## Abstract

From the genome sequence of hazelnut (*Corylus avellana*), 192 new polymorphic simple sequence repeat (SSR) markers were developed, characterized, and used to investigate genetic diversity in 50 accessions. Next-generation sequencing allows inexpensive sequencing of plant genomes and transcriptomes, and efficient development of polymorphic SSR markers, also known as microsatellite markers, at low cost. A search of the genome sequence of ‘Jefferson’ hazelnut identified 9094 fragments with long repeat motifs of 4, 5, or 6 base pairs (bp), from which polymorphic SSR markers were developed. The repeat regions in the ‘Jefferson’ genome were used as references to which genomic sequence reads of seven additional cultivars were aligned in silico. Visual inspection for variation in repeat number among the aligned reads identified 246 as polymorphic, for which primer pairs were designed. Polymerase chain reaction (PCR) amplification followed by agarose gel separation indicated polymorphism at 195 loci, for which fluorescent forward primers were used to amplify the DNA of 50 hazelnut accessions. Amplicons were post-PCR multiplexed for capillary electrophoresis, and allele sizes were determined for 50 accessions. After eliminating three, 192 were confirmed as polymorphic, and 169 showed only one or two alleles in each of the 50 cultivars, as expected in a diploid. At these 169 SSRs, a total of 843 alleles were found, for an average of 4.99 and a range of 2 to 17 alleles per locus. The mean observed heterozygosity, expected heterozygosity, polymorphism information content, and the frequency of null alleles were 0.51, 0.53, 0.47, and 0.03, respectively. An additional 25 primer pairs produced more than two bands in some accessions with an average of 6.8 alleles. The UPGMA dendrogram revealed a wide genetic diversity and clustered the 50 accessions according to their geographic origin. Of the new SSRs, 132 loci were placed on the linkage map. These new markers will be useful for diversity and parentage studies, cultivar fingerprinting, marker-assisted selection, and aligning the linkage map with scaffolds of the genome sequence.

Hazelnut, one of the world’s major nut crops, is an important crop in the Willamette Valley of Oregon where 99% of the U.S. hazelnut crop is produced. Over a 10-year period (2005–14), Turkey produced 67.2% of the world crop, followed by Italy (12.6%), the United States (4.0%), Azerbaijan (3.4%), and Republic of Georgia (3.1%) (Food and Agriculture Organization of the United Nations, 2017). In Oregon, hazelnuts are planted on 27,000 ha and plantings are expanding at 1200 ha per year (Oregon Hazelnuts, 2018). There are also many new plantings and production is increasing in China, Chile, Republic of Georgia, and other countries.

Hazelnut is diploid (2*n* = 2*x* = 22) and belongs to the family Betulaceae. Of the 13 commonly recognized species of *Corylus*, only the European species (*C. avellana*) is widely planted for commercial production. Hazelnuts are monoecious, dichogamous, and self-incompatible, and thus cross-pollinated in nature. Cultivars are highly heterozygous and clonally propagated. Most of the world's cultivars are selections from local wild vegetation. Based on simple sequence repeat markers, most cultivars have been assigned to one of four major geographical groups: Central European, Black Sea, English or Spanish-Italian (Boccacci et al., 2006; Gökirmak et al., 2009).

