Genetic Characterization Of Liriodendron Seed Orchards With EST SSR Markers LT157 2050 2389 4 1
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Journal of Plant Science & Molecular Breeding ISSN 2050-2389 | Volume 4 | Article 1 Research Open Access Genetic characterization of Liriodendron seed orchards with EST-SSR markers Xinfu Zhang1, Alanna Carlson1, Zhenkun Tian2, Margaret Staton3, Scott E. Schlarbaum4, John E. Carlson5 and Haiying Liang1* *Correspondence: hliang@clemson.edu CrossMark ← Click for updates Department of Genetics and Biochemistry, Clemson University, Clemson SC 29634, USA. Beijing Forestry University, Beijing, China. 3 Department of Entomology and Plant Pathology, The University of Tennessee, Knoxville, TN 37996-4563, USA. 4 Department of Forestry, Wildlife & Fisheries, The University of Tennessee, Knoxville, TN 37996-4563, USA. 5 Department of Ecosystem Science and Management and The Department of Plant Science, Pennsylvania State University, University Park, PA 16802, USA. 1 2 Abstract Liriodendron tulipifera L., is a wide-spread, fast-growing pioneering tree species native to eastern North America. Commonly known as yellow-poplar, tulip tree, or tulip-poplar, the species is valued, both ecologically and economically. It is perhaps the most commonly used utility hardwood in the USA, and is planted widely for reforestation and, in varietal forms, as an ornamental. Although most seedlings used for reforestation today derive from collections in natural populations, two known seed orchards, established from plus-tree selections, i.e. superior phenotypes, in the 1960’s and 1970’s have been used for local and regional planting needs in Tennessee and South Carolina. However, very little is known about the population genetics of yellow-poplar nor the genetic composition of the existing seed orchards. In this study, 194 grafted yellow-poplar trees from a Clemson, SC orchard and a Knoxville, TN orchard were genetically characterized with 15 simple sequence repeat (SSR) markers developed from expressed sequence tags (ESTs). Of the 15 EST-SSR markers, 14 had a polymorphic information content (PIC) of at least 0.5. There was no significant difference between the Clemson and Knoxville orchards in average effective number of alleles (5.93 vs 3.95), observed and expected heterozygosity (Ho: 0.64 vs 0.58; He: 0.74 vs 0.70), Nei’s expected heterozygosity (0.74 vs 0.58), or Shannon’s Information index (1.84 vs 1.51). The larger Clemson orchard exhibited a significantly greater number of observed alleles than the Knoxville orchard (15.3 vs7.4). Overall, substantial genetic diversity is captured in the Clemson and Knoxville orchards. Keywords: Genetic diversity, seed orchard, SSR markers, species Introduction Liriodendron tulipifera L., commonly known as yellow-poplar or tulip-poplar, is a wide-spread, fast-growing pioneering hardwood species of considerable economic value in the forests of eastern North America. Yellow-poplar is distributed predominantly east of the Mississippi River from the gulf coast to southern Canada (28° to 43° north latitude) [35]. According to the forest inventory analysis [11], as surveyed from 20062012, the total saw log volume of L. tulipifera on timberland in the United States was 25.9 billion cubic feet, with the majority (65%) located in the southeastern United States. The species is shade intolerant and highly competitive, growing faster than Acer rubrum L. (red maple) and Quercus rubra L. (northern red oak) seedlings under a variety of silvicultural understory treatments (Beckage and Clark 2003). Yellow-poplar is often seen as a pioneering species in