify mitochondrial DNA haplogroups by multiplex PCR-RFLPs and capillary electrophoresis

Mitochondrial DNA (mtDNA) genotyping provides a valuable tool for forensic studies and population genetics. Nevertheless, current methodologies for mtDNA genotyping are laborious and expensive. Here, we describe a detailed new multiplex PCR-RFLPs and capillary electrophoresis approach to simultaneously identify six mtDNA haplogroups: A, B, C, D, H & L. 38 samples previously typed by sequencing of the complete mtDNA control region (CR) were analyzed by this methodology. Haplogroups determined by RFLP-PCR for all samples corresponded with the results obtained by sequencing. The results show that this approach effectively allows the simultaneous determination of mtDNA haplogroups commonly present in Latin American populations in a relatively fast and economical way. This methodology offers a good resolution and sensitive detection procedure to assess mtDNA ancestry.

X-STR decaplex study in the population of Imbabura-Ecuador

X chromosome STR markers are a useful tool in population genetics, they provide supplementary information for forensic and kinship analysis where autosomal and Y chromosome information is not enough. A total of 100 unrelated individuals from Imbabura-Ecuador were genotyped using the X-STR Decaplex system (DXS8378, DXS9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7133, GATA172D05, GATA31E08, and DXS7423). Allele frequencies and population parameters were calculated for each STR. The most informative marker was DXS6809 and the least informative was DXS7133. High discrimination power values were obtained (greater than 99.99%), as well as high mean exclusion chance in trios (mother / daughter / father) 0.9999948011 and duos (father/daughter) 0.9992880836. The genetic distance (FST) obtained comparing the Ecuadorian population and the Pichincha province population show low genetic distances. The data analyzed confirmed the potential of these 10 X-STRs.

Biogeographically significant units in conservation: a new integrative concept for conserving ecological and evolutionary processes

SummaryWe combined tools of phylogeography, population genetics and biogeographical interpretation to analyse a group of phylogenetically independent lineages (animals and plants) that coexist within the same geographical region, yet under markedly different environments, in order to identify generalized barriers for gene flow. We tested the hypothesis that major geographic features have produced a concordant genetic structure in phylogenetically independent lineages. A rigorous bibliographic search was performed, selecting available molecular information from six taxa occupying distinct southern biomes of South America: Yungas, Prepuna, Puna and northern Monte. We estimated within-population genetic diversity, the genetic structure and haplotype phylogenies to assemble distribution maps of genetic barriers for each species. We found a strong association between genetic variation and latitudinal distribution of populations. We detected a major barrier for six taxa at 27°S latitude and a second one for a group of three species at 25–26°S. Two alternative non-exclusive hypotheses – geology and/or climate – explain concordant genetic barriers in divergent lineages. We suggest that the term ‘biogeographically significant units’ portrays a group of populations of phylogenetically unrelated taxa that inhabit the same geographic region that have been similarly impacted by major physical events, which can be used to identify priority areas in landscape conservation.

Gene flow across host-associated populations of the rice stem borer Chilo suppressalis Walker (Lepidoptera: Crambidae): implications for Bt resistance management in rice.

The rice stem borer, Chilo suppressalis, is a serious pest of rice, but also damages an aquatic vegetable, water oats (Zizania latifolia Turcz.). The time at which mating occurs is different between populations of rice stem borer associated with rice and those associated with water-oats, which suggests that undetected cryptic species may be associated with these plant hosts. If true, this would have significant management implications. This study is the first empirical test of this idea, using population genetic tools from two sampling cohorts. We genotyped 320 rice stem borer individuals from 2014, collected from rice and water-oats across five locations (where they exist in sympatry), using seven microsatellite loci. We found no genetic structuring associated with host plant species. On water oats, some rice stem borers were found that had a similar mating time to the rice population, so in 2016, a second cohort of samples was screened by their timing of mating to get 'pure rice feeders' and 'pure water oats feeders'. These samples were genotyped with microsatellites, mitochondrial DNA (mtDNA) (COI and COII), and a nuclear gene (EF1-α). Our mtDNA data suggest a relatively low amount of population subdivision associated with plant host, but the microsatellite data revealed no such genetic structure, and we were only able to identify one haplotype of EF1-α. Our results indicate gene flow between rice and water oats populations of rice stem borer, indicating that water oats will likely provide a refuge for resistance management of Bt rice. © 2019 Society of Chemical Industry.

