agronomy

Article
Construction of Soybean Mutant Diversity Pool
(MDP) Lines and an Analysis of Their Genetic
Relationships and Associations Using TRAP Markers
Dong-Gun Kim 1,2,† , Jae Il Lyu 1,† , Min-Kyu Lee 1,3 , Jung Min Kim 1,3 , Nguyen Ngoc Hung 1,3 ,
Min Jeong Hong 1 , Jin-Baek Kim 1 , Chang-Hyu Bae 2, * and Soon-Jae Kwon 1, *
1 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 56212, Korea;
[email protected] (D.-G.K.); [email protected] (J.I.L.); [email protected] (M.-K.L.);
[email protected] (J.M.K.); [email protected] (N.N.H.); [email protected] (M.J.H.);
[email protected] (J.-B.K.)
2 Department of Life-resources, Graduate School, Sunchon National University, Suncheon 57922, Korea
3 Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University,
Gwangju 61186, Korea
* Correspondence: [email protected] (S.-J.K.); [email protected] (C.-H.B.); Tel.: +82-63-570-3312
(S.-J.K.); +82-61-750-3214 (C.-H.B.)
† These authors contributed equally to this work.




Received: 30 December 2019; Accepted: 6 February 2020; Published: 10 February 2020


Abstract: Mutation breeding is useful for improving agronomic characteristics of various crops.
In this study, we conducted a genetic diversity and association analysis of soybean mutants to assess
elite mutant lines. On the basis of phenotypic traits, we chose 208 soybean mutants as a mutant
diversity pool (MDP). We then investigated the genetic diversity and inter-relationships of these MDP
lines using target region amplification polymorphism (TRAP) markers. Among the different TRAP
primer combinations, polymorphism levels and polymorphism information content (PIC) values
averaged 59.71% and 0.15, respectively. Dendrogram and population structure analyses divided
the MDP lines into four major groups. According to an analysis of molecular variance (AMOVA),
the percentage of inter-population variation among mutants was 11.320 (20.6%), whereas mutant
intra-population variation ranged from 0.231 (0.4%) to 14.324 (26.1%). Overall, intra-population
genetic similarity was higher than that of inter-populations. In an analysis of the association between
TRAP markers and agronomic traits using three different statistical approaches based on the single
factor analysis (SFA), the Q general linear model (GLM), and the mixed linear model (Q+K MLM),
we detected six significant marker–trait associations involving five phenotypic traits. Our results
suggest that the MDP has great potential for soybean genetic resources and that TRAP markers are
useful for the selection of soybean mutants for soybean mutation breeding.

Keywords: mutation breeding; soybean; mutant diversity pool (MDP); TRAP markers;
association analysis

1. Introduction
Soybeans (Glycine max L.), used for food, livestock feed, and biofuel, is one of the most important
agricultural crops worldwide. Soybeans are consumed directly by humans, especially in many Asian
countries, in the form of traditional food products such as tofu, soy flour, and soymilk [1,2]. Soybean
seeds are composed of 40%–42% protein, 18%–22% oil (85% unsaturated and 15% saturated fatty acids),
28% carbohydrates, and abundant quantities of other nutrients, such as phosphorus, calcium, iron,

Agronomy 2020, 10, 253; doi:10.3390/agronomy10020253 www.mdpi.com/journal/agronomy