old fields. As a component of 16 forest cover types, this species’ degree of dominance has created differentiation between the ecological communities [46]. In addition, yellow-poplar is valued as a nectar source for honey production, as a source of wildlife food (mast), and as a large shade tree in urban plantings [3]. The wood of yellow-poplar is used in a diverse range of products, such as in furniture, pallets and framing construction as well as pulp [12,41]). Chemical extracts from yellow-poplar wood or leaves have proven useful, © 2015 Liang et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf such as sesquiterpenes which have an anti-tumor effect and antifeeding for herbivores [27], and antimicrobial alkaloids [2]. L. tulipifera has been cultivated since 1663 [5] and is currently widely planted in eastern forests. Although seed orchards have been established to meet local or regional planting needs in the U.S.A. [6,36], genetic diversity of Liriodendron seed orchards in relation to natural stands has not been studied. Because seed orchards is the bridge between breeding and silvicultural activities, genetic diversity of tree seeds orchards determines the genetic quality of future forest stands and forms the basis for further improving the management of genetic resources and for the genetic modification of cultivars to meet new environmental challenges. Thus, the lacking information limits utilization of these Liriodendron orchards in a tree improvement program. The primary goal of our study was to determine the genetic composition and diversity in two Liriodendron seed orchards in the southeastern USA. The orchard residing in Knoxville, Tennessee, was established in 1966 and contains 100 grafted ramets, representing 31 genotypes or clones. The Clemson orchard in South Carolina was established in 1976 by grafting multiple ramets of 150 plus trees selected from throughout the 17,500-acre Clemson Experimental Forest by Dr. Roland E. Schoenike (http://www.clemson.edu/trails/history/schoenike. html#top). Seeds from this orchard have been used for reforestation efforts for a number of years. Currently there are 165 surviving trees in the Clemson orchard. Besides L. tulipifera, the only other Liriodendron species is Liriodendron chinense, which is native to China and Vietnam. Although the two species separated 10~16 million years ago [32], they are quite similar morphologically and are cross fertile [26,34], and the hybrids exhibit heterosis [31,39]. Because the incomplete records suggest that the Clemson orchard may contain L. chinense or hybrids, we first used the sequence of a chloroplast gene, maturase K (matK), to discriminate the two Liriodendron species and their hybrids. Then we investigated the genetic diversity and allele richness among selections of this unique native species in each orchard as a first step toward contrasting orchard-produced seedling diversity with natural diversity. We chose simple sequence repeat (SSR) markers (also called microsatellites) in the study, because SSR markers are co-dominant, easily reproduced and scored, highly polymorphic, abundant through the genome, and have higher information content than isoenzyme and dominant markers [45]. Materials and methods Plant materials and DNA isolation Fresh leaves of all Liriodendron trees (165) from the Clemson seed orchard and 31 trees from the Knoxville seed orchard were collected in the spring of 2013 and stored in plastic bags at -80°C prior to DNA isolation. All these trees represented different clones as validated by the SSR markers used in this study. Leaves from a Liriodendron tulipifera tree (accession