Simple sequence repeat markers are DNA segments comprised of tandem repeat motifs that are 1 to 8 nucleotides in length. SSRs are polymerase chain reaction (PCR)-based markers for which primers are designed from conserved sequences that flank the repeat region. At polymorphic loci, PCR amplification results in different product sizes. SSR markers are preferred over several other marker types because of their high level of polymorphism, multiple alleles, codominant inheritance, good genome coverage, interspecific and intergeneric transferability, high reproducibility, amenability to automated high-throughput genotyping, and ease of sharing among laboratories (Parida et al., 2009). SSR markers are widely used in plant genetics and breeding, including studies of genetic diversity and evolution, association, gene flow, genetic mapping, gene tagging, gene cloning, cultivar identification, parentage analysis, identification of duplicates, marker-assisted selection (MAS), and quantitative trait locus (QTL) analysis (Ellegren, 2004; Hearne et al., 1992; Parida et al., 2009). In hazelnut, SSR markers were initially developed from DNA libraries enriched for specific repeats (Bassil et al., 2005a, 2005b; Boccacci et al., 2005; Gürcan et al., 2010). Later, Gürcan and Mehlenbacher (2010a) developed SSRs from the sequences of inter simple sequence repeat (ISSR) markers and flanking regions. Sathuvalli and Mehlenbacher (2013) developed SSR markers from bacterial artificial chromosome (BAC) sequences. SSRs have also been developed from sequences of the hazelnut transcriptome (Colburn et al., 2017) and genome (Bhattarai and Mehlenbacher, 2017), and sequences in public databases (Boccacci et al., 2015; Gürcan and Mehlenbacher, 2010b). Although about 525 SSR markers are publicly available in hazelnut, the majority contain di-nucleotide motifs which suffer from PCR artifacts that prevent automated scoring (Testolin and Cipriani, 2010). In this study, we mined the ‘Jefferson’ genome sequence to develop new polymorphic SSR markers with repeat motifs of 4, 5, or 6 bp and further saturated the reference hazelnut genetic linkage map.

## Materials and Methods

#### Plant material.

A set of 48 diverse hazelnut accessions plus the two parents of a mapping population (Table 1) were used to characterize the new SSR markers. These accessions were a subset of the 198 unique types investigated by Gökirmak et al. (2009), and are phenotypically, genetically, and geographically diverse. The same 50 accessions were used in previous characterization studies (Bhattarai and Mehlenbacher, 2017; Colburn et al., 2017; Gürcan and Mehlenbacher, 2010a; Gürcan et al., 2010). Of these accessions, 24 were used to screen all primer pairs for polymorphism on agarose gels. For SSRs that appeared to be polymorphic on agarose gels, all 50 accessions and capillary electrophoresis were used to characterize them.

Hazelnut accessions used to identify and characterize tetra-, penta-, and hexa- repeat microsatellite markers mined from genome sequences. Accessions 1 to 24 were used to screen for polymorphism on agarose gels, while all 50 accessions were used for characterization of polymorphic simple sequence repeat loci. Some were imported as scions, and others as seed lots from which individuals were selected.

#### DNA extraction.

DNA was extracted from two to four young leaves collected during early spring from trees growing in fields at the USDA-ARS National Clonal Germplasm Repository (NCGR) and Smith Horticultural Research Farm of Oregon State University (OSU) in Corvallis. DNA extraction followed the method of Lunde et al. (2000) without RNAase treatment. The extracted DNA was quantified using ultraviolet spectrophotometry with a Synergy 2 Multi-Mode Reader with a Take 3 microplate reader and the data were analyzed with Gen5 software (all Biotek Instruments, Winooski, VT). The DNA was diluted with TE buffer to a concentration of 20 ng·µL^{−1}.

