American Journal of Plant Sciences, 2011, 2, 753-764
doi:10.4236/ajps.2011.26090 Published Online December 2011 (http://www.SciRP.org/journal/ajps)
753
Assessing Genetic Structure and Relatedness of
Jerusalem Artichoke (Helianthus tuberosus L.)
Germplasm with RAPD, ISSR and SRAP Markers
Preeya Puangs omlee W a ngsomn u k1*, Sudarat Khampa1, Sanun Jogloy2, Trin Srivong1,
Aran Patanothai2, Yong-Bi Fu3*
1Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; 2Department of Plant Science and Agri-
cultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand; 3Plant Gene Resources of Canada, Saska-
toon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Canada.
Received September 8th, 2011; revised October 7th, 2011; accepted October 21st, 2011.
ABSTRACT
Jerusalem artichoke (Helianthus tuberosus L.) is an old tuber crop with a recently renewed interest in multipurpose
improvement, but little effort has been made to characterize its genetic resources. A study was conducted to assess ge-
netic structure and genetic relatedness of 47 diverse Jerusalem artichoke accessions using RAPD, ISSR and SRAP
markers. A total of 296 (87.1%) polymorphic bands were detected from 13 RAPD markers; 92 (80%) from six ISSR
primers; and 194 (88.6%) for nine combinations of SRAP primers. Five optimal clusters were inferred by the STRUC-
TURE program from the RAPD or ISSR data, while six optimal clusters were found from the SRAP data or combined
marker data. Significant linear relationships between the distance matrices for all pairs of individual accessions were
detected for all marker pairs and the estimated correlation coefficient was 0.40 for RAPD-ISSR, 0.53 for RAPD-SRAP,
and 0.43 for ISSR-SRAP. Based on the combined data, the neighbor-joining clustering of the 47 accessions matched
closely with those inferred from the STRUCTURE program. Three ancestral groups were observed for the Canadian
germplasm. Most diverse germplasm harbored in the USA collection. These findings not only reveal the compatible
patterns of genetic structure and relatedness inferred with three marker types, but also are useful for managing Jerusa-
lem artichoke germplasm and utilizing diverse germplasm for genetic improvement.
Keywords: Jerusalem Artichoke, Genetic Structure, Germplasm Management, RAPD, ISSR, SRAP
1. Introduction
Recent years have seen an increased characterization effort
directed toward the assessments of genetic structure and
genetic relatedness in plant germplasm [1-3]. These as-
sessments can not only facilitate the conservation and
management of many plant germplasm collections, but
also enhance the utilization of existing germplasm diver-
sity in plant breeding [4]. The assessed genetic related-
ness is critical for selection of diverse parents from less
explored plant germplasm and for design of experimental
crosses to widen the breeding gene pool [5]. The inferred
genetic structure is essential to capture functional genetic
diversity by setting up core subsets of germplasm [6] and
association mapping of genes controlling complex traits
[7]. However, the marker-based assessments of genetic
structure and relatedness can vary, depending on the nature
of molecular markers used, as different markers may sam-
ple a plant genome in different ways [8]. The variation
could be substantial for a genetically heterogeneous plant
with highly outcrossing and variable ploidy. Thus, an
adequate attention should be paid to the performance of
different molecular markers in assessments of genetic
structure and relatedness [2,8]. Jerusalem artichoke (He-
lianthus tuberosus L.) has been cultivated mainly for tu-
bers since the 17th century [9]. Its inulin-containing tu-
bers are consumed as vegetable and can be used as raw
material to produce various value-added products such as
healthy food products, animal additive feed [10] and bio-
ethanol [11]. The crop has been largely abandoned after
the Second World War [12], but recently it has received
a renewed interest in genetic improvement for multiple
purposes [9]. Also, Jerusalem artichoke is a cold-hardy
North American wild relative of the cultivated sunflower

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
754
with RAPD, ISSR and SRAP Markers
(H. annuus L.) and will play an increasing role in the
genetic improvement of economically important traits in
sunflower such as oil characters and disease resistance
[13,14]. However, insufficient effort has been made to
characterize and conserve Jerusalem artichoke genetic
resources, in contrast to sunflower germplasm [15].
Currently, only several hundred Jerusalem artichoke
accessions are maintained in plant germplasm collections
worldwide [9,16,17]. These accessions represent germ-
plasm only from a dozen or so countries and include wild
and weedy accessions, landraces, or traditional and ob-
solete cultivars, and advanced or improved cultivars.