doi: 10.7243/2050-2389-4-1 number 70921 H) from the US National Arboretum (collected by Kevin Conrad) were also included in the study. Total genomic DNA was isolated from leaves using a CTAB protocol as described in [16] and suspended in TE buffer (Tris base 6.1g/L, EDTA 0.37 g/L, pH 8). The quality and concentrations of genomic DNA from individual plants were determined with a NanoDrop 3300 (Thermo Scientific, Wilmington, Delaware, USA) and by electrophoresis on 0.8% agarose gels. Distinguishing between two Liriodendron species based on maturase K sequence The record of the 165 surviving Liriodendron trees in the Clemson orchard is not complete. Therefore, the sequence of a chloroplast gene, maturase K (matK) was used to discriminate between the species/hybrids. The matK sequence was amplified with forward (5’-CGATCTATTCATTCAATATTTC-3’) and reverse primers (5’-TCTAGCACACGAAAGTCGAAGT-3’) in a 12.5-μl reaction containing 6.875 uL ddH2O, 1 uL MgCl2 (25 mM), 0.5 uL forward primer (10uM), 0.5 uL reverse primer (10uM), 0.25 uL dNTPs (10 mM each), 0.25 uL BSA (0.8ug/uL), 0.125 uL Taq Pololymerase (5u/uL), 0.5 uL DNA (~20ng/ul), 2.50 uL 5X PCR buffer (-Mg). The conditions for polymerase chain reactions (PCR) were as follows: 5 minutes of initial denaturation at 94°C, 35 cycles of touch-down PCR with 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60-50°C (first cycle 60°, then each subsequent cycle 1°C lower than the previous until 51°C annealing temperature, followed by 25 cycles each with a 50°C annealing temperature), and 3 minutes of extension at 72°C, and a final extension at 72°C for 10 minutes. Before being sequenced with 1 ul of 10 uM forward or reverse primer, PCR products were cleaned with ExoAP mix (89 uL H2O+ 10 uL 5000U/mL Antarctic Phosphatase +1 uL 20000U/ mL Exonuclease I) for 30 minutes in a reaction containing 1 uL of PCR product and 1uL of ExoAP mix, followed by a heat inactivation step at 80°C for 15min. An 834 bp-segment of maturase K gene from each tree was used for alignment with MUSCLE and curated with Gblocks, and a phylogenetic tree was built with maximum likelihood (PhyML) (http://www. phylogeny.fr/) [7]. The maturase K gene sequence of L. tulipifera (GI: 5731451), L. chinense (GI: 7239759), and a hybrid (GI: 389955358) available in GenBank were included in the analysis. L. tulipifera EST-SSR markers, PCR amplification, and allele sizing Twenty simple sequence repeat (SSR) markers (also called microsatellites) were used to investigate the genetic composition of the Liriodendron seed orchards. These markers included seven Expressed Sequenced Tags (EST)SSR markers (LT002, LT015, LT021, LT086, LT096, LT131, LT157) previously characterized by electrophoresis on 8% polyacrylamide gels [42] and thirteen new markers (LTCU19, LTCU40, LTCU51, LTCU53, LTCU125, LTCU139, LTCU142, LTCU143, 2 Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf LTCU145, LTCU150, LTCU151, LTCU152, LTCU154) mined from a comprehensive EST dataset [22]. PCR amplification for each marker was performed with genomic DNA of Liriodendron trees from the Clemson and Knoxville seed orchards and the US National Arboretum. For a more cost-effective 153 primer screening, a M13 tail (5’-CACGACGTTGTAAAACGAC-3’) was added to the 5’-end of the forward primer of each marker pair in order to amplify the fragments using a complementary adapter with a fluorescent dye (6-FAM, VIC, NED, or PET) at its 5’-end (Applied Biosystems, Foster City, California, USA). Polymerase chain reactions were carried out in a 12.5-μl solution comprising: approximate 75 ng DNA template, 0.052 U/μL Promega Taq DNA polymerase, 0.16 nM