Development and characterization of 19 polymorphic microsatellite loci from the Red-cowled Cardinal (Paroaria dominicana, Passeriformes, Aves) using next-generation sequencing.

The Red-cowled Cardinal (Paroaria dominicana) is an endemic passerine of the Caatinga biome in Brazil, and is one of the most traded passerines in the country. Illegal trade can have serious impacts on wild populations, such as reduced population sizes, the introduction of the species to areas outside their historical range or mixing individuals from different populations. Microsatellites constitute an important tool for population genetics and forensics studies, and hold great potential to help authorities manage illegal trafficking and inspect commercial breeders. We developed new microsatellite loci using massive parallel sequencing and characterized them in 23 seized Red-cowled Cardinals with unknown geographic origin. The DNA sequencing generated 2,068,684 paired-reads of which we identified 10,322 tri- to hexanucleotide loci. We selected 30 loci for amplification and polymorphism tests, of which 21 successfully amplified and 19 were polymorphic. The number of alleles ranged from 7 to 18 and the mean expected heterozygosity was 0.863. Six loci deviated from Hardy-Weinberg equilibrium probably due to null alleles and/or the Wahlund effect. Polymorphic loci in Hardy-Weinberg equilibrium showed low identity probability and high paternity exclusion probability. Our results indicate that this new set of microsatellite loci constitutes an important tool for both population genetic and forensic studies, with ultimate potential for assisting authorities in managing animal victims of illegal trafficking and the inspection of commercial breeders of the Red-cowled Cardinal.

Gene flow and genetic structure in Acacia stenophylla (Fabaceae): Effects of hydrological connectivity

AbstractAimRiparian ecosystems are regarded as vulnerable to the effects of climate change. Because of their reliance on passive dispersal to migrate from areas where conditions have become unfavourable, plants are particularly susceptible. On dryland river floodplains, the species diversity of herbaceous annuals is often high while that of structurally dominant woody perennials is low. We examined gene flow genetic structure and dispersal in Acacia stenophylla, a small perennial tree widely distributed throughout river systems of inland Australia. The role of the river corridor in shaping patterns of gene flow and genetic structure is also investigated.LocationMurray‐Darling Basin, south eastern AustraliaMethodsA total of 127 individuals, from 12 subpopulations located on seven rivers were genotyped at 13 microsatellite loci. Several population and landscape genetic tools were applied to the microsatellite data to evaluate spatial patterns of gene flow and genetic structure and make inferences regarding possible modes of dispersal.ResultsHigh gene flow and weak genetic structure was identified for the 12 subpopulations of A. stenophylla sampled, a surprising result given large distances between subpopulations. Pairwise genetic distance between subpopulations was low to moderate and could largely be explained (R2 = 0.68) by two variables: distance along the river and the proportion of no flow days. structure analysis revealed two genetic clusters. Subpopulations located on the Darling and Lower Balonne rivers were dominated by cluster one while subpopulations from the Warrego and Paroo rivers showed largely mixed ancestry with individuals descending from both clusters one and two.Main ConclusionsThese results indicate that the river corridor facilitates extensive gene flow between subpopulations of A. stenophylla in this system. Hydrochory appears to be the dominant process; however, upstream movements of propagules most probably via animal movement are sufficient to negate effects expected under unidirectional dispersal.

Multilocus Sequence Typing and Population Genetic Analysis of Enterocytozoon bieneusi: Host Specificity and Its Impacts on Public Health.