#### SSR identification.

The hazelnut genome sequences of Rowley (2016) generated by Illumina (San Diego, CA) technology were used in this study. The ‘Jefferson’ genome sequence (93 × coverage with 333,492 sequences comprising 224.58 Mb) was used as the reference. Seven other cultivars (Barcelona, Ratoli, Tonda Gentile delle Langhe, Tonda di Giffoni, Daviana, Hall's Giant, and Tombul) were resequenced at lower coverage (≈20×). The ‘Jefferson’ genome sequences were searched for SSRs using the MISA tool (Thiel et al., 2003) with the search criteria for minimum number of repeats set at six for dinucleotide repeats, five for trinucleotide repeats, and four each for tetra-, penta-, hexa-, hepta- and octanucleotide motifs. This study pursued only the tetra-, penta-, and hexanucleotide repeat SSRs. Repeats that contained only As and Ts were removed, as they tend to be very difficult to score. Short fragments (<400 bases) and fragments with repeats near the ends of the contigs were also removed, the latter because they lacked a flanking sequence sufficient for primer design. Paired-end Illumina reads from the seven other cultivars were concatenated into a single file, and aligned with SSR-containing sequences in the ‘Jefferson’ reference using the MAQ program (Li et al., 2008). The aligned reads were imported into Tablet software (Milne et al., 2013) for visualization and identification of SSRs with variation in the number of repeat units but conserved flanking sequences. Tablet software displayed the reference sequence at the top of the screen, with the aligned reads from the seven cultivars shown in subsequent rows without identification of the originating cultivar. The four nucleotides were displayed with unique colors for ease of visualization. After alignment and visualization, each sequence was classified as “clearly polymorphic,” “slightly polymorphic,” “not polymorphic,” “poor alignment,” or “no reads.” Of these categories, only the “clearly polymorphic” sequences were pursued, and forward and reverse primers were designed using the Websat (Martins et al., 2009) and Primer 3 (Untergasser et al., 2012) programs with lengths of 18 to 27 bp, annealing temperature of 60 °C, and a range of expected product sizes of 90–400 bp to facilitate post-PCR multiplexing. Non-fluorescent forward and reverse primers were ordered from Integrated DNA Technologies (Coralville, IA). The “slightly polymorphic” sequences, for which <2% of the aligned reads showed variation in the number of repeats, were not pursued.

#### PCR amplification for polymorphism screening.

PCR was performed with each primer pair using DNA of 24 accessions in the diversity panel (Table 1). PCRs were in 10-µL volumes containing 0.3 µM each of forward and reverse primers, 1 × Biolase NH_{4} reaction buffer, 2 mm MgCl_{2}, 200 µM each of dATP, dCTP, dGTP, and dTTP, 20 ng template DNA, and 0.25 units of DNA polymerase (Biolase; Bioline USA, Taunton, MA). PCR amplification was performed in thermal cyclers (GeneAmp PCR system 9700; Applied Biosystems, Foster City, CA) in 96-well plates using the following program: denaturation at 95 °C for 5 min followed by 40 cycles of at 94 °C for 40 s, 60 °C for 40 s, 72 °C for 40 s, a final extension at 72 °C for 7 min, and an infinite hold at 4 °C. The PCR products were separated by electrophoresis on 3% agarose gels in TBE buffer at 90V for 3.5 h, stained for 30 min in ethidium bromide and then destained in distilled water for 25 min. The gels were then photographed under ultraviolet light using an imaging system (BioDoc-It^{®}; UVP, Upland, CA), and the images were inspected for size polymorphism among the 24 genotypes.

#### Genotyping at polymorphic SSRs.

For SSR loci that were polymorphic on the agarose gels, fluorescent forward primers labeled with 6-FAM or 5-HEX were ordered from Integrated DNA Technologies and fluorescent forward primers labeled with NED were ordered from Applied Biosystems. The fluorescent forward and non-fluorescent reverse primers (Supplemental Table 1) were used to amplify DNA from the 48 accessions and the two parents of the mapping population (Table 1). The use of fluorescent primers and PCR products of different sizes allowed efficient post-PCR multiplexing of 5 to 7 primer pairs in a single well. For multiplexing, 2 µL of the PCR products from each primer pair were mixed and diluted with water to make a final volume of 150 µL. An aliquot of 1 to 1.5 µL of multiplexed PCR products was submitted to the Core Laboratories of OSU's Center for Genome Research and Biocomputing (CGRB) for fragment sizing by a capillary electrophoresis instrument (ABI 3730; Life Technologies, Carlsbad, CA) using ROX-500 as the size standard. GeneMapper^{®} software (Life Technologies) was used for allele sizing and sizes were recorded in a spreadsheet. PCR amplification and capillary electrophoresis were repeated if the initial PCR failed or the result was ambiguous, and to verify unique allele sizes.