forward primer, 0.4 nM reverse primer, 0.4 nM fluorescent M13 primer, 0.24 mM each dNTPs, and 1.2×Promega PCR buffer. The PCR profile consisted of an initial denaturation at 94°C for 3 minutes followed by 10 cycles of 1 minute at 94°C, 1 minute at annealing temperature (Table 1), and 1 minute 15 seconds at 72°C, and then 35 cycles of 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C, with a final extension of amplified DNA at 72°C for 5 minutes. An aliquot of 1.5 μl PCR products were treated with 1.5 μl of 10-fold diluted ExoSAP-IT (Affymetrix Inc. Cleveland, OH, USA) to remove single stranded primers which might influence fragment analysis at 37°C for 30 minutes and then at 80°C for 15 minutes. After being diluted to 100 ng/μl, 1μl of each sample was mixed with 0.1 μl of LIZ600 and 8.9 μl of Hi-Di Formamide, denatured at 95°C for 5 minutes, and then put on ice for 10 minutes before being separated on an ABI 3730 Genetic Analyzer. The Dye set was DS-33 (6-FAM, VIC, NED, PET and LIZ). Allele sizes were scored with GeneMapper (4.0) (Applied Biosystems, Foster City, California, USA). Functional annotation of EST-SSRs was performed by applying a homology search of reassembled ESTs against the non-redundant (nr) NCBI database using the BLASTx algorithm [1]. Data analysis MICRO-CHECKER [38] was employed to check for potential genotyping errors arising from large allele drop-out and stuttering. Observed and expected heterozygosities and polymorphic information content (PIC) were calculated using Cervus 2.0 [25]. Deviations from Hardy–Weinberg equilibrium and the Shannon’s Information index were calculated with GENEPOP (http://genepop.curtin.edu.au/, Raymond and Rousset 1995). doi: 10.7243/2050-2389-4-1 lobed leaves and smaller flowers. However, our attempt to tell these two species apart by morphology failed: the leaf shape varied depending on age (Supplementary figure S1) and the flowers were located at a too high for sampling. Molecular techniques including biochemical analysis [34], isozymes [14], and fingerprinting with random amplified polymorphic DNA (RAPD) [21] have been explored in discrimination of Liriodendron species and their hybrids. In 2012, Zhang et al., reported an SSR marker that amplified a 190-bp fragment from L. chinense, a 180-bp fragment from L. tulipifera, and both 190- and 180-bp fragments from hybrid. In this study, matK sequence was employed. The matK gene locates within the intron of the trnK and codes for maturase like protein involved in Group II intron splicing [37]. The trnKUUU-matK region, ranging from approximately 2.2 kb (liverworts) to 2.6 kb (seed plants) in size, is universally present in land plants and only few exceptions of a secondary loss or reorganizations are known to date [40]. Because the matK gene evolves more rapidly, compared to other plastid genes, it has become a valuable marker for lower-level phylogenetic reconstruction of systematic and evolutionary studies. The Clemson seed orchard contains 165 surviving trees. The matK sequence was amplified from each of the 165 trees in the Clemson orchard (Supplementary figure S2). When the amplicons were pair-end sequenced, an 834-bp segment of high quality was obtained for each tree, representing 55% of the full-length gene. There were only eight nucleotides different between the two Liriodendron species within the 834bp segment. As shown in Figure 1 and Supplementary figure S3, only Tree#CU24 and #134 were not L. tulipifera. Their hybrid status was established by having seven nucleotides different from L. tulipifera and 2 nucleotides different from L. Chinense. These results confirm the record of