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*
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
© 2015 Liang et al; licensee Herbert Publications Ltd. is is an Open Access article distributed under the terms of Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0). is permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 2006-
2012, 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 treat-
ments (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,
*Correspondence: hliang@clemson.edu
1Department of Genetics and Biochemistry, Clemson University, Clemson SC 29634, USA.
2Beijing Forestry University, Beijing, China.
3Department of Entomology and Plant Pathology, The University of Tennessee, Knoxville, TN 37996-4563, USA.
4Department of Forestry, Wildlife & Fisheries, The University of Tennessee, Knoxville, TN 37996-4563, USA.
5Department of Ecosystem Science and Management and The Department of Plant Science, Pennsylvania State University,
University Park, PA 16802, USA.
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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
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 amplication, 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,
Zhang et al. Journal of Plant Science & Molecular Breeding 2015,
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doi: 10.7243/2050-2389-4-1
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 Shannons Information index were calculated with
GENEPOP (http://genepop.curtin.edu.au/, Raymond and
Rousset 1995).
Results and discussion
Distinguishing between two Liriodendron species with
maturase K (matK) sequence
It is not clear when L. chinense was first introduced to U.S.A.,
but the two Liriodendron species hybridize readily [26] and
efforts in crossing have been well-documented [e.g., 31,34].
The two species are similar morphologically, except that L.
chinense is smaller in stature and has larger, more deeply
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 gure 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 gure 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 834-
bp segment. As shown in
Figure 1
and
Supplementary gure 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.
Amplication 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
L. chinense tree from the US National Arboretum. Eleven loci
were heterozygous and one locus failed in the L. tulipifera
tree. PCR amplification for all 20 markers was successful in
the L. Chinense tree, although sizing in an ABI 3730 Genetic
Analyzer failed for LT157 and LTCU142, due to stuttering.
Fourteen loci were heterozygous in the L. chinense tree. This
indicates a high frequency of transferability of L. tulipifera
EST-SSR markers in L. Chinense, supporting the previous
Zhang et al. Journal of Plant Science & Molecular Breeding 2015,
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doi: 10.7243/2050-2389-4-1
Marker
name
Repeat
motif
Forward primer
sequence (5’-3’)
Reverse primer
sequence
(5’-3’)
Expected size
(bp)
StuteringaAnnealing
temperature C
LT002
(GCA)8
CCTACCACCAGCA TCTCGTCGCTGAAGAT
189
N
59
ATACCTA
ATG
LT015
(CCGAAC)5
TCCGTTATCTCTCT
CTAGACAGGTGCTCGG
110
N
59
CCAAAA ATAC
LT021
(TTC)8
CAAATACCATTGC
ACGCATCCTCTTCCAC
180
N
57
ACCTTGT
TAC
LT086
(CTT)10
AAGACAGGACTTT
GAACGAACCTAACCA
274
N
55
CCACTGA
AATGA
LT096 (CT)20
TGCAACCTAACAA
TGAAAAGCAACCAAG
272
N
55
GATGTGT
TTACC
LT131
(AC)22
GCAGCATCTCCTC
TTGCAGTTGAGCTATT
240
Y
55
ATATTCT
GTTG
LT157
(TTC)6
AGTTGCCCTTTAGC
GCCACAGAGTTTTGGA
222
Y
55
TTCTTT
AGTA
LTCU19
(AG)10
GTGGATTGCAAAG
AAAACAAAAGCAAGC
183
N
57
GCAGAGT
AAGCC
LTCU40
(ATG)8 TTGCGTAAATGCA
GAAGCCtaTGCAAGAT
181
Y
55
TCCAAAA
GCAA
LTCU51
(CT)18
ATCACCATCTTCCT
AAACCATTCCAACCAT
198
N
55
CATCGC
CCAA
LTCU53
(TG)14
CGGATCTTTCTCTT
AAGAAGATTGCAGAG
223
N
55
TCCATCC
GCAGAA
LTCU12
5
(TC)8
CGAAAGACATTCC
CCATTACAATCCACAG
205
N
55
CATCACA
CCAA
LTCU139
(TCT)10
GAATAACCGCTCT AAGCCAAGTGGCAAA
164
Y
55
TTTGGGA
GAAGA
LTCU142
(AAT)8
TGGTGCATATGGG TATTCCCCCAGCTTCT
171
Y
55
CTTAGAA
CCTT
LTCU143
(TG)13
AAAAATGCTAATC
TATCCAACCGATCACC
160
N
55
CAATAACTTTCG CATT
LTCU145
(GA)18
TTGAAGTCCAGAT
GCCTAGGGaGATGtTTT
157
N
55
TGATTGATTG
TGG
LTCU150
(TC)10
TCTTCAAACCAAG
GCACTACATCCCTTTTc
167
N
55
GCTGTTG CCA
LTCU151
(TC)11
TGAGGTGACTTTG GACCCgaGCTGTAAAA
189
N
55
GCTTTTG
TGGA
LTCU152
(CA)17
CATCCAAATGCAG
ATTCCCACTCGGTTGA
177
N
55
CAGAAAT
ACAC
LTCU154
(CT)10
GATGAAGGAGAAT CCAGCCAAGAAAGAA
156
N
55
TCTATATTTTCTGA
AATGG
Table 1. Characteristics of 20 EST-SSR loci.
aY:Yes; N:No.