Agronomy 2020, 10, 253 2 of 16

lysine, and vitamins A, B, and D [3]. In addition, soybeans play an important role in crop diversification
and improve other crops through its addition of nitrogen to the soil during crop rotation [4].
Because the rate of spontaneous mutations in higher plants is quite low (10−5 to 10−8 ) [5], physical
and chemical mutagens can be used to induce mutations in cultivated plants [6]. Gamma radiation is a
very effective tool to induce genetic variation in many plant characters, with the resulting changes
dependent on the irradiation dose. Various plant organisms, such as seeds, pollen, whole plants, and
embryoid bodies, can be irradiated [7]. Because gamma rays can also cause various types of DNA
damage, including single- or double-strand breaks and substitutions [8,9], agronomic traits, such as
flowering, maturation date, seed coat color, chloroplast number, and biomass yield, are frequently
altered in soybean [10,11]. At present, 3200 mutant varieties of more than 210 plant species have
been produced for commercial use. Approximately 170 mutant varieties of soybean, the second-most
registered species after rice, are found in the FAO/IAEA Mutant Variety Database (http://mvd.iaea.org).
The use of molecular marker-based techniques in genetic studies, such as estimation of genetic
diversity and population structure, has advanced remarkably in recent years. Among the different types
of DNA markers, restriction fragment length polymorphisms (RFLPs), random amplified polymorphic
DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and inter-simple sequence repeats
(ISSRs) have been extensively used in soybeans, each with their own advantages and limitations [12].
In addition, SNPs, which are widely distributed throughout genomes in both non-coding and coding
regions, constitute the most abundant molecular markers recently used in plant genetic breeding [13],
but their development is time-consuming and costly. The target region amplification polymorphism
(TRAP) is a relatively new, simple, polymerase chain reaction (PCR)-based marker system that takes
advantage of the available EST database sequence information to generate polymorphic markers
targeting candidate gene [14]. Essentially, it derives an 18-mer primer from the EST sequence and
pairs it with an arbitrary primer that targets the intron and/or exon region (AT- or GC-rich core).
Because it can be used to generate markers for specific gene sequences, the TRAP technique is useful
for genotyping germplasm and generating markers associated with desirable crop agronomic traits
for marker-assisted breeding [15]. In recent years, the TRAP marker technique has been applied for
genetic diversity analyses [16,17] and genetic mapping [18]. In addition, Im et al. [19] have developed
a transposable element-based TRAP (TE-TRAP) marker system that is reportedly suitable for the
mutation breeding of sorghum. Although TRAP markers have most commonly been used for genetic
mapping and phylogenetic studies, they have also recently been applied to detect DNA mutations [20].
Rapid advances in the field of molecular biology and its allied sciences have led to the routine
use of molecular markers, thereby providing plant breeders with a precise genetic-diversity analysis
tool for plant improvement [21,22]. A combined molecular and morphological analysis is one of the
most widely used approaches for the estimation of genetic distances within a group of genotypes,
and molecular markers serve as an excellent tool for obtaining genetic information. Molecular markers
are also of great value to plant breeders for assessment of genetic divergence among genotypes for
various agronomic traits [23]. Another recent strategy for analyzing agronomic traits, association
analysis based on molecular-marker linkage disequilibrium (LD), can reduce experimental time and
costs. Association analysis has therefore been widely applied to study a variety of crops, such as
rice [24], maize [25], and soybeans [26].
In this study, we constructed 208 mutant diversity pool (MDP) lines based on agronomic traits
and investigated their genetic diversity and relationships using TRAP markers. Finally, we performed
an association analysis between agronomic traits and polymorphic TRAP amplicons.

2. Materials and Methods

2.1. Plant Materials and Phenotypic Evaluation

A total of 1000 seeds each of one soybean landraces, KAS360-22, and six representative Korean
soybean cultivars [27], 94Seori, BangSa (BS), PalDal (P), DanBaek (DB), DaePung (DP), and HwangKeum
 Agronomy 2020, 10, 253 3 of 20