hybrids being planted in the Clemson Orchard. Thus, these two hybrids were excluded in the genetic composition analysis. Further, our study indicates that L. tulipifera, L. Chinense, and their hybrids contain unique nucleotide compositions in matK sequence that can be utilized in distinguishing the species and hybrids. Amplification of EST-SSR loci in Liriodendron No evidence for large allele dropout was found for any of the 20 markers. Stuttering occurred in five markers: LT131, LT157, LTCU40, LTCU142, and LTCU143, and these five markers were excluded in further analyses. All of the remaining 15 markers were polymorphic in both Clemson and Knoxville orchards. The 20 markers were also tested on one L. tulipifera and one Results and discussion L. chinense tree from the US National Arboretum. Eleven loci Distinguishing between two Liriodendron species with were heterozygous and one locus failed in the L. tulipifera maturase K (matK) sequence tree. PCR amplification for all 20 markers was successful in It is not clear when L. chinense was first introduced to U.S.A., the L. Chinense tree, although sizing in an ABI 3730 Genetic but the two Liriodendron species hybridize readily [26] and Analyzer failed for LT157 and LTCU142, due to stuttering. efforts in crossing have been well-documented [e.g., 31,34]. Fourteen loci were heterozygous in the L. chinense tree. This The two species are similar morphologically, except that L. indicates a high frequency of transferability of L. tulipifera chinense is smaller in stature and has larger, more deeply EST-SSR markers in L. Chinense, supporting the previous 3 Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf doi: 10.7243/2050-2389-4-1 Table 1. Characteristics of 20 EST-SSR loci. Marker name Repeat motif Forward primer sequence (5’-3’) LT002 (GCA)8 CCTACCACCAGCA TCTCGTCGCTGAAGAT ATACCTA ATG LT015 (CCGAAC)5 LT021 (TTC)8 LT086 (CTT)10 LT096 (CT)20 LT131 (AC)22 LT157 (TTC)6 LTCU19 (AG)10 LTCU40 LTCU51 (ATG)8 (CT)18 LTCU53 (TG)14 LTCU125 (TC)8 LTCU139 (TCT)10 LTCU142 (AAT)8 LTCU143 (TG)13 LTCU145 (GA)18 LTCU150 (TC)10 LTCU151 (TC)11 LTCU152 (CA)17 LTCU154 (CT)10 Reverse primer sequence (5’-3’) Expected size Stuteringa Annealing (bp) temperature ⁰C 189 N 59 110 N 59 180 N 57 274 N 55 272 N 55 TCCGTTATCTCTCT CTAGACAGGTGCTCGG CCAAAA ATAC CAAATACCATTGC ACGCATCCTCTTCCAC ACCTTGT TAC AAGACAGGACTTT GAACGAACCTAACCA CCACTGA AATGA TGCAACCTAACAA TGAAAAGCAACCAAG GATGTGT TTACC GCAGCATCTCCTC TTGCAGTTGAGCTATT 240 Y 55 ATATTCT GTTG AGTTGCCCTTTAGC GCCACAGAGTTTTGGA 222 Y 55 TTCTTT AGTA GTGGATTGCAAAG AAAACAAAAGCAAGC 183 N 57 GCAGAGT AAGCC TTGCGTAAATGCA GAAGCCtaTGCAAGAT 181 Y 55 TCCAAAA GCAA ATCACCATCTTCCT AAACCATTCCAACCAT 198 N 55 CATCGC CCAA CGGATCTTTCTCTT AAGAAGATTGCAGAG 223 N 55 TCCATCC GCAGAA CGAAAGACATTCC CCATTACAATCCACAG 205 N 55 CATCACA CCAA 164 Y 55 171 Y 55 160 N 55 157 N 55 167 N 55 189 N 55 177 N 55 156 N 55 GAATAACCGCTCT AAGCCAAGTGGCAAA TTTGGGA GAAGA TGGTGCATATGGG TATTCCCCCAGCTTCT CTTAGAA CCTT AAAAATGCTAATC TATCCAACCGATCACC CAATAACTTTCG CATT TTGAAGTCCAGAT GCCTAGGGaGATGtTTT TGATTGATTG TGG TCTTCAAACCAAG GCACTACATCCCTTTTc GCTGTTG CCA TGAGGTGACTTTG GACCCgaGCTGTAAAA GCTTTTG TGGA CATCCAAATGCAG ATTCCCACTCGGTTGA CAGAAAT ACAC GATGAAGGAGAAT CCAGCCAAGAAAGAA TCTATATTTTCTGA AATGG Y:Yes; N:No. a 4 Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf 1 1 0.003 Chinese_GI_7239759 Hybrid_GI_389955358 CU24 CU134 CU16 Tul_GI_5731451 Tul_ACC70921H CU2 CU3 CU4 CU5 CU6 CU7 CU8 CU9 CU10 CU13 CU14 CU17 CU18 CU19 CU21 CU22 CU23 CU75 CU76 CU78 CU96 CU118 CU119 CU124 Figure 1. Sequence comparison of maturase K gene. The sequences were aligned with MUSCLE the phylogenetic tree was built with