Some efforts have been made to characterize existing Je-
rusalem artichoke germplasm [12,18-20]. However, these
characterizations were mainly focused on phenotypic and
genotypic data and would be more informative with sup-
plementary applications of informative molecular mark-
ers. Many molecular markers have been developed for
plant genetic research over the recent decades [4,21]. The
random amplified polymorphism DNA (RAPD) [22] and
inter simple sequence repeats (ISSR) [23] were among
the earliest developed molecular tools used to assess
plant genetic diversity due to technical simplicity and
practical feasibility. For example, the RAPD and ISSR
markers require no prior sequence information for the
survey of plant genomes, but generally suffer from low
resolution due to various issues associated with repro-
ducibility, dominance and non-homologous DNA frag-
ment [8], of which issues are similar to other dominant
markers [24]. The sequence-related amplified polymor-
phism (SRAP) [25] represents another simple and re-
liable PCR-based marker tool for genetic diversity ana-
lysis [26,27]. However, these molecular markers have
rarely been applied to assess genetic variation of Jerusa-
lem artichoke [28-30]. A study was conducted to assess
the genetic structure and genetic relatedness of 47 di-
verse Jerusalem artichoke accessions using RAPD, ISSR
and SRAP markers and to compare the congruency of the
structural and relatedness assessments. It was our hope
that this study can provide a useful set of diversity in-
formation not only for genetic improvement of Jerusalem
artichoke, but also for understanding to what extent the
different classes of molecular markers provide concor-
dant information about the structure of populations and
the relationships among individuals.
2. Materials and Methods
2.1. Plan t Ma teria l
Forty-seven Jerusalem artichoke accessions were used
for this study (Ta ble 1). The studied germplasm was ob-
tained from Plant Gene Resources of Canada (PGRC),
Saskatoon, Canada and originated from Canada, USA,
France, and the former Union of Soviet Socialist Repub-
lics (USSR). It also included six accessions collected
directly from the wild populations in Texas, USA.
2.2. DNA Extraction
Young leaf tissue was collected from at least three indi-
vidual plants of one accession and bulked for DNA ex-
traction following the Tai and Tanksley’s modified me-
thod [31], which was shown to be the most effective
DNA extraction method for Jerusalem artichoke [32].
The bulked tissue (300 mg) was ground with a homoge-
nizer and 0.7 ml of extraction buffer (100 mM Tris-HCl;
pH 8, 50 mM EDTA pH 8, 0.5 M NaCl, 1.25% SDS, 8.3
mM NaOH, 0.38% Na bisulfite) was added and mixed by
vortexing. The sample was incubated at 65˚C for 20 min
and 0.22 ml of 5 M potassium acetate added and mixed
well. The tube was placed on ice for 40 min, followed by
centrifugation for 3 min. The supernatant was transferred
to the new tube. The DNA was precipitated by adding
0.7 volume of isopopanol, mixed well and centrifuged
for 3 min. The supernatant was poured off and the pellet
rinsed with 70% ethanol. The pellet was re-suspended in
300 µl of T5E (50 mM Tris-HCl pH 8, 10 mM EDTA)
by briefly vortexing, and incubated at 65˚C for 5 min,
followed by vortexing again. 150 µl of 7.4 M ammonium
acetate were added and mixed well before centrifugation
for 3 min and removal of the supernatant to the new tube.
The DNA was precipitated by mixing with 330 µl of iso-
propanol and centrifuged for 3 min. The pellet was rin-
sed with 70% ethanol and re-suspended in 100 µl of T5E,
incubated at 65˚C for 5 min, and then vortexing. The
DNA was re-suspended in 150 µL of TE (10 mM Tris-
HCl, pH 8.0, 1 mM EDTA). The purity and quality of
genomic DNA were assessed after digested with RNaseA
(Sigma), and quantified on 1% agarose gel against know
concentration of 100 bp DNA ladder plus (Vivantis). The
extracted genomic DNAs were stored at –20˚C until fur-
ther use.
2.3. RAPD Analysis
Thirty-one decamer primers (Operon Technologies, Ala-
meda, CA) were initially screened using two sets of bul-
ked DNAs of Jerusalem artichoke to determine the suit-
ability of each primer for the study. The first bulk con-
sisted of 34 accessions (JA29, JA30, JA31, JA32, JA34,
JA35, JA36, JA42, JA43, JA44, JA45, JA46, JA47, JA48,
JA49, JA50, JA54, JA55, JA58, JA59, JA60, JA61, JA66,
JA69, JA70, JA71, JA72, JA73, JA74, JA78, JA87, JA88,
JA91, JA92) and the second bulk included 11 accessions
(JA95, JA97, JA98, JA100, JA105, JA106, JA107,
JA108, JA109, JA110, JA111). Based on their ability to

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
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755
Table 1. List of 47 Jerusalem artichoke accessions studied, along with some description, country origin and the cluster in-
ferred with the STRUCTURE program.