#### Characterization of polymorphic SSRs.

For polymorphic primer pairs that showed the expected one or two PCR products in all cultivars, PowerMarker software (Liu and Muse, 2005) was used to calculate number of alleles (*n*), observed heterozygosity (H_{o}), expected heterozygosity (H_{e}), and polymorphism information content (PIC). Observed heterozygosity is the number of heterozygous genotypes divided by the total number of genotypes. Expected heterozygosity estimates the probability that a randomly chosen individual is heterozygous at a locus and is calculated as *p*_{i} is the frequency of the *i*^{th} allele and *n* is the number of alleles (Nei, 1973). The PIC value is a measure of marker informativeness and is calculated as *p*_{i} is the frequency of the *i*^{th} allele, *p*_{j} is the frequency of the *j*^{th} allele, and *n* is the number of alleles (Botstein et al., 1980). The frequency of null alleles (r) was calculated using Cervus software (Field Genetics, London, UK), which uses the formula of Kalinowski and Taper (2006). Genetic similarities between pairs of accessions were calculated as the proportion of shared alleles (P_{s}) and genetic distance was calculated as (1 – P_{s}), resulting in a frequency-based distance matrix. For SSRs with one or two bands per accession, dendrograms were constructed using PowerMarker (Liu and Muse, 2005) and the unweighted pair-group method using arithmetic averages (UPGMA) algorithms. To include SSRs with more than two PCR products in some accessions, the genotype data for the 50 accessions at all new SSR loci were converted to binary format (i.e., dominant scoring) using an Excel macro (Rinehart, 2004). The dendrograms were visualized using Mega7 software (Kumar et al., 2016).

#### Segregation and linkage mapping.

For the newly developed SSR marker loci at which the parent genotypes of the mapping population [OSU 252.146 x OSU 414.062 (Mehlenbacher et al., 2006)] predicted segregation, all 138 available seedlings were genotyped as described above. The expected allele sizes were known, and genotyping was in multiplex sets of 10 to 14 primer pairs. Each allele was scored as present or absent in each seedling, scores were tallied, and the expected ratio (1:1 or 1:1:1:1 or 1:2:1) was noted. Scores for the new SSR markers were added to those for previously mapped random amplified polymorphic DNA (RAPD) and SSR markers (Bhattarai and Mehlenbacher, 2017). Linkage maps were constructed using the two-way pseudo-testcross method and the BC1 function in JoinMap 4.1 (Kyazma; Wageningen, Netherlands). Markers segregating in a ratio of 1:1 or 1:1:1:1 were scored as h, a or u for presence, absence, or unknown status, respectively, for each allele in each seedling. Similarly, markers segregating in a ratio of 1:2:1 were scored as h if present in homozygous state, a if absent (homozygous), and u if heterozygous or unknown status. Heterozygotes at loci segregating in a 1:2:1 ratio provide no information useful for mapping. Markers were assigned to linkage groups (LGs) using a median logarithm of odds (LOD) score of 12 (range 9 to 15). Maps were constructed separately for each linkage group using the maximum likelihood (ML) algorithm and distances shown in Haldane units (centimorgans). Markers that clustered loosely with the others and fell out at LOD scores <9 were removed. Markers present in repulsion phase were included in the map by creating “dummy variables,” the use of which allowed the merger of the repulsion phase and coupling phase markers, and generation of a single map for each linkage group in each parent. The JoinMap output was inspected for “Fit and Stress” and markers were removed in stepwise manner until the “Nearest Neighbor Stress” value for all markers was less than an arbitrarily set value of 7.60. The “Nearest Neighbor Fit (cM)” values were also inspected, as high values indicate blocks of markers that fit poorly with adjacent markers. In addition, the length of the gaps between markers was inspected, with gaps of >20 cM considered suspicious.