maximum likelihood (PhyML). The maturase K gene sequence of L. tulipifera (GI: 5731451), L. chinense (GI: 7239759), and a hybrid (GI: 389955358) available in GenBank were included in the analysis. The complete illustration of the tree is included in the Supplementary figure S1. doi: 10.7243/2050-2389-4-1 Table 2. Statistics of the 15 markers analyzed by cervus. Locus K N HO HE LT002 6 174 0.72 0.69 0.65 PIC LT015 6 172 0.55 0.60 0.54 LT021 3 173 0.17 0.19 0.18 LT086 7 170 0.36 0.53 0.50 LT096 15 165 0.65 0.76 0.73 LTCU19 12 174 0.60 0.71 0.69 LTCU51 18 172 0.74 0.87 0.85 LTCU53 13 167 0.63 0.84 0.82 LTCU125 18 164 0.88 0.89 0.88 LTCU143 14 164 0.74 0.80 0.76 LTCU145 11 172 0.84 0.86 0.84 LTCU150 15 172 0.51 0.74 0.72 LTCU151 11 158 0.51 0.76 0.73 LTCU152 19 143 0.65 0.93 0.92 LTCU154 26 160 0.74 0.93 0.92 Average 13 167 0.62 0.74 0.71 K: number of alleles; N: number of individuals; H0: observed heterozygosity; He: expected heterozygosity; PIC: polymorphic information content. Genetic composition of the L. tulipifera orchards While there were only two loci (LT002 and LT015) significantly deviating from Hardy-Weinberg proportions in the Clemson population, there were 10 deviating loci in the Knoxville findings of 72.4% success rate by [42] and 82.1% by [43]. This population (p>0.05) (Supplementary Table S1 and S2). This is expected because EST-SSRs have generally demonstrated may be due to insufficient sample size from the Knoxville a high frequency of cross-species transferability despite less population. As shown in Tables 3 and 4, the Clemson orchard polymorphism compared to genomic SSRs [9,12,44]. Among had higher values for observed number of alleles (15.3 vs the 194 L. tulipifera trees included in the study, the number 7.4), effective number of alleles (5.9 vs 4.0), observed (Ho, of alleles per locus ranged from 3 to 26 (mean=13.0) (Table 2). 0.64 vs 0.58) and expected heterozygosity (He, 0.74 vs 0.70), The observed and expected heterozygosities (Ho and He) Nei’s expected heterozygosity (0.74 vs 0.58), and Shannon’s ranged from 0.17 to 0.89 and from 0.19 to 0.93, with averages Information index (1.85 vs 1.51) than the Knoxville orchard. of 0.62 and 0.74, respectively. The polymorphic information However, the differences were not statistically significant content (PIC) ranged from 0.17 to 0.92, with an average of (p=0.05, t-Test) except for observed number of alleles. The 0.71. Overall, 14 of the 15 markers had a PIC≥0.5. different number of trees from the orchards included in the Many genomic resources, such as expressed sequence study, 163 from the Clemson vs 31 from the Knoxville, may tag (EST) databases [15,22,23] and genomic DNA libraries be a contributing factor. [24], have been developed for L. tulipifera. Through these This is the first report of genetic composition of Liriodendron resources, several thousand putative SSR markers have been cultivated populations in North America and has provided identified by in silico mining. However, only 345 L. tulipifera the basic data of genetic diversity and allele richness among SSR markers having been tested for polymorphism by selections of this unique native species. [43] examined 27 polyacrylamide denaturing gels [42,43]. Compared to other trees from a cultivated population of L. tulipifera in the Jurong, species, Liriodendron has lacked development of polymorphic Jiangsu Province of China with 39 polymorphic EST-SSR loci and informative SSR markers. through electrophoreses in 6% polyacrylamide denaturing As a result, no genetic linkage maps of Liriodendron have gels and visualization with silver nitrate staining. It was found been