Acc Description Origin StCAcc Description Origin StC
JA27 DHM-7 Canada 6 JA66 FR. MAMMOTH WHITE USA 5|1
JA28 DHM-13 Canada 6 JA69 TUB-364 USDA-ARS-SR USA, Taxas 3
JA29 DHM-14 Canada 6 JA70 TUB-365 USDA-ARS-SR USA, Taxas 3
JA30 DHM-16 Canada 6 JA71 TUB-675 USDA-ARS-SR USA, Taxas 3
JA31 DHM-18 Canada 6 JA72 TUB-676 USDA-ARS-SR USA, Taxas 1
JA32 DHM-19 Canada 6 JA73 TUB-709 USDA-ARS-SR USA, Taxas 2
JA34 DHM-22 Canada 6 JA74 TUB-847 USDA-ARS-SR USA, Taxas 2
JA35 W-97 Canada 6 JA78 FUSEA U 60 France 3
JA36 W-106 Canada 6 JA87 242-63 France 1
JA42 75005 Canada 6 JA88 TOPINSOL 63 USSR 1
JA43 75004-52 Canada 6 JA91 KIEVSKII USSR 1
JA44 A-3-6 Canada 6 JA92 INDUSTRIE USSR 5|1
JA45 HM hybrid-A-4 Canada 2 JA95 NACHODKA USSR 1
JA46 DHM-14-3 Canada 6 JA97 D19-63340 France 5
JA47 DHM-14-6 Canada 2 JA98 242-62 France 1
JA48 DHM-15 Canada 6 JA100 105-62G2 France 1
JA49 7513A Canada 2|6 JA105 357303 VOLGA 2 USSR 1
JA50 W-97 Canada 1 JA106 83-001-1 (37 × 6) Canada 5
JA54 Unknown USA 2 JA107 83-001-2 (37 × 6) Canada 5
JA55 Unknown USA 2 JA108 83-001-3 (37 × 6) Canada 5
JA58 Intress USSR 3 JA109 83-001-4 (37 × 6) Canada 5
JA59 VOLZSKIJ-2 USSR 3 JA110 83-001-5 (37 × 6) Canada 5
JA60 Jamcovskij Krashyj USSR 4 JA111 83-001-6 (37 × 6) Canada 1
JA61 VADIM USSR 4
Acc = accession and label following those described in [9]. USSR = the former Union of Soviet Socialist Republics. Six accessions from Texas, USA, were
collected from wild populations. StC = the most likely cluster inferred with STRUCTURE based on the combined marker data; the accession with two clusters
means that the ancestry levels for both clusters were less than 0.5, but the first cluster had the larger ancestry than the other cluster.
detect distinct, clearly resolved, and reproducible ampli-
fied products in the initial screening, 13 most informative
primers (Table 2) were selected for further analysis.
The polymerase chain reaction (PCR) was run in final
volume of 50 ng DNA template, 0.4 U Taq DNA poly-
merase (Vivantis), 1.0 µl 10x buffer (750 mM NH4(SO2)4,
0.1% Tween 20, Fermantas), 1.5 mM MgCl2 (Fermantas),
0.2 mM dNTPs (Vivantis), 1.0 µM RAPD primer in 0.20
ml PCR tube. The amplification was performed in a ther-
mocycler called “CG1-96” (Corbett Research, Germany).
The amplification regime consisted of 95˚C for 2 min;
then 45 cycles at 94˚C for 30 s, annealing temperature
40˚C (36˚C for OPS04) for 30 s, and 72˚C for 90 s; and a
final extension at 72˚C for 5 min.
The RAPD amplification products were analyzed by
electrophoresis on 1.2% agarose gels, run in 1× TBE,
visualized under UV transilluminator, and photographed.
The PCR reactions were done three times independently.
Only repeatable amplified DNA fragments were manu-
ally scored as 1 or 0 for presence or absence, respectively,
for each sample.
2.4. ISSR Analysis
A total of 25 primers were initially screened using two
sets of bulked DNAs described above to determine the
suitability of each primer for the study. Based on their
ability to detect distinct, clearly resolved, and reproduci-
ble amplified products from the initial screening, six
most informative primers (Table 2) were selected for
further analysis. The polymerase chain reaction (PCR)
was run in final volume of 50 ng DNA template, 0.4 U
Taq DNA polymerase (Vivantis), 1.0 µl 10× buffer (750
mM NH4(SO 2)4, 0.1% Tween 20; Fermantas) 1.5 mM
MgCl2 (Fermantas), 0.2 mM dNTPs (Vivantis), 1.0 µM

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
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Table 2. List of 13 RAPD, 6 ISSR and 9 SRAP markers used and polymorphism detected in the 47 Jerusale m artichoke ac-
cessions.