Zhang et al. Journal of Plant Science & Molecular Breeding 2015,
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doi: 10.7243/2050-2389-4-1
Figure 1. Sequence comparison of maturase K gene. e
sequences were aligned with MUSCLE the phylogenetic tree
was built with maximum likelihood (PhyML). e 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. e complete illustration of the
tree is included in the Supplementary gure S1.
findings of 72.4% success rate by [42] and 82.1% by [43]. This
is expected because EST-SSRs have generally demonstrated
a high frequency of cross-species transferability despite less
polymorphism compared to genomic SSRs [9,12,44]. Among
the 194 L. tulipifera trees included in the study, the number
of alleles per locus ranged from 3 to 26 (mean=13.0) (Table 2).
The observed and expected heterozygosities (Ho and He)
ranged from 0.17 to 0.89 and from 0.19 to 0.93, with averages
of 0.62 and 0.74, respectively. The polymorphic information
content (PIC) ranged from 0.17 to 0.92, with an average of
0.71. Overall, 14 of the 15 markers had a PIC≥0.5.
Many genomic resources, such as expressed sequence
tag (EST) databases [15,22,23] and genomic DNA libraries
[24], have been developed for L. tulipifera. Through these
resources, several thousand putative SSR markers have been
identified by in silico mining. However, only 345 L. tulipifera
SSR markers having been tested for polymorphism by
polyacrylamide denaturing gels [42,43]. Compared to other
species, Liriodendron has lacked development of polymorphic
and informative SSR markers.
As a result, no genetic linkage maps of Liriodendron have
been reported. This is in contrast with the species’ecological
and economic value and phylogenetic position as a basal
angiosperm.
Locus K N HO HE PIC
LT002 6 174 0.72 0.69 0.65
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
Table 2. Statistics of the 15 markers analyzed by cervus.
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
population (p>0.05) (Supplementary Table S1 and S2). This
may be due to insufficient sample size from the Knoxville
population. As shown in
Tables 3
and
4
, the Clemson orchard
had higher values for observed number of alleles (15.3 vs
7.4), effective number of alleles (5.9 vs 4.0), observed (Ho,
0.64 vs 0.58) and expected heterozygosity (He, 0.74 vs 0.70),
Nei’s expected heterozygosity (0.74 vs 0.58), and Shannons
Information index (1.85 vs 1.51) than the Knoxville orchard.
However, the differences were not statistically significant
(p=0.05, t-Test) except for observed number of alleles. The
different number of trees from the orchards included in the
study, 163 from the Clemson vs 31 from the Knoxville, may
be a contributing factor.
This is the first report of genetic composition of Liriodendron
cultivated populations in North America and has provided
the basic data of genetic diversity and allele richness among
selections of this unique native species. [43] examined 27
trees from a cultivated population of L. tulipifera in the Jurong,
Jiangsu Province of China with 39 polymorphic EST-SSR loci
through electrophoreses in 6% polyacrylamide denaturing
gels and visualization with silver nitrate staining. It was found
that the number of alleles per locus ranged from three to 18
and the average Ho and He were 0.68 and 0.78, respectively.