2. Materials and Methods

2.1. Plant Materials and Phenotypic Evaluation

Agronomy 2020, 10, 253 3 of 16
A total of 1000 seeds each of one soybean landraces, KAS360-22, and six representative Korean
soybean cultivars [27], 94Seori, BangSa (BS), PalDal (P), DanBaek (DB), DaePung (DP), and
HwangKeum
(HK), (HK), were
were irradiated withirradiated
250 Gy ofwith 250 Gy
gamma of using
rays gamma a 60
rays
Cousing a 60Co gamma-irradiator
gamma-irradiator (150
(150 TBq capacity;
TBq capacity;
ACEL, Ottawa,ACEL, Ottawa, at
ON, Canada) ON,theCanada) at the Korea
Korea Atomic Energy Atomic Energy
Research Research
Institute Institute
in 2008. Theinirradiated
2008.
MThe irradiated
1 and control M 1 and control (non-irradiated)
(non-irradiated) seeds were immediately
seeds were immediately sown in the
sown in the research research
field field of
of the Advanced
the Advanced
Radiation Radiation
Technology Technology
Institute. Institute. MDP
To construct To construct
lines, weMDP lines,1000
sowed we sowed 1000 seeds
irradiated irradiated
of each
ofseeds of eachsoybean
the seven of the seven soybean
cultivars andcultivars
harvestedandaharvested a total
total of 1695 of 1695
M1:2 M1:2 individual
individual seeds,
seeds, only only
excluding
excluding
those thosegrowth
exhibiting exhibiting growth aberrations,
aberrations, such as stuntedsuchgrowth,
as stunted growth,
pollen pollen
sterility, sterility,
and no and nodue
germination
germination due to the degree of radiosensitivity (Figure 1). Next, we generated
to the degree of radiosensitivity (Figure 1). Next, we generated 1695 individual gamma-irradiated 1695 individual
gamma-irradiated mutants during M1–M5 generations by single-seed descent and then continued as
mutants during M1 –M5 generations by single-seed descent and then continued as bulks until M12
bulks until M12 generation. In a first selection phase, we selected 523 mutant lines from the M5
generation. In a first selection phase, we selected 523 mutant lines from the M5 generation that
generation that possessed at least 30% superior agricultural characteristics related to various
possessed at least 30% superior agricultural characteristics related to various environmental factors,
environmental factors, such as grain yield, growth type, and climate adaptability. In a second
such as grain yield, growth type, and climate adaptability. In a second selection phase, we investigated
selection phase, we investigated the morphological phenotypes of the 523 mutant lines in the M12
the morphological phenotypes of the 523 mutant lines in the M12 generation to eliminate redundant
generation to eliminate redundant phenotypes. Overall, we selected 208 genetically fixed mutant
phenotypes. Overall,
lines (201 mutants we their
with selected
wild208 genetically
types), which fixed mutant lines
we designated (201
as the mutants
mutant with their
diversity poolwild
(MDP),types),
which we designated
and assessed as thefour
the following mutant diversity
agronomic pool
traits: (MDP),
days and assessed
of flowering the following
(DF), maturation datefour
(MD),agronomic
seed
traits: days of flowering (DF), maturation date (MD), seed index (SI), node
index (SI), node number (NN), and the following seven morphological traits: growth type (GT), number (NN), and the
following seven
flower color morphological
(FC), seed coat colortraits:
(SCC),growth type (GT),
seed hilum flower stem
color (SHC), coloranthocyanin
(FC), seed coat color
(SA), plant (SCC),
heightseed
hilum
(PH),color (SHC), stemnumber
and ramification anthocyanin
(RN). (SA), plant height (PH), and ramification number (RN).

Figure1.1.Schematic
Schematic illustration
illustration of
of the
thebreeding
breedingofofMM1–M 12 generations of 208 soybean mutant diversity
Figure 1 –M12 generations of 208 soybean mutant diversity
pool(MDP) lines. *Information
(MDP)lines. Informationofofcultivars was described in in
LeeLee et [27]
et al.
*
pool cultivars was described al. [27]