Primer Sequence (5' - 3') Total No. of bands Polymorphic bands (%) Entropy-based diversity
content
RAPD
OPA02 TGCCGAGCTG 28 85.7 5.61
OPA10 GTGATCGCAG 25 88.0 5.56
OPA20 GTTGCGATCC 25 84.0 5.69
OPE01 CCCAAGGTCC 29 82.8 5.92
OPE02 GGTGCGGGAA 31 90.3 7.65
OPE08 TCACCACGGT 22 95.5 4.64
OPE09 CTTCACCCGA 20 90.0 3.87
OPS01 CTACTGCGCT 20 90.0 3.89
OPS02 CCTCTGACTG 33 90.9 8.67
OPS04 CACCCCCTTG 28 64.3 4.23
OPS06 GATACCTCGG 26 96.2 6.10
OPS12 CTGGGTGAGT 32 84.4 7.03
OPS15 CAGTTCACGG 21 95.2 4.95
Total/Mean 340 87.1 5.68
ISSR
P03 HVHTCCTCCTCCTCCTCC 19 78.9 4.25
P06 CACACACACACACACART 16 68.8 1.92
P07 TGTGTGTGTGTGTGTGRT 6 50.0 0.70
P18 CACACACACACACACAG 27 85.2 5.92
P40 GAGAGAGAGAGAGAGAGAYT 21 71.4 4.24
P76 GATAGATAGACAGACA 26 96.2 6.75
Total/Mean 115 80.0 3.97
SRAP
ME2/EM5 ME2: TGAGTCCAAACCGGAGC 19 100.0 4.55
EM5: GACTGCGTACGAATTCAA
ME2/EM6 ME2: TGAGTCCAAACCGGAGC 37 97.3 9.08
EM6: GACTGCGTACGAATTCCA
ME2/EM8 ME2: TGAGTCCAAACCGGAGC 20 90.0 3.33
EM8: GACTGCGTACGAATTCAC
ME5/EM5 ME5: TGAGTCCAAACCGGAAG 24 79.2 4.23
EM5: GACTGCGTACGAATTCAA
ME5/EM6 ME5: TGAGTCCAAACCGGAAG 20 80.0 2.51
EM6: GACTGCGTACGAATTCCA
ME5/EM8 ME5: TGAGTCCAAACCGGAAG 33 93.9 7.85
EM8: GACTGCGTACGAATTCAC
ME7/EM5 ME7: TGAGTCCTTTCCGGTCC 12 83.3 3.37
EM5: GACTGCGTACGAATTCAA
ME7/EM6 ME7: TGAGTCCTTTCCGGTCC 26 69.2 4.25
EM6: GACTGCGTACGAATTCCA
ME7/EM8 ME7: TGAGTCCTTTCCGGTCC 28 96.4 6.03
EM8: GACTGCGTACGAATTCAC
Total/Mean 219 88.6 5.02

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm 757
with RAPD, ISSR and SRAP Markers
ISSR primer in 0.20 ml PCR tube. The amplification was
performed in a thermocycler called “CG1-96” (Corbett
Research, Germany). The amplification regime consisted
of 95˚C for 2 min; then 45 cycles at 94˚C for 30 s, an-
nealing temperature 45, 49 or 51˚C for 30 s, and 72˚C for
90 s; and a final extension at 72˚C for 5 min.
The ISSR amplification products were analyzed by
electrophoresis on 1.2% agarose gels, run in 1xTBE, vis-
ualized under UV transilluminator, and photographed.
The PCR reactions were done three times independently.
Only repeatable amplified DNA fragments were manually
scored as 1 or 0 for presence or absence, respectively, for
each sample.
2.5. SRAP Analysis
The SRAP primers were selected based on previous re-
ports [25]. Nine SRAP primer combinations (ME2-EM5,
ME2-EM6, ME2-EM8, ME5-EM5, ME5-EM6, ME5-
EM8, ME7-EM5, ME7-EM6 and ME7-EM8) were ini-
tially screened using two sets of bulked DNAs described
above and confirmed on their suitability for further ana-
lysis (Table 2).