reported. This is in contrast with the species’ecological that the number of alleles per locus ranged from three to 18 and economic value and phylogenetic position as a basal and the average Ho and He were 0.68 and 0.78, respectively. angiosperm. Compared to this cultivated population in China, the two 5 Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf doi: 10.7243/2050-2389-4-1 Table 3. Genetic variation at 15 EST-SSR loci characterized in the clemson orchard. Clemson orchard (163 trees) Sample size 326 324 324 318 308 326 324 310 308 306 324 322 294 258 302 312 -- Locus LT002 LT015 LT021 LT086 LT096 LTCU19 LTCU51 LTCU53 LTCU125 LTCU143 LTUCU145 LTUCU150 LTUCU151 LTUCU152 LTUCU154 Mean St. Dev. Na 6.00 8.00 6.00 9.00 18.00 15.00 18.00 13.00 26.00 14.00 12.00 14.00 9.00 25.00 37.00 15.33 8.53 Ne 3.16 2.30 1.99 1.84 3.75 3.20 7.74 4.84 11.46 4.84 7.49 2.90 2.99 15.89 14.49 5.93 4.59 Obs_Hom 0.28 0.46 0.54 0.69 0.38 0.40 0.26 0.30 0.05 0.23 0.17 0.42 0.54 0.33 0.28 0.36 0.16 Obs_Het 0.72 0.54 0.46 0.31 0.62 0.60 0.74 0.70 0.95 0.77 0.83 0.58 0.46 0.67 0.72 0.64 0.16 Exp_Hom 0.31 0.43 0.50 0.54 0.26 0.31 0.13 0.20 0.08 0.20 0.13 0.34 0.33 0.06 0.07 0.26 0.15 Exp_Het 0.69 0.57 0.50 0.46 0.74 0.69 0.87 0.80 0.92 0.80 0.87 0.66 0.67 0.94 0.93 0.74 0.15 Nei’s 0.68 0.57 0.50 0.46 0.73 0.69 0.87 0.79 0.91 0.79 0.87 0.66 0.67 0.94 0.93 0.74 0.15 I 1.35 1.11 1.01 1.00 1.80 1.71 2.26 1.89 2.75 1.81 2.18 1.61 1.38 2.91 3.03 1.85 0.66 Na: Observed number of alleles. Ne: Effective number of alleles (Kimura and Crow 1964). Obs_Hom/Obs_ Het: Observed homozygosity/heterozygosity. Ext_Het/Exp_Het: expected homozygosity/heterozygosity (Levene 1949). Nei's (1973) expected heterozygosity. I=Shannon's Information index (Lewontin 1972). St. Dev.: Standard deviation. Table 4. Genetic variation at 15 EST-SSR loci characterized in the knoxville orchard. Locus Knoxville orchard (31 trees) Sample size Na Ne Obs_Hom Obs_Het Exp_Hom Exp_Het Nei’s I LT002 62 5 3.65 0.32 0.68 0.26 0.74 0.73 1.43 LT015 60 5 3.38 0.43 0.57 0.28 0.72 0.7 1.36 LT021 62 2 1.17 0.84 0.16 0.85 0.15 0.15 0.28 LT086 62 6 2.81 0.35 0.65 0.35 0.65 0.64 1.22 LT096 62 10 4.75 0.32 0.68 0.2 0.8 0.79 1.85 LTCU19 62 9 3.59 0.52 0.48 0.27 0.73 0.72 1.68 LTCU51 60 12 5.84 0.27 0.73 0.16 0.84 0.83 2 LTCU53 62 6 2.77 0.74 0.26 0.35 0.65 0.64 1.32 LTCU125 60 15 6.14 0.3 0.7 0.15 0.85 0.84 2.17 LTCU143 60 8 5.26 0.33 0.67 0.18 0.82 0.81 1.8 LTUCU145 60 8 4.64 0.1 0.9 0.2 0.8 0.78 1.71 LTUCU150 62 5 3.29 0.68 0.32 0.29 0.71 0.7 1.33 LTUCU151 56 3 2.26 0.39 0.61 0.43 0.57 0.56 0.89 LTUCU152 62 9 5.88 0.42 0.58 0.16 0.84 0.83 1.93 LTUCU154 58 8 3.83 0.24 0.76 0.25 0.75 0.74 1.62 Mean 61 7.4 3.95 0.42 0.58 0.29 0.70 0.58 1.51 St. Dev. -- 3.4 1.44 0.20 0.20 0.17 0.17 0.16 0.46 Na: Observed number of alleles. Ne: Effective number of alleles (Kimura and Crow 1964). Obs_ Hom/Obs_Het: Observed homozygosity/heterozygosity. Ext_Het/Exp_Het: expected homozygosity/ heterozygosity (Levene 1949). Nei's (1973) expected heterozygosity. I=Shannon's Information index (Lewontin 1972). St. Dev.: Standard deviation. 6 Zhang et al. Journal of Plant Science & Molecular Breeding 2015, http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf doi: 10.7243/2050-2389-4-1 US orchards had slightly lower values of average Ho and L.tulipifera in Clemson orchard He, with 0.64 (Ho) and 0.74 (He) in the Clemson orchard and L.tulipifera in Knoxville orchard L.tulipifera L.tulipifera in National Arboretum 0.58 (Ho) and 0.70 (He) in the Knoxville orchard. These values Hybrids in Clemson orchard L.tulipifera X chinense are comparable to those reported in a L. chinense cultivated L.chinense in