Use Of Simple Sequence Repeats Research Articles

Morphological and molecular diversity of eggplant accessions (Solanum melongena L) using Simple Sequence Repeats (SSR) markers

The evaluation of various desirable characteristics in eggplant genotypes has facilitated the efficient process of selecting and improving their traits. Morphological parameters have proven to be valuable in assessing the similarities or differences among different accessions, while molecular data have been used to support the conclusions drawn from the morphological analysis. This study was conducted to evaluate the performance of 42 eggplant genotypes collected from Malaysia, China, and Thailand. Data for growth, yield, and related traits were collected and subjected to analysis of variance (ANOVA) using SAS 9.4. The field trial experiment was designed as a completely randomized block design with three replications. Analysis of variance (ANOVA) showed that the traits under study were highly significant (p ≤ 0.01). MV18 (5.97 kg) and TV17 (5.59 kg) were observed to have high yields per plant. The SSR markers used exhibited moderate average values for the number of alleles (2.53). The major allele frequency displayed a high average value (0.53) and a moderate average number of effective alleles (2.31). Additionally, the observed Shannon's information index, expected heterozygosity, and PIC were high (0.84, 0.54, and 0.45, respectively). 42 accessions were grouped into five major clusters based on similarities using the unweighted pair-group method with arithmetic averages based on similarity matrices UPGMA Dendrogram and the EIGEN module. The findings of this study indicate that the use of simple sequence repeat (SSR) markers can effectively estimate genetic diversity and analyze phylogenetic relationships. Moreover, these markers can assist eggplant breeders in selecting desirable quantitative traits within their breeding program.

Multiple-Genome-Based Simple Sequence Repeat Is an Efficient and Successful Method in Genotyping and Classifying Different Jujube Germplasm Resources.

Jujube (Ziziphus jujuba Mill.) is a commercially important tree native to China, known for its high nutritional value and widespread distribution, as well as its diverse germplasm resources. Being resilient to harsh climatic conditions, the cultivation of jujube could provide a solution to food insecurity and income for people of arid and semi-arid regions in and outside of China. The evaluation of germplasm resources and genetic diversity in jujube necessitates the use of Simple Sequence Repeat (SSR) markers. SSR markers are highly polymorphic and can be used to evaluate the genetic diversity within and between cultivars of Chinese jujube, and are important for conservation biology, breeding programs, and the discovery of important traits for Chinese jujube improvement in China and abroad. However, traditional methods of SSR development are time-consuming and inadequate to meet the growing research demands. To address this issue, we developed a novel approach called Multiple-Genome-Based SSR identification (MGB-SSR), which utilizes the genomes of three jujube cultivars to rapidly screen for polymorphic SSRs in the jujube genome. Through the screening process, we identified 12 pairs of SSR primers, which were then used to successfully classify 249 jujube genotypes. Based on the genotyping results, a digital ID card was established, enabling the complete identification of all 249 jujube plants. The MGB-SSR approach proved efficient in rapidly detecting polymorphic SSRs within the jujube genome. Notably, this study represents the first successful differentiation of jujube germplasm resources using 12 SSR markers, with 4 markers successfully identifying triploid jujube genotypes. These findings offer valuable information for the classification of Chinese jujube germplasm, thereby providing significant assistance to jujube researchers and breeders in identifying unknown jujube germplasm.

Characterization of the Aspergillus flavus Population from Highly Aflatoxin-Contaminated Corn in the United States.

Aflatoxin contamination of corn is a major threat to the safe food and feed. The United States Federal Grain Inspection Service (FGIS) monitors commercial grain shipments for the presence of aflatoxin. A total of 146 Aspergillus flavus were isolated from 29 highly contaminated grain samples to characterize the visual phenotypes, aflatoxin-producing potential, and genotypes to explore the etiological cause of high aflatoxin contamination of US corn. Five of the isolates had reduced sensitivity (43-49% resistant) to the fungicide azoxystrobin, with the remainder all being over 50% resistant to azoxystrobin at the discriminating dose of 2.5 µg/mL. Only six isolates of the highly aflatoxigenic S morphotype were found, and 48 isolates were non-aflatoxigenic. Analysis of the mating type locus revealed 45% MAT 1-1 and 55% MAT 1-2. The A. flavus population originating from the highly aflatoxin contaminated grain samples was compared to a randomly selected subset of isolates originating from commercial corn samples with typical levels of aflatoxin contamination (average < 50 ppb). Use of simple sequence repeat (SSR) genotyping followed by principal component analysis (PCoA) revealed a similar pattern of genotypic distribution in the two populations, but greater diversity in the FGIS-derived population. The noticeable difference between the two populations was that genotypes identical to strain NRRL 21882, the active component of the aflatoxin biocontrol product Afla-Guard™, were ten times more common in the commercial corn population of A. flavus compared to the population from the high-aflatoxin corn samples. The other similarities between the two populations suggest that high aflatoxin concentrations in corn grain are generally the result of infection with common A. flavus genotypes.