A total of 10 l PCR reaction mixture was composed
of 1x Taq buffer (75 mM Tris-HCl (pH 8.4), 20 mM
(NH4)2SO4, and 0.01% Tween 20: Fermentas), 0.2 mM
dNTP mix, 1.5 mM MgCl2, 0.5 µM of each primer, 0.4
unit/10 l of Taq DNA polymerase (Fermentas, U.S.A),
and 30 ng of template DNA. PCR amplification was car-
ried out in a thermocycler called “CG1-96” (Corbett Re-
search, Germany) programmed for pre-denaturalization
of 3 min at 95˚C and 5 cycles (or otherwise stated) of 1
min at 95˚C, 1 min at 35˚C, and 2 min at 72˚C, followed
by 35 cycles of 1 min at 95˚C, 1 min at 50˚C, and 2 min
at 72˚C, finally by one cycle of 5 min at 72˚C.
The SRAP products were analyzed by a 1.5% agarose
gel electrophoresis, ethidium bromide stained and visu-
alized by Electrophoresis Gel Photodocumentation Sys-
tem (Vilber Lourmat, Japan). In addition, the PCR pro-
ducts also were analyzed by electrophoresis on 10% (w/v)
polyacrylamide gel and revealed DNA bands by a gel
silver staining. 100 bp DNA ladder plus (Vivantis) was
used as a molecular size standard. The PCR reactions
were done three times independently. Only repeatable
amplified DNA fragments were manually scored as 1 or
0 for presence or absence, respectively, for each sample.
2.6. Data Analysis
The RAPD, ISSR and SRAP data were separately ana-
lyzed for the levels of polymorphism with respect to
primer by counting the number of polymorphic bands
and generating summary statistics of band frequencies.
Shannon’s entropy was calculated following the appro-
ach of Russell et al. [33] to estimate the diversity content
per locus, as this estimate does not require strict genetic
assumptions such as marker inheritance and sample ploi-
dy. The entropy-based diversity content provides a mea-
sure of the effective number of alleles per marker locus
[34]. These analyses were performed by using a SAS
program written in SAS IML [35].
The model-based Bayesian method available in the
program STRUCTURE version 2.2.3 [36-38] was used
to detect population structure and to assign accessions to
subpopulations. The STRUCTURE program was run 40
times for each subpopulation (K) value, ranging from 2 -
10, using the admixture model with 10,000 replicates for
burn-in and 10,000 replicates during analysis. The final
population subgroups were determined based on 1) like-
lihood plot of these models, 2) the change in the second
derivative (∆K) of the relationship between K and the
log-likelihood [39], and 3) stability of grouping patterns
across 30 runs. For a given K with 30 runs, the run with
the highest likelihood value was selected to assign the
posterior membership coefficients to each accession. A
graphical bar plot was then generated with the posterior
membership coefficients. These structural data inferen-
ces were made separately for each marker type and the
combined marker data.
The genetic relationships of the Jerusalem artichoke
accessions were assessed using two approaches. Distance
matrices based on band sharing for all pairs of the 47 in-
dividual accessions were constructed using GenAIEx 6
[40]. The relationship between the distance matrices was
assessed using the Mantel’s test [41] with 9999 random
permutations and plotted. A neighbor-joining analysis of
the 47 accessions was also made using PAUP* [42] and
a radiation tree was displayed using MEGA 3.01 [43] to
confirm the genetic relationships of individual accessions
and to identify any genetic clustering without restriction
to known characteristics. These relationship assessments
were performed separately for each marker type and the
combined marker data.
3. Results and Discussion
3.1. Marker Polymorphism
The characterization effort revealed variable, but compa-
tible, polymorphism in the 47 Jerusalem artichoke acces-
sions for three marker types, as summarized in Table 2
and Figure 1. A total of 340 RAPD bands were obtained
from 13 RAPD primers; 115 ISSR bands from six ISSR
primers; and 219 bands from nine combinations of SRAP
primers. The number of polymorphic bands was 296
(87.1%), 92 (80%) and 194 (88.6%) for RAPD, ISSR
and SRAP markers, respectively. Based on the estimates

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
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with RAPD, ISSR and SRAP Markers
Band frequency
0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95
Percentage of total bands
0
2
4
6
8
10
12
14
RAPD
ISSR
SRAP
Figure 1. Band frequency spectra for RAPD, ISSR and
SRAP markers detected in the 47 Jerusalem artichoke ac-
cessions.
of the Shannon’s entropy per primer (or primer pair), the
most informative marker type was RAPD with the Shan-
non entropy of 5.68, followed by SRAP with the Shan-
non’s entropy of 5.02 and ISSR with the Shannon’s en-
tropy of 3.97. The low information value for ISSR mark-
ers may reflect the use of a smaller number of ISSR
primers.
The range of the band frequencies observed in the 47
accessions was roughly the same from 0.021 to 0.979 for
three marker types, but their average band frequencies
differed with 0.46, 0.40 and 0.60 for RAPD, ISSR and
SRAP markers, respectively. Interestingly, an average of
five RAPD bands was observed for each 0.05 interval of
band frequency ranging from 0 to 1 (Figure 1). Slightly
more ISSR bands with a frequency of 0.5 or smaller were
observed, while slightly more SRAP bands with a fre-
quency greater than 0.5 were found (Figure 1).