## Results

#### SSR discovery and characterization.

A search of the ‘Jefferson’ genome sequence identified 51,268 SSRs with repeat motifs of 2 to 8 bp (Table 2). Dinucleotide repeats (64.90%) were most abundant, followed by trinucleotide (16.99%), tetranucleotide (12.49%), pentanucleotide (3.43%), hexanucleotide (1.82%), heptanucleotide (0.37%), and octanucleotide (0.001%) repeats. The number of repeats in these SSRs ranged from 6 to 14 in the dinucleotide repeats, and decreased as the motif length increased. Among the dinucleotide repeats, AG and AT repeats were most common, representing 49.4% and 40.2% of the total, respectively (Supplemental Table 2). Among the trinucleotide repeats, AAT and AAG were most common, representing 43.1% and 28.4% of the total, respectively. The most common motifs were AAAT (55.0%) among the tetranucleotide repeats, AAAAT (36.3%) and AAAAG (25.7%) among the pentanucleotide repeats, and AAAAAG (18.3%) and AAAAAT (12.6%) among the hexanucleotide repeats. The focus of this study was the tetra-, penta- and hexanucleotide repeats, for which the maximum number of repeats was 8, 6, and 6, respectively. Among the 192 polymorphic SSRs markers developed in this study, the most common motifs were ACGT/TGCA, AAAGG/TTTCC, and AGTTGG/TCAACC for the tetra-, penta-, and hexanucleotide repeats, respectively.

Counts of SSR in the ‘Jefferson’ hazelnut genome by type and number of repeats.

Initially, the number of sequences containing tetra-, penta-, and hexanucleotide repeats was 9094. The number of sequences was reduced in stepwise manner, resulting in the final set of 192 polymorphic SSR markers (Table 3). Of the 9094 repeats, 4774 were removed because the repeat motifs contained only As and Ts. The removal of short fragments (<400 bp) and those with the repeats at the fragment ends further reduced the number to 1013. After alignment with the reference genome, 246 were scored as “polymorphic” as they showed variation in number of repeat units but conserved flanking sequences. An additional 17 were scored as “slightly polymorphic,” and 616 showed no polymorphism in number of repeat motifs. For 114 sequences, the reads of the seven cultivars aligned poorly with the reference, and for 20 sequences none of the reads from the seven cultivars aligned with the reference. Primers were designed for the 246 scored as polymorphic based on visual inspection of aligned sequences. After amplification of 24 diverse accessions followed by separation on agarose gels, 195 were scored as polymorphic. Fluorescent forward primers were ordered for these 195, which were then used to amplify and score allele sizes in 50 accessions after separation by capillary electrophoresis. Two primer pairs gave poor amplification in many cultivars, and one was monomorphic, while 192 new polymorphic SSR markers were pursued, of which 89 were labeled with 6-FAM, 70 with 5-HEX, and 33 with NED (Supplemental Table 1). Of the newly developed SSRs, 60, 66, and 66 consist of tetra-, penta-, and hexanucleotide repeats, respectively. Allele sizes at the 192 new marker loci in the 50 accessions are presented (Supplemental Tables 3 and 4). At 169 SSR loci (88.0%), all 50 accessions showed the one or two alleles expected in a diploid, and diversity parameters were estimated for them (Table 3; Supplemental Table 3). However, at 23 SSRs (12.0%), more than one accession generated more than two PCR products; for these the presence of each allele was scored in each accession (Table 3; Supplemental Table 4). In the 169 characterized markers (Table 3), 846 alleles were detected. The number of alleles per locus ranged from 2 to 17 with an average of 4.99. Expected heterozygosity had a mean of 0.53 and a range of 0.04 to 0.85, while observed heterozygosity had a mean of 0.51 and a range of 0.04 to 0.90. The PIC value averaged 0.47 and ranged from 0.04 to 0.84. GB726 was the most polymorphic marker (H_{o} = 0.81), while the four least informative markers (GB404, GB405, GB446, and GB616) had PIC values less than 0.10. Of these, GB405 had four alleles while the other three had only two alleles each. The average frequency of null alleles was 0.03, with values for individual loci ranging from –0.20 to 0.59. At the 23 primer pairs that gave more than two PCR products in one or more accessions, 170 alleles were present with an average of 7.39 alleles per primer pair (Table 3). Allele size distributions at the 192 polymorphic SSR markers are shown as histograms (Supplemental Fig. 1).