Genetic diversity based on molecular analysis for Asam Gelugur (Garcinia atroviridis griff. ex t. anders) some districts in North Sumatra

Garcinia atroviridis Griff. known as asam gelugur, it is native to Peninsular Malaysia. The distribution area of Asam gelugur in Indonesia is from Aceh to South Sumatra. The use of Simple Sequence Repeats (SSR) markers aims to understanding of the genetic diversity of G.atroviridis. This study aims to observed at the genetic diversity of asam gelugur from Deli Serdang, Langkat, Karo, Serdang Bedagai and Asahan Districts, North Sumatera, Indonesia. The research was conducted at the Molecular Genetics Laboratory, Agriculture Faculty, Universitas Sumatera Utara, Medan. The research method used of the molecular marking method using 2 SSR primers (M1s75 and Vsur) based on PCR (Polymerase Chain Reaction) which amplifies the Specific DNA sequence. The results showed that primers M1s75 and Vsur were able to amplify 25 samples. The size of the bands was around 150-285 bp and showed monomorphic bands.

Use of Simple Sequence Repeat Marker to Assess the Segregating Population of Rice (Oryza sativa) in field for Grain Length Quality Parameter

Rice grain quality is of paramount importance in the consumer preference for rice varieties and therefore the development of rice varieties should match to the consumer preference. Assessment of segregating population of breeding material in rice for quality parameter made it possible by the use of simple sequence repeat(SSR) marker. The F2 mapping population of a cross between a long slender rice genotype VDN 1137-1-1 and a short slender rice landrace Badshabhog was studied for the inheritance of grain length and to identify the marker associated with grain type. The F2 population showed 3:1 ration for long slender to short slender grain type. This segregating population was assessed by 24 SSR markers using bulk segregant analysis. Among these 24 SSR Markers, a marker RM 147 reported polymorphism between the long slender and short slender parents and respective bulks. Genetic analysis indicated goodness of fit for single gene model indicating the association of RM147 with grain length gene in the segregating population. Therefore, SSR marker RM 147 is of immense value in the assessment of grain quality parameter in rice during the development of rice varieties.

Transferability of apple and pear SSRs to other temperate pome fruit crops of family Rosaceae

Extensive use of simple sequence repeat (SSR) is facilitated if loci would be transferable across species even in closely related genera to overcome high cost and efforts involved in their development as major constraints. In the present study, apple and pear genomic microsatellite primer pairs were used to amplify SSR loci in apple, pear, quince and loquat genotypes, respectively. Already reported SSRs were selected based on their polymorphic survey for successful amplification with at least one polymerase chain reaction (PCR) product of the approximate size expected for a homologous locus screened among apple and pear genotypes for further transferability exploration across other temperate pome fruit crops, respectively. Highest transferability of apple and pear SSR, 61.53 % and 73.33 % was observed in closely related quince and apple genotypes, respectively. This indicated that primer binding sites between these two closely related genera, Malus and Pyrus, are fairly well conserved. Maximum transferability rate was found to be 93.33 % and 80.00 % across all the subjected genotypes for primer CH05D11 and TSUenh016 in apple and pear, respectively. The transferability of markers is based on genomic similarity, and can reflect the relationship of genome collinearity and even evolution between species. This high level of transferability of apple and pear SSRs to other temperate pome fruit crops indicated their promise for application to future molecular screening, map construction, and comparative genomic studies, etc.