As expected for Jerusalem artichoke, an outcrossing,
hexaploidy (2n = 6x = 102) plant [44], a high level of
genetic polymorphism was observed for these marker
types [45]. Such a level of polymorphism was consistent
with some reports based on various molecular markers
[28-30], and compatible with those results reported in the
cultivated sunflower [46,47]. Interestingly, the overall
polymorphism was compatible over the three marker ty-
pes, but the SRAP marker appeared to display a slightly
higher polymorphism and detect more DNA fragments
with frequencies larger than 0.5.
3.2. Genetic Structure
The model-based inference of genetic structure within
the 47 Jerusalem artichoke accessions by STRUCTURE
considered K = 2 to 10 clusters and revealed five to six
optimal clusters with the highest log-likelihoods for these
three marker types and their combined data (Figure 2).
First, the RAPD or ISSR markers revealed five most
likely clusters with more than 80% memberships shared
in corresponding clusters (Figure 2(a)). Similarly the
SRAP markers or the combined marker data displayed
six most likely clusters with more than 85% member-
ships shared in corresponding clusters (Figure 2(a)).
Note that the colors or labels used for inferred clusters
may differ for different markers, but a corresponding clu-
ster inferred from two marker types was defined based
on the share of the membership majority. For example,
the blue and green SRAP clusters are corresponding to
the red and yellow clusters in the combined data, respec-
tively (Figure 2(a)).
The inferences of the optimal number of clusters for
three marker types gained further support from the pat-
terns of log-likelihood of the data (Figure 2(b)) and from
the change in the second derivative (∆K) of the relation-
ship between K and the log-likelihood (Figure 2(c)). The
largest average log-likelihood of –6556.3 was observed
for the RAPD markers when K = 5; –2024.8 for ISSR
when K = 5; and –3946.0 for SRAP when K = 6 (Figure
2(b)). When the three marker data were combined, the
highest average log-likelihood of –18,004.8 was obtained
when K = 6. Similarly, the first dramatic change in the
second derivative of the log-likelihoods over various Ks
analyzed was occurred when K = 5 for the RAPD and
ISSR markers, and when K = 6 for the SRAP markers
(Figure 2(c)).
Interestingly, the RAPD and ISSR data carried similar
signals of genetic structure in this germplasm set, while
the SRAP data were more compatible with the combined
data in the structural inference. The reason for such a
discrepancy remains unknown, but it was clear that the
SRAP data carried more unique information on the ge-
netic structure of this germplasm set. In spite of this, the
overall patterns of genetic structure inferred from these
three marker types were highly compatible, as one of the
six optimal clusters obtained from SRAP or the com-
bined data had only two members from the former Soviet
Union (JA60 and JA61) and removing these two acces-
sions generated compatible patterns of genetic structure
for all marker types (results not shown).
Based on the combined data, the average distance be-
tween individual accessions in the same cluster for six
clusters were 0.346, 0.254, 0.138, 0.158, 0.239 and 0.261,
respectively, for clusters 1 to 6. The mean value of cluster-
specific Fst was 0.113, 0.390, 0.670, 0.710, 0.439, and
0.323, respectively, for clusters 1 to 6. The overall pro-
portions of membership of the sample in each of the six
clusters were 0.248, 0.153, 0.132, 0.059, 0.128 and 0.279,
respectively, for clusters 1 to 6. The detailed member-

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
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759
K
246810
0
1
2
3
4
K
246810
-14000
-12000
-10000
-8000
-6000
-4000
-2000
0
p
(b): In (pr(Data/K)) (c): ΔK
Figure 2. The genetic structure of the 47 Jerusalem artichoke accessions inferred with STRUCTURE and the sensitivity as-
sessment of inference w i th ST RUCTURE with respect to marker ty pe. (a): th e most likelihood genetic structures inferred with
STRUCTURE for three marker data and the combine d marker data. Each column represents an accession and the column labels
from 1 to 47 matched the sequence of the accessions given in Table 1. For example, the column 4 is JA30 and the column 26 is JA69;
(b) and (c): the log-likelihood profiles and the rates of change in log-likelihood for models with K = 2 to 10 for RAPD, ISSR
and SRAP markers labeled with filled circle, open circle, and open square, respectively. Note that the standard deviations of
the log-likelihoods for RAPD markers when K = 8 and 10 were reduced in half for ease of illustration.
ships of the 47 accessions in each cluster were given in
Table 1, which was based on the highest level of inferred
ancestry for one cluster from one STRUCTURE run with
the highest log-likelihood of data (–11,792.9). Three
accessions had the cluster membership with an ancestry
level of 0.5 or less and 24 accessions displayed an ance-
stry level of 0.80 or higher (Figure 2(a)).