2.2.DNA
2.2. DNAExtraction
Extraction
Thecontrol
The controland
andtreated
treatedseeds
seedsofof the
the 208
208 genetically
genetically fixed
fixed mutant
mutant lines
lines were
wereimmediately
immediatelysownsown in
in 50-cell (5 × 10) vegetable nursery trays containing bio-bed soil (Dongbu
50-cell (5 × 10) vegetable nursery trays containing bio-bed soil (Dongbu Farm Hannong, Farm Hannong, Gimje,
Gimje, Korea)
Korea)
and thenand then incubated
incubated in a greenhouse
in a greenhouse at 20 ± 5 at◦ C20 ± 5 °C
under underlight
natural natural
for light for 1 Fresh
1 month. month. Fresh
leaf leaf
tissue from
tissue from seedlings of each mutant line was collected and subjected to total
seedlings of each mutant line was collected and subjected to total genomic DNA extraction using a genomic DNA
extraction
DNeasy usingkit
96 Plant a DNeasy
(Qiagen,96 Plant kit
Leipzig, (Qiagen,following
Germany) Leipzig, Germany) followingprotocol.
the manufacturer’s the manufacturer’s
The extracted
protocol. The extracted DNAs ◦ were stored at −20 °C until use. For PCR analysis, DNA concentrations
DNAs were stored at −20 C until use. For PCR analysis, DNA concentrations were determined using
were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific.,
a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific., Waltham, MA, USA) were and
Waltham, MA, USA) were and then adjusted to 10 ng/µl.
then adjusted to 10 ng/µl.
2.3. TRAP Analysis
2.3. TRAP Analysis
Four fixed primers and four arbitrary primers were used to generate TRAP markers (Table 1).
Four fixed primers and four arbitrary primers were used to generate TRAP markers (Table 1).
All four arbitrary primers and one of the fixed primers were designed from other studies of monocot
All four arbitrary primers and one of the fixed primers were designed from other studies of monocot
plants [14,16]. The three other fixed primers (with ’MIR’ prefixes) were designed based on Arabidopsis
plants [14,16]. The three other fixed primers (with ’MIR’ prefixes) were designed based on Arabidopsis
thaliana microRNA sequences [28] using the Primer3 program (http://frodo.wi.mit.edu/primer3/). PCR
amplifications with 16 primer combinations were carried out on all DNA samples according to the
protocol of Hu et al. [29] with slight modification. Briefly, reactions were performed in 20-µl volumes
containing 2 µl genomic DNA (10 ng/µl), 1 µl fixed primer (10 pmol/µl), 1 µl of each arbitrary primer
(10 pmol/µl), 0.8 µl of dNTPs (2.5 mM), 2.0 µl 10 × PCR buffer, and 0.3 µl Phoenix Taq DNA polymerase
Agronomy 2020, 10, 253 4 of 16

(5 U/µl; cat no. Phoenix2013). DNA amplification was performed in a thermocycler (G Storm, UK)
according to the following program: initial denaturation at 94 ◦ C for 2 min, followed by 5 cycles of 94
◦ C for 45 s, 35 ◦ C for 45 s, and 72 ◦ C for 60 s, then 35 cycles of 94 ◦ C for 45 s, 53 ◦ C for 45 s, and 72 ◦ C

for 60 s, and a final extension at 72 ◦ C for 7 min. The amplified products were analyzed separately
using a fragment analyzer automated capillary electrophoresis instrument (FA; Advanced Analytical
Technologies, Ankeny, USA), and the collected images were scored manually.

Table 1. List of soybean target region amplification polymorphism- (TRAP) marker primers.

Primer name Sequence (5’–3´)

Fixed primers
B14G14B AAT CTC AAG GAC AAA AGG
MIR 156A GAT CTC TTT GGC CTG TC
MIR 157B GAT CAT TGT CCA GAT TC
MIR 159A GAT CCT TGG TTC TTT GG
Arbitrary primers
Sa4 TTA CCT TGG TCA TAC AAC ATT
Sa12 TTC TAG GTA ATC CAA CAA CA
Ga3 TCA TCT CAA ACC ATC TAC AC
Ga5 GGA ACC AAA CAC ATG AAG A