The genetic diversity and relationships of cauliflower (Brassica oleracea var. botrytis) inbred lines assessed by using SSR markers.

Inbred lines are important germplasm in cauliflower breeding programs. To understand the genetic diversity and relationships of cauliflower inbred lines, the use of simple sequence repeat (SSR) markers will be of great value for parental line selection and breeding strategy design. In this study, the genetic diversity and relationships of 165 cauliflower inbred lines primarily derived from southeast China were assessed using SSR markers. Forty-three SSR markers were polymorphic across these inbred lines and generated a total of 111 alleles. The mean values of the number of alleles (Na), effective number of alleles (Ne), Shannon’s Information index (I), and polymorphism information content (PIC) per locus were 2.581, 1.599, 0.517 and 0.316, respectively. Genetic distance values among all pairs of the inbred lines varied from 0 to 0.67 with an average of 0.30. On the basis of genetic distance data estimated with the SSR markers, the 165 cauliflower inbred lines were classified into four main clusters (from group Ⅰ to group Ⅳ) by cluster analysis and four subpopulations (from POP 1 to POP 4) by structure analysis. The classification patterns of most cauliflower inbred lines were not consistent with their curd maturity, curd solidity or geographic origins. These results based on estimates by the SSR markers, suggested the genetic diversity of the 165 cauliflower inbred lines was relatively narrow. Therefore, pyramiding the valuable genes among different types of the cauliflower inbred lines is important to increase the genetic diversity to obtain desirable hybridization combinations. The information generated in this report will be useful for assessing germplasm and breeding in cauliflower.

Development and Use of Simple Sequence Repeats (SSRs) Markers for Sugarcane Breeding and Genetic Studies

Recently-developed molecular markers are becoming powerful tools, with applications in crop genetics and improvement. Microsatellites, or simple sequence repeats (SSRs), are widely used in genetic fingerprinting, kinship analysis, and population genetics, because of the advantages of high variability from co-dominant and multi-allelic polymorphisms, and accurate and rapid detection. However, more recent evidence suggests they may play an important role in genome evolution and provide hotspots of recombination. This review describes the development of SSR markers through different techniques, and the detection of SSR markers and applications for sugarcane genetic research and breeding, such as cultivar identification, genetic diversity, genome mapping, quantitative trait loci (QTL) analysis, paternity analysis, cross-species transferability, segregation analysis, phylogenetic relationships, and identification of wild cross hybrids. We also discuss the advantages and disadvantages of SSR markers and highlight some future perspectives.

Extremophiles as a Model of a Natural Ecosystem: Transcriptional Coordination of Genes Reveals Distinct Selective Responses of Plants Under Climate Change Scenarios.

The goal of this research was to generate networks of co-expressed genes to explore the genomic responses of Rhizophora mangle L. populations to contrasting environments and to use gene network analysis to investigate their capacity for adaptation in the face of historical and future perturbations and climatic changes. RNA sequencing data were generated for R. mangle samples collected under field conditions from contrasting climate zones in the equatorial and subtropical regions of Brazil. A gene co-expression network was constructed using Pearson’s correlation coefficient, showing correlations among 78,364 transcriptionally coordinated genes. Each region exhibited two distinct network profiles; genes correlated with the oxidative stress response showed higher relative expression levels in subtropical samples than in equatorial samples, whereas genes correlated with the hyperosmotic salinity response, heat response and UV response had higher expression levels in the equatorial samples than in the subtropical samples. In total, 992 clusters had enriched ontology terms, which suggests that R. mangle is under higher stress in the equatorial region than in the subtropical region. Increased heat may thus pose a substantial risk to species diversity at the center of its distribution range in the Americas. This study, which was performed using trees in natural field conditions, allowed us to associate the specific responses of genes previously described in controlled environments with their responses to the local habitat where the species occurs. The study reveals the effects of contrasting environments on gene expression in R. mangle, shedding light on the different abiotic variables that may contribute to the genetic divergence previously described for the species through the use of simple sequence repeats (SSRs). These effects may result from two fundamental processes in evolution, namely, phenotypic plasticity and natural selection.

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family.

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.

Ricebase: a breeding and genetics platform for rice, integrating individual molecular markers, pedigrees and whole-genome-based data.