The optimal clusters detected here might reflect the
current Jerusalem artichoke gene pool, as some clusters
reflected either the wild populations sampled or the con-
sequence of long term Jerusalem artichoke breeding,
particularly in Canada. However, the sampling bias may

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
760
with RAPD, ISSR and SRAP Markers
exist, as this germplasm set is small and may not well re-
present the worldwide Jerusalem artichoke germplasm.
Adding other representative samples to such structural
analysis would help to verify and correct the sampling
bias. Thus, some caution is needed in interpretation of
these optimal clusters.
3.3. Genetic Distance
Distance matrices based on band sharing were constru-
cted for all pairs of the 47 individual accessions for three
marker types and the relationship between the distance
matrices was plotted in Figure 3. The pairwise distances
based on the RAPD markers ranged from 0.091 to 0.432
and averaged 0.328; the pairwise distances based on the
ISSR markers ranged from 0.065 to 0.5 and averaged
0.326; and the pairwise distances based on the SRAP
markers ranged from 0.036 to 0.474 and averaged 0.339.
Obviously, highly significant (P < 0.0001) linear rela-
tionships between the distance matrices were detected for
three pairs of marker type. The relationship for the pair
of the RAPD and ISSR markers explained 16.4% varia-
tion with a correlation coefficient of 0.40. The relation-
ship for the pair of the RAPD and SRAP markers ex-
plained 28.4% variation with a correlation coefficient of
0.53. The relationship for the pair of the ISSR and SRAP
markers explained 19.2% variation with a correlation co-
efficient of 0.44. However, the estimated correlation coef-
ficients of distance matrices among these marker types are
relatively low.
The revealed correlations of pairwise distance matri-
ces, although relatively weak, were compatible among
three marker types. The extent of distance matrix corre-
lation reported here was consistent with those reported
from similar studies of other plants [48,49]. The low co-
rrelations reflect the relatively low resolution of sam-
pling genetic variation of a genome with these marker
types, in contrast to the most informative markers such
as microsatellite or simple-nucleotide polymorphism av-
ailable in well-studied plant species [1,2]. However, the
SRAP marker seemed to be slightly more informative
than the other two marker types, as the correlations be-
tween SRAP and the other marker types were higher.
The neighbor-joining (NJ) analysis of the combined
data revealed several interesting patterns of genetic re-
latedness (Figure 4). First, up to six clusters could be
identified, but they were not well separated, even based
on the information from a total of 582 DNA fragments.
In spite of the low resolution, the NJ clustering matched
relatively well with those inferred from the STRUC-
TURE program, as illustrated in Figure 4.
Two discrepancies were identified: the accessions JA45
and JA47 for Cluster 6 and Cluster 2 and the accession
of JA72 for Cluster 4 and Cluster 1. Second, the six wild
accessions from Texas, USA, were clustered into three
groups and may reflect the different levels of ancestry
among them. Third, the 24 accessions from Canada lar-
gely represented cultivated materials and were clustered
into three groups, two of which the wild accessions from
USA were also present. This suggests at least three ancestral
ISSR distance
0.0 0.1 0.2 0.30.40.5
0.0
0.1
0.2
0.3
0.4
0.5
RAPD dista n ce
0.0 0.1 0.2 0.30.40.5
0.0
0.1
0.2
0.3
0.4
0.5
RAPD dista n ce
0.0 0.1 0.2 0.30.40.5
0.0
0.1
0.2
0.3
0.4
0.5
Y=0.8031X+0.0756
R2=0.2835, P<0.0001
Y=0.5115X+0.1587
R2=0.1636, P<0.0001
Y=0.5227X+0.1683
R2=0.192, P<0.0001
A
C
B
(a)
(b)
(c)
ISSR distance
SRAP distance
SRAP distance
Figure 3. Correlation between genetic distances based on RAPD,
ISSR and SRAP markers in the 47 Jerusalem artichoke acces-
sions. Each point represents the genetic distance between a pair
of acces sions, based on band sharing of either m arker.