2.4. Data Analysis

TRAP marker alleles were scored as binary data, with ‘00 indicating the absence of a given allele and
‘10 indicating its presence. The binary data were entered into a Microsoft Excel spreadsheet, and genetic
similarities were computed. Using the 0/1-matrix, we calculated gene diversity, percent polymorphism,
polymorphism information content (PIC), and genetic distance with the genetic analysis package
PowerMarker. A dendrogram was constructed according to the unweighted pair group method with
arithmetic mean (UPGMA) algorithm based on the Nei distance method in PowerMarker v3.25 as well as
the embedded MEGA7 program. To analyze population structure, a Bayesian population analysis was
performed in STRUCTURE 2.3.4. A graphical determination of the optimal number of populations (K)
was carried out using STRUCTURE HARVESTER (http://taylor0.biology.ucla.edu/structureHarvester/).
The number of putative populations (K), assumed according to a set of allele frequencies at each locus,
can provide the degree of admixture of mutant lines. Each individual was assigned to one or more
populations based on membership (q) using a Q-matrix derived from STRUCTURE 2.3.4. Ideally, the
average estimated log probability of the data Pr(x|k) should plateau at the most appropriate value of K.
To determine the optimal K, we calculated the average probability of K for values of K = 2 − 15 based on
10 Markov chain Monte Carlo runs, each consisting of 10,000 burn-in (initiation) iterations followed by
100,000 iterations under a population admixture model. We then conducted an analysis of molecular
variance (AMOVA) and calculated genetic distances to support the genetic diversity information. An
AMOVA of 999 permutations was completed to assess inter- and intra-population variance (wild type
and mutants) using GenALEx v6.501. Pairwise fixation index (Fst) values were also estimated by
AMOVA. The hypothesis of an association of molecular markers with phenotypic data was tested using
three different methods in the software program TASSEL 4. The first association method, a single factor
analysis (SFA) of variance that does not consider population structure, was performed using each
marker as the independent variable. The mean performance of each allelic class was compared using
the general linear model (GLM) function in TASSEL. Next, a Q GLM method was carried out using
the same software. This method applies population structure detected by STRUCTURE (Q matrix) as
co-factors. To obtain an empirical threshold for marker significance and an experiment-wise P-value,
10,000 permutations of the data were performed. The final marker–trait association test, Q + K MLM,
considers both the kinship matrix and the population structure Q-matrix. The K matrix of pairwise
kinship coefficients for all pairs of lines was calculated from the TRAP data by TASSEL. Basic statistics
and a correlation analysis of agronomic traits were performed using Microsoft Excel and Python v2.7.
Agronomy 2020, 10, 253 5 of 16

3. Results

3.1. Phenotypic Analysis and Correlation Analysis

A summary of agronomic and morphological traits of the 208 MDP lines is shown in Table 2
and Supplementary Table S1. With respect to growth characteristics, a variety of phenotypes were
observed. DF ranged from 42 (mutant numbers; S87, S88, and S149) to 64 (S138), while MD varied
from 112 (S6) to 150 (S8). In addition, the seed index (SI) ranged from 7.9 (S13) to 28.8 (S7) g, and PH
ranged from 23 (S14) to 92.2 (S76) cm. NN and RN varied from 8.4 (S14) to 24.6 (S78) and 2.6 (S81)
to 8.8 (S12) cm, respectively. As shown in the histogram in Figure 2, the phenotypic values of these
six quantitative traits in the 208 soybean MDP lines followed a Gaussian distribution. The SI was
relatively well distributed, whereas DF and MD had dynamic distributions. Substantial variation was
observed in agronomic traits between mutants and wild types of the 208 MDP lines (Figure 3, Figure S1).
Compared with their wild types, P- and DB-derived mutants possessed a wider distribution of PH and
NN phenotypes, whereas the phenotypes of the six quantitative traits of the HK-derived mutants were
only slightly changed. DB-derived mutants, in particular, had mostly increased values of agronomic
traits, such as DF, SI, PH, NN, and RN, compared with the wild type. In contrast, HK-derived mutants
tended to have reduced values relative to the wild type, albeit only very slightly smaller (Figure 3).
With regards to the five qualitative traits, altered phenotypes, such as changes in growth type and
color-related traits, were confirmed in the MDP lines (Figure S1). The seven wild-type plants exhibited
determinate growth, whereas 46 P-, DB-, and DP-derived mutants were indeterminate. In addition,
changes were observed in color-related traits, including FC, SCC, SHC, and SA. These results indicate
that MDP lines were successfully constructed through multiplex genetic and phenotypic mutation
induced by gamma irradiation. In addition, we calculated the pairwise correlation coefficients of the 11
agronomic traits in the 208 soybean MDP lines. The strongest positive correlations were between PH
and NN (0.912*), GT and NN (0.824*), and GT and PH (0.749*), while the most negative correlations
were those between MD and SI (−0.512*) and between SI and NN (−0.357*) (Table 3).

Table 2. Summary of quantitative trait values in 208 soybean MDP lines.