Characteristics and linkage group assignments of new simple sequence repeat markers developed from the ‘Jefferson’ hazelnut genome sequence.

#### Segregation and linkage mapping.

A total of 135 loci segregated in the mapping population, of which 70 (51.9%) segregated in a 1:1 ratio, 35 (25.9%) in a 1:1:1:1 ratio, and 30 (22.2%) in a 1:2:1 ratio (Supplemental Table 5). New markers were placed in each of the 11 linkage groups, and the resulting genetic map included 132 new and 297 previously mapped SSR loci (Fig. 2). Three loci (GB733, GB737, and GB746) showed severe segregation distortion and could not be assigned to a LG (Supplemental Table 4). The lengths of the linkage groups ranged from 63.5 to 277 cM.

#### Genetic diversity in hazelnut accessions.

A dendrogram was constructed for the 50 accessions using the UPGMA algorithm (Fig. 1) and allele sizes at 192 loci. The dendrogram confirmed the wide genetic diversity in hazelnut, grouped the accessions according to their geographic origins, and showed the four major groups seen in previous studies: Spanish-Italian, Black Sea, Central European, and English (Bhattarai and Mehlenbacher, 2017; Boccacci et al., 2006; Colburn et al., 2017; Gökirmak et al., 2009; Gürcan et al., 2010). The Central European group includes four cultivars of German origin (Gunslebert, Hall’s Giant, Gustav’s Zellernuss, and Early Long Zellernuss), as well as ‘Bergeri’ from Belgium and ‘Alli’ from Estonia. The Black Sea group includes six accessions from Turkey and one from Republic of Georgia. The English group includes ‘Cosford’ from Reading, England, ‘Rote Zellernuss’ from the Netherlands, and ‘Buttner's Zellernuss’ from Landsberg, Germany. Their adjacent placement in the dendrogram suggests that the latter two might be seedlings of ‘Cosford’. The ornamental ‘Contorta’ originated in England, as did ‘Du Chilly’, which is grown as ‘Kentish Cob’ in southeastern England. ‘Pellicule Rouge’, obtained from France, has leaves and pellicles with anthocyanin, but all other traits and its DNA fingerprint are identical to the English cultivar White Filbert (Gökirmak et al., 2009). ‘Des Anglais’ was obtained from Bordeaux, France, but its name indicates an English origin. All of the Spanish and Italian cultivars were included in the large Spanish-Italian group.

## Discussion

The high throughput and low cost of next-generation sequencing allows plant genomes to be sequenced and SSR markers to be developed efficiently as a by-product of genome sequencing. In hazelnut, we identified 9094 ‘Jefferson’ genome sequences that contained tetra-, penta- or hexanucleotide repeats and proceeded in a stepwise manner to develop polymorphic SSR markers from them. Of the 1013 sequences for which aligned sequences were visually inspected, 246 showed variation in number of repeat units but conserved flanking sequences. After primer design, amplification, allele sizing, and characterization, this number was reduced to the 192 new polymorphic SSR loci described in this paper, of which 169 amplified only one or two bands in all cultivars, as expected in a diploid species. The ratio of single-locus polymorphic SSRs developed to number of primer pairs designed was 68.7% in this study, compared with 28.5% for the mostly trinucleotide repeat SSRs developed from transcriptome sequences (Colburn et al., 2017) without in silico evaluation. The comparison illustrates the improved efficiency that results from this evaluation. For the dinucleotide repeat SSRs developed from enriched libraries, the ratio was an intermediate 57% (Gürcan et al., 2010). Although single nucleotide polymorphism (SNP) markers can be generated in large numbers by procedures including genotyping-by-sequencing [GBS (Elshire et al., 2011)] and double digest RADseq (Peterson et al., 2012), SSR markers continue to have many research and breeding applications in plants. In hazelnut, for example, Özturk et al. (2017) studied associations of SSR markers with nut and kernel traits in a Slovenian hazelnut collection, and Marinoni et al. (2018) used SSR and SNP markers to identify QTL in a seedling population from ‘Tonda Gentile delle Langhe’ x ‘Merveille de Bollwiller’.