Microsporidia comprise a large class of unicellular eukaryotic pathogens that are medically and agriculturally important, but poorly understood. There have been nearly 1,500 microsporidian species described thus far, which are variable in biology, genetics, genomics, and host specificity. Among those, Enterocytozoon bieneusi is the well-known species responsible for the most recorded cases of human microsporidian affections. The pathogen can colonize a broad range of mammals and birds and most of the animals surveyed share some genotypes with humans, posing a threat to public health. Based on DNA sequence analysis of the ribosomal internal transcribed spacer (ITS) and phylogenetic analysis, several hundreds of E. bieneusi genotypes have been defined and clustered into different genetic groups with varied levels of host specificity. However, single locus-based typing using ITS might have insufficient resolution to discriminate among E. bieneusi isolates with complex genetic or hereditary characteristics and to assess the elusive reproduction or transmission modes of the organism, highlighting the need for exploration and application of multilocus sequence typing (MLST) and population genetic tools. The present review begins with a primer on microsporidia and major microsporidian species, briefly introduces the recent advances on E. bieneusi ITS genotyping and phylogeny, summarizes recent MLST and population genetic data, analyzes the inter- and intragroup host specificity at the MLST level, and interprets the public health implications of host specificity in zoonotic or cross-species transmission of this ubiquitous fungus.

Beyond Biodiversity: Can Environmental DNA (eDNA) Cut It as a Population Genetics Tool?

Population genetic data underpin many studies of behavioral, ecological, and evolutionary processes in wild populations and contribute to effective conservation management. However, collecting genetic samples can be challenging when working with endangered, invasive, or cryptic species. Environmental DNA (eDNA) offers a way to sample genetic material non-invasively without requiring visual observation. While eDNA has been trialed extensively as a biodiversity and biosecurity monitoring tool with a strong taxonomic focus, it has yet to be fully explored as a means for obtaining population genetic information. Here, we review current research that employs eDNA approaches for the study of populations. We outline challenges facing eDNA-based population genetic methodologies, and suggest avenues of research for future developments. We advocate that with further optimizations, this emergent field holds great potential as part of the population genetics toolkit.

Taraxacum kok-saghyz (rubber dandelion) genomic microsatellite loci reveal modest genetic diversity and cross-amplify broadly to related species

Taraxacum kok-saghyz (TKS) carries great potential as alternative natural rubber source. To better inform future breeding efforts with TKS and gain a deeper understanding of its genetic diversity, we utilized de novo sequencing to generate novel genomic simple sequence repeats markers (gSSRs). We utilized 25 gSSRs on a collection of genomic DNA (gDNA) samples from germplasm bank, and two gDNA samples from historical herbarium specimens. PCR coupled with capillary electrophoresis and an array of population genetics tools were employed to analyze the dataset of our study as well as a dataset of the recently published genic SSRs (eSSRs) generated on the same germplasm. Our results using both gSSRs and eSSRs revealed that TKS has low- to- moderate genetic diversity with most of it partitioned to the individuals and individuals within populations, whereas the species lacked population structure. Nineteen of the 25 gSSR markers cross-amplified to other Taraxacum spp. collected from Southeastern United States and identified as T. officinale by ITS sequencing. We used a subset of 14 gSSRs to estimate the genetic diversity of the T. officinale gDNA collection. In contrast to the obligatory outcrossing TKS, T. officinale presented evidence for population structure and clonal reproduction, which agreed with the species biology. We mapped the molecular markers sequences from this study and several others to the well-annotated sunflower genome. Our gSSRs present a functional tool for the biodiversity analyses in Taraxacum, but also in the related genera, as well as in the closely related tribes of the Asteraceae.

Development and Characterization of Genic Microsatellites for the Ornamental Plant Green and Gold (Chrysogonum virginianum)

The genus Chrysogonum is native to the eastern United States. Three entities have been recognized—either as three varieties of Chrysogonum virginianum or as two species, one of them with two varieties. The current study suggests that a fourth entity should be recognized. Several forms of the complex are in commercial trade as ornamentals. As very limited molecular information on Chrysogonum is available, we developed a set of genic simple sequence repeat markers (eSSRs) from de novo transcriptome sequencing. We tested a set of 17 eSSRs on a collection of C. virginianum genomic DNA samples from the three botanical varieties, and a new putative type observed in Tennessee, dubbed “Ocoee-type” for its geographic origin. The polymerase chain reaction and capillary electrophoresis analyses with downstream population genetics tools verified the usefulness of the eSSRs. By applying this approach, we showed recognizable variation within Chrysogonum, although it did not correspond exactly to previous infraspecific classifications. Finally, as demonstrated for the commercial cultivar Pierre included in the study, the eSSRs can be used for enhancing the future breeding or hybridization efforts of this ornamental plant.