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
with RAPD, ISSR and SRAP Markers
761
C6
C1
C5
C4
C2
C3
JA27
JA28
JA29
JA30
JA31
JA32
JA34
JA44
JA46
JA35
JA36
JA42
JA43
JA48
JA45
JA47
JA54
JA55
JA73*
JA74*
JA49
JA50
JA58
JA59
JA69*
JA70*
JA71*
JA78
JA60
JA61
JA66
JA72*
JA87
JA88
JA91
JA105
JA95
JA98
JA100
JA92
JA97
JA106
JA108
JA107
JA109
JA110
JA111
0.05
Figure 4. The Neighbor-J oining (NJ) tree display ing the genetic asso ciations of the 47 J erusale m articho ke accessi ons re pre-
senting four countries. Each accession is labeled with its country origin: open circle for Canada; filled circle for USA; open
diamond for France; and filled diamond for the former Union of Soviet Socialist Republics (USSR). The accession with a star
was collected from a wild population in USA. Six most likely clusters inferred with STRUCTURE from the combined data
were labeled with C1 - C6, except two i nconsistent cases t hat J A 50 should be located in C1 and JA66 in C5 (see Table 1) .
groups for these selected Canadian accessions. Fourth,
the accessions from France and the former Soviet Union
were closely related to the accessions from USA, and
less to the accessions from Canada. This may reflect the
independent ancestral selections for Jerusalem artichoke
breeding from the USA wild collection.
3.4. Prac t i c al Implic ation s
The molecular markers applied here knowingly have limita-
tions with low resolution of sampling a plant genome due to
various issues associated with reproducibility, dominance
and non-homologous DNA fragment [4,8]. Typically,
RAPD has low reproducibility; ISSR may include non-
homologous fragments of similar size; and SRAP has a
sampling bias toward the DNA fragments with an open
reading frame. These technical issues are expected to
introduce more deviations of sampling genetic variation
among these marker types for Jerusalem artichoke with
highly outcrossing and variable ploidy [27,48,49].
Surprisingly, however, only some deviations were ob-
served in this study and the revealed deviations seemed
to slightly favor SRAP markers in the marker choice for
a diversity analysis. For example, the SRAP markers
displayed a slightly higher percentage of polymorphism
(Table 2), more compatible inference of genetic struc-
ture with the combined marker data (Figure 2(a)), and
the higher correlations of pairwise genetic distances with
the other two markers (Figure 3). Interestingly, the three
different markers revealed similarly high levels of ge-
netic polymorphism and compatible patterns of genetic
structure and genetic relatedness in these Jerusalem arti-
choke accessions.
The compatible diversity patterns revealed by the three
different markers are encouraging for a diversity analysis

Assessing Genetic Structure and Relatedness of Jerusalem Artichoke (Helianthus tuberosus L.) Germplasm
762
with RAPD, ISSR and SRAP Markers
of an under-explored plant species like Jerusalem arti-
choke with limited genomic resources available. The
three types of molecular markers applied here are tech-
nically simple and practically feasible and could still
play a role in the sampling of genetic variation in poorly
known or less characterized plant species. The SRAP
marker appeared to be slightly more informative than the
other assayed markers and favored for further diversity
analysis, but its limitation in sampling bias should also
be considered in the marker choice.
The revealed patterns of genetic structure are useful
for managing worldwide Jerusalem artichoke germplasm
by taking the structural patterns into account in the de-
velopment of diverse core subsets for further germplasm
characterization and utilization. The specific core subsets
[6] can facilitate the association mapping of genes con-
trolling ecologically important traits such as inulin, oil
characters and disease resistance. The revealed patterns
of genetic relatedness are informative for parental selec-
tions and experimental design of productive crosses in
Jerusalem artichoke breeding. Three ancestral lines were
detected for the Canadian germplasm and quite distin-
guished from the germplasm from other countries. As
expected with its species distribution, more genetically
diverse accessions remain in the USA germplasm collec-
tion and a further comprehensive characterization of the
USA collection would yield more useful diversity infor-
mation for utilizing Jerusalem artichoke germplasm.
3.5. Conclusive Remarks
The multi-marker characterization of the 47 Jerusalem
artichoke accessions revealed compatible patterns of ge-
netic polymorphism, genetic structure and genetic rela-
tedness for three marker types. The SRAP marker ap-
peared to be slightly more informative and thus favored
for further diversity analysis. A high level of genetic
polymorphism was detected and six optimal groups were
identified in this germplasm set. Three ancestral groups
were identified for the Canadian germplasm. Most di-
verse germplasm harbored in the USA collection. These
results are useful for managing Jerusalem artichoke ger-
mplasm and utilizing diverse germplasm for genetic im-
provement.
4. Acknowledgements
We gratefully acknowledge the Thailand Research Fund
(TRF), the commission for High Education (CHE) and
Khon Kaen University (KKU) for providing financial
supports to this research through the Distinguish Re-
search Professor Grant to Professor Dr. Aran Patanothai
and an anonymous reviewer for his/her helpful com-
ments on an early version of the manuscript.
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