National Arboretum L.chinense population in China, which had a Ho and He of 0.48 and 0.74 [43]. Other forest tree species have similar heterozygosities 0 50 40 30 20 10 as well, for example, a Pinus merkusii parental and seedling Figure 2. The UPGMA dendrogram based on Nei's (1978) populations had a He of 0.55 and 0.49, respectively [10,30]. genetic distance. Bootstrap replicates=1,000. Reported 0.48 (Ho) and 0.63 (He) in a white spruce plantation and 0.49 (Ho) and 0.63 (He) in a white spruce improvement selection population. It is noteworthy that genetic diversity of and provides a foundation for further genetic and breeding natural L. tulipifera populations has been reported [e.g., 18,32]. exploration. The polymorphic markers developed in this However these studies utilized either allozymes or amplified study will serve as a resource enabling the future study of fragment length polymorphism (AFLP) markers, which usually population dynamics and adaptive variation in Liriodendron. have lower information content than SSR markers. None of the reported expected heterozygosities from these studies Additional files exceeded 0.29. Overall, substantial genetic diversity is captured Supplementary Table S1 in the Clemson and Knoxville seed orchards. Conclusion The data obtained in this study will be useful in future applications such as prediction of genetic gain and gene diversity in the seed orchards. Nei’s genetic distance between the two orchards was 0.39, which was the lowest among all comparisons (Table 5). The L. chinense and L. tulipifera trees from the National Arboretum exhibited the largest genetic distance (1.17). The two orchards and the L. tulipifera sample from the US National Arboretum grouped together in the UPGMA dendrogram. The genetic distance of the hybrids in the Clemson orchard was closest to the Clemson orchard (0.50), followed by the Knoxville orchard (0.80) and L. chinense from the National Arboretum (0.88), and then by the L. tulipifera from the National Arboretum (1.17) (Figure 2). With a widespread range of distribution, L. tulipifera has adapted to many different ecological conditions and is one of the species becoming increasingly dominant in forests due to its quick respond to increases in light to the forest floor and rapid initial growth rate [8]. Its increasingly important roles in forestry and wood products is making studying Liriodendron of great interest. Our study provides a first look at the genetic diversity and allele richness among selections of this unique native species, Table 5. Nei's (1978) unbiased identity (above diagonal) and distance (below diagonal). Clemson Clemson Knoxville NA L. chinense -0.6792 0.6234 Hybrids in NA L. Clemson chinense 0.6051 0.4051 Knoxville 0.3869 Pop ID NA L. 0.4725 tulipifera Hybrids in 0.5025 clemson NA L. 0.9037 chinense -- 0.4648 0.4484 0.3495 0.7662 -- 0.3608 0.3097 0.8021 1.0195 -- 0.4138 1.0513 1.1721 0.8823 -- Supplementary Table S2 Supplementary figure S1 Supplementary figure S2 Supplementary figure S3 Competing interests The authors declare that they have no competing interests. Authors’ contributions Authors’ contributions XZ AC ZT MS SES JEC HL Research concept and design -- -- -- -- -- ✓ ✓ Collection and/or assembly of data ✓ ✓ ✓ ✓ ✓ -- -- Data analysis and interpretation ✓ -- -- -- -- -- ✓ Writing the article ✓ -- -- -- -- -- ✓ Critical revision of the article -- -- -- ✓ ✓ -- ✓ Final approval of article ✓ ✓ ✓ ✓ ✓ ✓ ✓ Statistical analysis ✓ -- -- -- -- -- ✓ Acknowledgement The authors thank Nick Wheeler for his guidance and thorough review of the manuscript. 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