Ricebase (http://ricebase.org) is an integrative genomic database for rice (Oryza sativa) with an emphasis on combining datasets in a way that maintains the key links between past and current genetic studies. Ricebase includes DNA sequence data, gene annotations, nucleotide variation data and molecular marker fragment size data. Rice research has benefited from early adoption and extensive use of simple sequence repeat (SSR) markers; however, the majority of rice SSR markers were developed prior to the latest rice pseudomolecule assembly. Interpretation of new research using SNPs in the context of literature citing SSRs requires a common coordinate system. A new pipeline, using a stepwise relaxation of stringency, was used to map SSR primers onto the latest rice pseudomolecule assembly. The SSR markers and experimentally assayed amplicon sizes are presented in a relational database with a web-based front end, and are available as a track loaded in a genome browser with links connecting the browser and database. The combined capabilities of Ricebase link genetic markers, genome context, allele states across rice germplasm and potentially user curated phenotypic interpretations as a community resource for genetic discovery and breeding in rice.

Cross-amplification of Vicia sativa subsp. sativa microsatellites across 22 other Vicia species.

The temperate and herbaceous genus Vicia L. is a member of the legume tribe Fabeae of the subfamily Papilionoideae. The genus Vicia comprises 166 annual or perennial species distributed mainly in Europe, Asia, and North America, but also extending to the temperate regions of South America and tropical Africa. The use of simple sequence repeat (SSR) markers for Vicia species has not been investigated as extensively as for other crop species. In this study, we assessed the potential for cross-species amplification of cDNA microsatellite markers developed from common vetch (Vicia sativa subsp. sativa). For cross-species amplification of the SSRs, amplification was carried out with genomic DNA isolated from two to eight accessions of 22 different Vicia species. For individual species or subspecies, the transferability rates ranged from 33% for V. ervilia to 82% for V. sativa subsp. nigra with an average rate of 52.0%. Because the rate of successful SSR marker amplification generally correlates with genetic distance, these SSR markers are potentially useful for analyzing genetic relationships between or within Vicia species.

SSR genotyping.

SSR genotyping involves the use of simple sequence repeats (SSRs) as DNA markers. SSRs, also called microsatellites, are a type of repetitive DNA sequence ubiquitous in most plant genomes. SSRs contain repeats of a motif sequence 1-6 bp in length. Due to this structure SSRs frequently undergo mutations, mainly due to DNA polymerase errors, which involve the addition or subtraction of a repeat unit. Hence, SSR sequences are highly polymorphic and may be readily used for detection of allelic variation within populations. SSRs are present within both genic and nongenic regions and are occasionally transcribed, and hence may be identified in expressed sequence tags (ESTs) as well as more commonly in nongenic DNA sequences. SSR genotyping involves the design of DNA-based primers to amplify SSR sequences from extracted genomic DNA, followed by amplification of the SSR repeat region using polymerase chain reaction, and subsequent visualization of the resulting DNA products, usually using gel electrophoresis. These procedures are described in this chapter. SSRs have been one of the most favored molecular markers for plant genotyping in the last 20 years due to their high levels of polymorphism, wide distribution across most plant genomes, and ease of use and will continue to be a useful tool in many species for years to come.

Association Analysis for Vegetative Propagation Traits in Eucalyptus tereticornis and Eucalyptus camaldulensis Using Simple Sequence Repeat Markers

Eucalyptus, one of the most widely planted forestry species, is an introduced species to India which is mainly exploited for its pulpwood. Presently, the largest clonal forestry programs are in practice with species of Eucalyptus and the variable rooting potential among the selections are considered to be a hindrance to the success of clonal propagation. Many breeding programs target intra- and inter-specific hybridization for the transfer of vegetative propagation traits and hence SSR markers linked with vegetative propagation traits gained importance for practicing marker assisted selection. Eucalyptus species show high synteny and marker correspondence across genome of different species favoring use of simple sequence repeats (SSRs) linked to quantitative trait loci (QTLs) for association analysis. In this study, 43 accessions of E. tereticornis and 40 accessions of E. camaldulensis were examined for their rooting parameters and subjected to association analysis. The rooting percentage of Eucalyptus accessions showed continuous variation (0–100 %). Association analysis with 62 loci showed that two SSR loci (Embra40 and Embra7) were associated with rooting and mortality percent and shoot length in E. tereticornis. Two SSR loci (Embra167 and Embra39) were associated with shoot length and root length in E. camaldulensis. This study validated the presence of generic genomic regions through SSR markers, which enabled the identification of orthologous QTL regions for vegetative propagation properties in E. tereticornis and E. camaldulensis.