Agronomic Traits Morphological Traits

Values
Days of Maturity Node Plant Ramification
Seed Index (g)
Flowering Days Number (ea) Height (cm) Number (ea)
Min 42 112 7.9 8.4 23.0 2.6
Mean 52.83 133.58 18.06 13.40 42.73 4.96
Max 64 150 28.8 24.6 92.2 8.8
Line No.
S87, S88,
Min S6 S13 S14 S14 S81
S149
Max S138 S8 S7 S78 S76 S12
Min, minimum; max, maximum; Line No., see Supplementary Table S1. Data were investigated in 2019 at the
KAERI research field, Jeongup, Korea. Traits are all means of five biological replicates.

Table 3. Matrix of correlation coefficients between 11 agronomic traits in 208 soybean MDP lines.

DF MD GT FC SCC SHC SI SA PH NN RN
DF (days of flowering) – 0.419 * 0.266 * 0.436 * 0.038 0.060 −0.327 * 0.435 * 0.272 * 0.323 * 0.248 *
MD (Maturity days) – 0.269 * 0.369 * 0.042 0.034 −0.512 * 0.491 * 0.407 * 0.381 * 0.224 *
GT (Growth type) – 0.031 0.129 * 0.237 * 0.308 * 0.117 0.749 * 0.824 * 0.101
FC (Flower color) – 0.301 * 0.280 * 0.369 * 0.685 * 0.058 0.003 0.099
SCC (Seed coat color) – 0.354 * 0.071 0.243 * 0.166 * 0.134 * 0.068
SHC (Seed hilum color) – 0.005 0.197 * 0.241 * 0.260 * 0.124 *
SI (Seed index) – 0.554 * −0.295 * −0.357 * −0.211 *
SA (Stem anthocyanin) – 0.027 0.103 0.009
PH (Plant height) – 0.912 * 0.177 *
NN (Node number) – 0.202 *
RN (Ramification
–
number)
* Significant at the 0.05 probability level.
Agronomy 2020, 10, 253 6 of 16
Agronomy 2020, 10, 253 7 of 20

Figure 2. Distribution of agronomic traits among 208 soybean MDP lines. Data are presented for (a)
Figure 2. Distribution of agronomic traits among 208 soybean MDP lines. Data are presented for (a) six
six quantitative traits (DF, MD, SI, PH, NN, and RN) with gaussian fitting curve and (b) five
quantitative traits (DF, MD, SI, PH, NN, and RN) with gaussian fitting curve and (b) five qualitative
qualitative traits (GT, FC, SCC, SHC, and SA).
traits (GT, FC, SCC, SHC, and SA).
Agronomy 2020, 10, 253 7 of 16
Agronomy 2020, 10, 253 8 of 20

Figure 3. Changes in the phenotypes of six quantitative traits in 208 MDP lines. The box plots of
Figure 3. Changes in the phenotypes of six quantitative traits in 208 MDP lines. The box plots of
phenotypic distributions in seven MDP lines and their wild types are shown. The data shown are the
phenotypic distributions in seven MDP lines and their wild types are shown. The data shown are the
mean values of individual mutants (gray) and wild types (red).
mean values of individual mutants (gray) and wild types (red).

3.2.
3.2. TRAPMarker
TRAP MarkerPolymorphism
Polymorphism

AAsummary
summaryofofthetheTRAP
TRAP markers
markers produced
produced byby 16
16 primer
primercombinations
combinations(four fixed
(four forward
fixed forward
primers in combination with arbitrary reverse primers) across all 208 soybean mutants is
primers in combination with arbitrary reverse primers) across all 208 soybean mutants is given given in in
Table 4. Sixteen primer combinations amplified a total of 551 fragments. The number of amplified
Table 4. Sixteen primer combinations amplified a total of 551 fragments. The number of amplified
fragments ranged from 25 (for primers MIR157B + Ga5) to 45 (for primers B14G14B + Ga5). A total of
fragments ranged from 25 (for primers MIR157B + Ga5) to 45 (for primers B14G14B + Ga5). A total of
551 amplicons were scored, of which 222 (40.29%) were monomorphic alleles and 329 (59.71%) were
551 amplicons were scored, of which 222 (40.29%) were monomorphic alleles and 329 (59.71%) were
Agronomy 2020, 10, 253 8 of 16

polymorphic. An average of 34.44 amplicons, 20.56 polymorphic, were scored per primer combination.
The highest (84.00%) and lowest (32.35%) polymorphism levels were obtained with primers MIR157B
+ Ga5 and B14G14B + Ga3, respectively. PIC varied among the primer combinations, ranging from 0.07
(B14G14B + Sa12) to 0.23 (MIR157B + Sa4), with a mean value of 0.15.