Most of the tetra-, penta-, and hexa- SSRs identified in this study were composed of only As and Ts (52%). The very high frequency of A and T bases in SSRs have been observed in many other tree species (Xia et al., 2017). Mononucleotide repeats, although abundant in the hazelnut genome, were not pursued as they tend to be difficult to score. Dinucleotide repeats were not pursued as large numbers had previously been developed (Bassil et al., 2005a, 2005b; Boccacci et al., 2005, 2015; Gürcan et al., 2010; Gürcan and Mehlenbacher, 2010a; Sathuvalli and Mehlenbacher, 2013) and stuttering is a common problem with dinucleotide repeats (Hearne et al., 1992). Colburn et al. (2017) developed 111 SSRs from transcriptome sequences, most of which were trinucleotide repeats, and Bhattarai and Mehlenbacher (2017) developed 150 trirepeat SSRs from the Illumina draft genome sequence for ‘Jefferson’. This study adds 192 markers to the 525 previously developed, and further saturates the genetic map. Previous studies (Bassil et al., 2005a, 2013; Boccacci et al., 2005, 2015; Gürcan and Mehlenbacher, 2010b) showed a high rate of transferability to other *Corylus* species, and lower transferability (≈30%) to the genera *Betula* and *Alnus*, which are also in the family Betulaceae. Thus, the SSR markers developed in this study could have applications beyond *C. avellana*.

Of the new polymorphic SSRs developed in this study, 23 primer pairs amplified more than two bands in some hazelnut accessions. SSRs of this type were previously reported in hazelnut (Bhattarai and Mehlenbacher, 2017; Gürcan and Mehlenbacher, 2010a) and other plant species (Chavarriaga-Aguirre et al., 1998; Kim et al., 2014). We attribute this to either nonspecific amplification or genome duplication events, including paralogous sequences.

The count of private or unique alleles (occurring in only one individual) is 201 at 103 SSR loci (Supplemental Table 6). These unique alleles were confirmed by repeating the PCR and allele sizing. Null alleles, which fail to amplify with PCR, are the result of a mutation in either one or both primer binding sites of the genome sequences. The presence of null alleles leads to errors in pedigree and segregation analysis (Dakin and Avise, 2004; Pemberton et al., 1995). The mean frequency of null alleles was low (r = 0.03), although a few loci had high values and 16 (GB405, GB409, GB421, GB454, GB461, GB465, GB505, GB532, GB602, GB624, GB640, GB646, GB649, GB671, GB718, and GB721) had values >0.20.

The UPGMA dendrogram, based on binary data of allele sizes at 192 SSRs, showed accessions clustered according to their geographic origin into four main groups: Central European, Black Sea, English, and Spanish-Italian. This grouping is similar to previous studies (Boccacci et al., 2006; Colburn et al., 2017; Gökirmak et al., 2009; Gürcan et al., 2010). The dendrogram (Fig. 1) shows a high level of diversity in the 50 accessions, including 13 diverse accessions at the top that did not cluster with any of the others (Fig. 1).