Linkage disequilibrium and population-structure analysis among Capsicum annuum L. cultivars for use in association mapping

Knowledge of population structure and linkage disequilibrium among the worldwide collections of peppers currently classified as hot, mild, sweet and ornamental types is indispensable for applying association mapping and genomic selection to improve pepper. The current study aimed to resolve the genetic diversity and relatedness of Capsicum annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes in samples collected in 2011 and 2012. The physical distance covered by the entire set of SSRs used was 2,265.9 Mb from the 3.48-Gb hot-pepper genome size. The model-based program STRUCTURE was used to infer five clusters, which was further confirmed by classical molecular-genetic diversity analysis. Mean heterozygosity of various loci was estimated to be 0.15. Linkage disequilibrium (LD) was used to identify 17 LD blocks across various chromosomes with sizes from 0.154 Kb to 126.28 Mb. CAMS-142 of chromosome 1 was significantly associated with both capsaicin (CA) and dihydrocapsaicin (DCA) levels. Further, CAMS-142 was located in an LD block of 98.18 Mb. CAMS-142 amplified bands of 244, 268, 283 and 326 bp. Alleles 268 and 283 bp had positive effects on both CA and DCA levels, with an average R(2) of 12.15 % (CA) and 12.3 % (DCA). Eight markers from seven different chromosomes were significantly associated with fruit weight, contributing an average effect of 15 %. CAMS-199, HpmsE082 and CAMS-190 are the three major quantitative trait loci located on chromosomes 8, 9, and 10, respectively, and were associated with fruit weight in samples from both years of the study. This research demonstrates the effectiveness of using genome-wide SSR-based markers to assess features of LD and genetic diversity within C. annuum.

Use of EST-SSR markers for evaluating genetic diversity and fingerprinting celery (Apium graveolens L.) cultivars.

Celery (Apium graveolens L.) is one of the most economically important vegetables worldwide, but genetic and genomic resources supporting celery molecular breeding are quite limited, thus few studies on celery have been conducted so far. In this study we made use of simple sequence repeat (SSR) markers generated from previous celery transcriptome sequencing and attempted to detect the genetic diversity and relationships of commonly used celery accessions and explore the efficiency of the primers used for cultivars identification. Analysis of molecular variance (AMOVA) of Apium graveolens L. var. dulce showed that approximately 43% of genetic diversity was within accessions, 45% among accessions, and 22% among horticultural types. The neighbor-joining tree generated by unweighted pair group method with arithmetic mean (UPGMA), and population structure analysis, as well as principal components analysis (PCA), separated the cultivars into clusters corresponding to the geographical areas where they originated. Genetic distance analysis suggested that genetic variation within Apium graveolens was quite limited. Genotypic diversity showed any combinations of 55 genic SSRs were able to distinguish the genotypes of all 30 accessions.

Survey of simple sequence repeats in woodland strawberry (Fragaria vesca)

The use of simple sequence repeats (SSRs), or microsatellites, as genetic markers has become popular due to their abundance and variation in length among individuals. In this study, we investigated linkage groups (LGs) in the woodland strawberry (Fragaria vesca) and demonstrated variation in the abundances, densities, and relative densities of mononucleotide, dinucleotide, and trinucleotide repeats. Mononucleotide, dinucleotide, and trinucleotide repeats were more common than longer repeats in all LGs examined. Perfect SSRs were the predominant SSR type found and their abundance was extremely stable among LGs and chloroplasts. Abundances of mononucleotide, dinucleotide, and trinucleotide repeats were positively correlated with LG size, whereas those of tetranucleotide and hexanucleotide SSRs were not. Generally, in each LG, the abundance, relative abundance, relative density, and the proportion of each unique SSR all declined rapidly as the repeated unit increased. Furthermore, the lengths and frequencies of SSRs varied among different LGs.