Compared to this cultivated population in China, the two
Chinese_GI_7239759
Hybrid_GI_389955358
CU24
CU134 CU16
CU2
CU3
CU4
CU5
CU6
CU7
CU8
CU9
CU10
CU13
CU14
CU17
CU18
CU19
CU21
CU22
CU23
CU75
CU76
CU78
CU96
CU118
CU119
CU124
0.003
Tul_GI_5731451
Tul_ACC70921H
1
1
Zhang et al. Journal of Plant Science & Molecular Breeding 2015,
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doi: 10.7243/2050-2389-4-1
Clemson orchard (163 trees)
Locus
Sample size
Na Ne
Obs_Hom
Obs_Het
Exp_Hom
Exp_Het
Nei’s
I
LT002
326
6.00 3.16
0.28
0.72 0.31 0.69
0.68 1.35
LT015
324
8.00 2.30
0.46
0.54 0.43 0.57
0.57 1.11
LT021
324
6.00 1.99
0.54
0.46 0.50 0.50
0.50 1.01
LT086
318
9.00 1.84
0.69
0.31 0.54 0.46
0.46 1.00
LT096
308
18.00
3.75
0.38
0.62 0.26 0.74
0.73 1.80
LTCU19
326
15.00
3.20
0.40
0.60 0.31 0.69
0.69 1.71
LTCU51
324
18.00
7.74
0.26
0.74 0.13 0.87
0.87 2.26
LTCU53
310
13.00
4.84
0.30
0.70 0.20 0.80
0.79 1.89
LTCU125
308
26.00 11.46
0.05
0.95 0.08 0.92
0.91 2.75
LTCU143
306
14.00
4.84
0.23
0.77 0.20 0.80
0.79 1.81
LTUCU145
324
12.00
7.49
0.17
0.83 0.13 0.87
0.87 2.18
LTUCU150
322
14.00
2.90
0.42
0.58 0.34 0.66
0.66 1.61
LTUCU151
294
9.00 2.99
0.54
0.46 0.33 0.67
0.67 1.38
LTUCU152
258
25.00 15.89
0.33
0.67 0.06 0.94
0.94 2.91
LTUCU154
302
37.00 14.49
0.28
0.72 0.07 0.93
0.93 3.03
Mean
312
15.33
5.93
0.36
0.64 0.26 0.74
0.74 1.85
St. Dev. --
8.53 4.59
0.16
0.16 0.15 0.15
0.15 0.66
Table 3. Genetic variation at 15 EST-SSR loci characterized in the clemson orchard.
Na: Observed number of alleles. Ne: Eective 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.
Knoxville orchard (31 trees)
Locus
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
Table 4. Genetic variation at 15 EST-SSR loci characterized in the knoxville orchard.
Na: Observed number of alleles. Ne: Eective 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.
Zhang et al. Journal of Plant Science & Molecular Breeding 2015,
http://www.hoajonline.com/journals/pdf/2050-2389-4-1.pdf
7
doi: 10.7243/2050-2389-4-1
US orchards had slightly lower values of average Ho and
He, with 0.64 (Ho) and 0.74 (He) in the Clemson orchard and
0.58 (Ho) and 0.70 (He) in the Knoxville orchard. These values
are comparable to those reported in a L. chinense cultivated
population in China, which had a Ho and He of 0.48 and 0.74
[43]. Other forest tree species have similar heterozygosities
as well, for example, a Pinus merkusii parental and seedling
populations had a He of 0.55 and 0.49, respectively [10,30].
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
natural L. tulipifera populations has been reported [e.g., 18,32].
However these studies utilized either allozymes or amplified
fragment length polymorphism (AFLP) markers, which usually
have lower information content than SSR markers. None of
the reported expected heterozygosities from these studies
exceeded 0.29. Overall, substantial genetic diversity is captured
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,
Pop ID
Clemson
Knoxville NA L.
chinense
Hybrids in
Clemson
NA L.
chinense
Clemson
--
0.6792 0.6234 0.6051 0.4051
Knoxville
0.3869
--
0.4648 0.4484 0.3495
NA L.
tulipifera
0.4725 0.7662
--
0.3608 0.3097
Hybrids in
clemson
0.5025 0.8021 1.0195
--
0.4138
NA L.
chinense
0.9037 1.0513 1.1721 0.8823
--
Table 5. Nei's (1978) unbiased identity (above diagonal) and
distance (below diagonal).
Figure 2. e UPGMA dendrogram based on Nei's (1978)
genetic distance. Bootstrap replicates=1,000.
and provides a foundation for further genetic and breeding
exploration. The polymorphic markers developed in this
study will serve as a resource enabling the future study of
population dynamics and adaptive variation in Liriodendron.
Additional les
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Acknowledgement
The authors thank Nick Wheeler for his guidance and thorough
review of the manuscript. The funding for the study came from
the NSF Plant Genome Research program (NSF 1025974) and
National Institute of Food and Agriculture, USDA SC-1700449 with
a Clemson University Experiment Station technical contribution
number of 6215).
Publication history
Editor: Shouan Zhang, University of Florida, USA.
Received: 21-Jan-2015 Revised: 24-Feb-2015
Accepted: 17-Mar-2015 Published: 27-Mar-2015
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 -- -- -- -- --
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Citation:
Zhang X, Carlson A, Tian Z, Staton M, Schlarbaum SE,
Carlson JE and Liang H. Genetic characterization of
Liriodendron seed orchards with EST-SSR markers.
J Plant Sci Mol Breed. 2015; 4:1.
http://dx.doi.org/10.7243/2050-2389-4-1

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