Of the 192 new SSR markers, 135 segregated in the mapping population, of which 132 were placed on the map and three with distorted segregation remained unlinked. Segregation ratios at 135 loci (Supplemental Table 4) were examined for goodness-of-fit to expected ratios. Poor fit to Mendelian expectations (*P* < 0.05) was observed at 20 loci, of which 11 showed very poor fit (*P* < 0.01). Several SSRs did not segregate in the reference mapping population. Their map locations may be determined in future research using alternate mapping populations, or by alignment with a mapped genome sequence contig. Linkage groups are presented separately for the female and male parents (Fig. 2). The female parent map consists of 11 distinct linkage groups, while the male parent map initially had 10 distinct groups, with LG2 and LG7 merged into a single large group, as first encountered by Mehlenbacher et al. (2006) and more recently by Beltramo et al. (2016) for a population from ‘Tonda Gentile delle Langhe’ x ‘Merveille de Bollwiller’. Reciprocal translocations are common in hazelnut (Salesses, 1973; Salesses and Bonnet, 1988), and may be responsible for this merger. We separated LG2 and LG7 in the male parent map following the method of Colburn et al. (2017) and present 11 groups. The newly developed markers were assigned to all 11 LGs (Fig. 2), but were not evenly distributed.

Linkage maps of the hazelnut reference mapping population (OSU 252.146 x OSU 414.062). The new simple sequence repeat markers are indicated by * and bold font.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04461-18

Linkage maps of the hazelnut reference mapping population (OSU 252.146 x OSU 414.062). The new simple sequence repeat markers are indicated by * and bold font.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04461-18

Linkage maps of the hazelnut reference mapping population (OSU 252.146 x OSU 414.062). The new simple sequence repeat markers are indicated by * and bold font.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 5; 10.21273/JASHS04461-18

Four markers (GB478, GB456, GB605, and GB671) were placed on LG6 < 20 cM from the locus from ‘Gasaway’ for resistance to eastern filbert blight (EFB) caused by *Anisogramma anomala* (Mehlenbacher et al., 2006), although none are closer than four SSRs developed from BAC sequences that cosegregate with the allele for resistance (Sathuvalli and Mehlenbacher, 2013). EFB resistance from five other sources maps to the same region (Bhattarai et al., 2017b; Colburn et al., 2015; Sathuvalli et al., 2012). Three other markers (GB467, GB482, and GB642), were assigned to LG7 in the region of the EFB resistance loci from ‘Ratoli’, ‘Yoder #5’, and *C. americana* ‘Rush’ (Bhattarai et al., 2017a; Sathuvalli et al., 2011a). Four markers (GB609, GB618, GB669, and GB740) were placed on LG2 near the EFB resistance locus from Georgian selection OSU 759.010 (Sathuvalli et al., 2011b). These new markers will be useful for mapping additional EFB resistance sources and developing breeder-friendly DNA markers in these regions for use in marker-assisted selection. Additionally, GB646 was placed on LG5 near the S-locus that controls pollen-stigma incompatibility (Ives et al., 2014; Mehlenbacher et al., 2006).

The genomic SSRs developed in this study add to those developed previously and will facilitate research in hazelnut genetics, including cultivar fingerprinting, parentage analysis, studies of genetic diversity, mapping of qualitative trait loci and QTL, marker-assisted breeding, and the fine mapping and cloning of genes. Because SSR markers map to the same location in different populations, they allow the alignment or merger of genetic maps from different crosses. In addition, the mapped SSR markers will allow alignment of the linkage map with the genome sequences and the physical map of BAC contigs.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Histograms showing allele frequencies at 194 new simple sequence repeat loci in hazelnut.

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Repeat motifs, amplicon size, primer sequences, and annealing temperatures (Tm) of simple sequence repeat markers developed from the ‘Jefferson’ hazelnut genome sequence.

Repeat motifs at SSR in the ‘Jefferson’ hazelnut genome by type and number of repeats.

Alleles at 169 simple sequence repeat marker loci in 50 hazelnut accessions. All cultivars showed one or two alleles at each locus.

Alleles at 23 simple sequence repeat marker loci in 50 hazelnut accessions. Some cultivars showed more than two alleles.

Segregation at new simple sequence repeat markers and linkage group assignments in the hazelnut mapping population (OSU 252.146 x OSU 414.062).

Private (unique) alleles at SSR loci in 50 hazelnut accessions. At 103 loci, a total of 201 unique alleles were observed.