Assessment of gene flow in white clover (Trifolium repens L.) under field conditions in Australia using phenotypic and genetic markers

White clover is one of the most important pasture legumes in global temperate regions. It is an outcrossing, insect-pollinated species with gene flow occurring naturally between plants. A 2-year study was conducted to assess the relationship between gene flow and physical distance in white clover under field conditions in southern Australia. White clover plants exhibiting a red leaf mark phenotypic trait acted as pollen donors to recipient plants lacking leaf markings at distances up to 200 m distant from the donor plants. Progeny were scored for the dominant red-leafed phenotype and gene flow was modelled. Paternity was confirmed using simple sequence repeat markers. A leptokurtic pattern of gene flow was observed under conditions designed to measure maximised gene flow with the majority of pollination occurring in the first 50 m from the donor pollen source. The combined use of simple sequence repeat and visual markers confirmed that there was also a white clover pollen source in addition to the donor plants. This research confirms the difficulty in ensuring absolute containment of gene flow in an outcrossing species grown in an environment when endemic populations are known to exist.

Species Discrimination, Population Structure and Linkage Disequilibrium in Eucalyptus camaldulensis and Eucalyptus tereticornis Using SSR Markers

Eucalyptus camaldulensis and E. tereticornis are closely related species commonly cultivated for pulp wood in many tropical countries including India. Understanding the genetic structure and linkage disequilibrium (LD) existing in these species is essential for the improvement of industrially important traits. Our goal was to evaluate the use of simple sequence repeat (SSR) loci for species discrimination, population structure and LD analysis in these species. Investigations were carried out with the most common alleles in 93 accessions belonging to these two species using 62 SSR markers through cross amplification. The polymorphic information content (PIC) ranged from 0.44 to 0.93 and 0.36 to 0.93 in E. camaldulensis and E. tereticornis respectively. A clear delineation between the two species was evident based on the analysis of population structure and species-specific alleles. Significant genotypic LD was found in E. camaldulensis, wherein out of 135 significant pairs, 17 pairs showed r2≥0.1. Similarly, in E. tereticornis, out of 136 significant pairs, 18 pairs showed r2≥0.1. The extent of LD decayed rapidly showing the significance of association analyses in eucalypts with higher resolution markers. The availability of whole genome sequence for E. grandis and the synteny and co-linearity in the genome of eucalypts, will allow genome-wide genotyping using microsatellites or single nucleotide polymorphims.

EVALUATION OF SIMPLE SEQUENCE REPEATS (SSR) FOR GENETIC ANALYSIS OF CHESTNUT TREES IN MICHIGAN ORCHARDS

In Michigan, non-grafted seedlings of Chinese descent commonly planted by growers predominate chestnut orchards because of the resistance to chestnut blight. In addition, grafted hybrids of European, Chinese, and Japanese descent, many with unknown pedigrees, are also present in orchards. The outcome of this genetic mixing in orchards is a chestnut product with variable size and quality. Therefore, the objective of this study is to evaluate the use of simple sequence repeats (SSRs) to genetically characterize promising chestnut trees and cultivars currently growing in Michigan orchards. In the summer of 2007, we began a series of crosses in four isolated orchards, where each orchard consisted of two cultivars. Trees of the C. sativa × C. crenata hybrid cultivar 'Colossal' were selected as the female parent for every cross due to its good yield and because it is planted in orchards throughout the state. The chestnut cultivars 'Benton Harbor' and 'Eaton', both with Asian backgrounds, and the European hybrid cultivars 'Nevada', and 'Okei' were selected as pollinizer trees. Fifty nuts per cross were randomly selected as the individuals representing the four populations. We were able to generate fingerprints using both universal and specific primers such as (GTG) 5 and CsCAT/EMCs loci, respectively. The SSRs data were analyzed using 'PeakScanner' and 'Identity' software. Complex polymorphic bands were observed among chestnut cultivars, which ranged from 1,200 to 300 base pairs in length when (GTG) 5 was used. Amplicon sizes ranged from 76 to 193 base pairs when specific SSRs were used and the number of alleles observed at a locus ranged from 4 to 6. Results obtained from this study will allow for the genetic characterization of the chestnut cultivars and seedling trees currently growing in Michigan, and subsequently will help us establish high quality chestnut plantings for food and fiber production.