Table 4. Summary of polymorphism of 16 TRAP marker sets in 208 soybean MDP lines.

Primer Total Number of Polymorphic

Polymorphism (%) PIC
Combination Fragments Fragments
B14G14B + Sa4 38 23 60.53 0.15
B14G14B + Sa12 38 17 44.74 0.07
B14G14B + Ga3 34 11 32.35 0.08
B14G14B + Ga5 45 29 64.44 0.16
MIR156A + Sa4 39 31 79.49 0.16
MIR156A + Sa12 37 22 59.46 0.16
MIR156A + Ga3 35 17 48.57 0.12
MIR156A + Ga5 35 25 71.43 0.20
MIR157B + Sa4 31 23 60.53 0.23
MIR157B + Sa12 31 19 61.29 0.16
MIR157B + Ga3 30 17 56.67 0.14
MIR157B + Ga5 25 21 84.00 0.21
MIR159A + Sa4 36 22 58.33 0.18
MIR159A + Sa12 37 18 48.65 0.10
MIR159A + Ga3 29 15 51.72 0.16
MIR159A + Ga5 31 20 64.52 0.16
Total 551 329 – –
Average 34.44 20.56 59.71 0.15

3.3. Genetic Relationships and Population Structure of the 208 MDP Lines
A dendrogram was constructed to clarify genetic relationships of the 208 MDP lines. At a genetic
distance of 0.097, the seven wild-type cultivars and their mutants could be divided into four major
groups (Figure 4). Group I included five mutants with their wild types KAS360-22 and 94seori. Group
II comprised 22 mutants originating from BS and P. Group III was made up of two subgroups: III-a,
which mainly contained DP mutants and their wild type DP as well as some HK mutants, and III-b,
which mainly included HK mutants and HK with a few DP mutants. Group IV was distinct from
the other three groups and consisted of all 64 DB mutants with DB and DP mutants. We performed
a population structure analysis with a predefined number of sub-populations (K) ranging from 2 to
15. The optimal K was determined using an ad-hoc statistic (∆K), which was based on the rate of
change in the log probability of the data between successive K-values (Figure S2). According to the
analysis, the optimal K value was 4, which corresponded to a division of the genetic composition into
four groups (Figure 4). Each accession was assigned to single or multiple membership depending on
whether its genotype indicated admixture. The result of this analysis was consistent with the topology
of the dendrogram.
Agronomy 2020, 10, 253 9 of 16

Agronomy 2020, 10, 253 10 of 19

Figure 4. Dendrogram revealed by unweighted pair group method with arithmetic mean (UPGMA)
Figure 4. Dendrogram revealed by unweighted pair group method with arithmetic mean (UPGMA)
cluster analysis and the population structure of 208 soybean MDP lines based on TRAP markers.
cluster analysis and the population structure of 208 soybean MDP lines based on TRAP markers.
*Indicated original cultivars.
* Indicated original cultivars.
Agronomy 2020, 10, 253 10 of 16

3.4. AMOVA
An AMOVA of the 208 MDP lines based on TRAP markers was performed to analyze the
distribution of inter- (among) and intra- (within) mutant population genetic diversity. According to
the AMOVA, the estimated inter-mutant population variance was 11.320 (20.6%), while approximately
79.4% of the variation in all variance positions was attributed to intra-mutant population variance.
These results indicate that the majority of the variance was intra-mutant populations. However,
notwithstanding the estimation of variance in each intra-mutant population, the variation of
intra-mutant population was mostly lower than inter-mutant population. The highest percentage
of observed intra-mutant population genetic variation was that of DB populations (26.1%) and the
lowest was intra KAS360-22 populations (0.4%). We also examined genetic differentiation between
soybean MDP lines using Fst data estimated from pairwise comparisons. Fst values varied from 0.065
(KAS360-22 and 94seori) to 0.351 (HK and 94seori), with an average of 0.248 (Table 5).

Table 5. Analysis of molecular variance results and pairwise Fst values estimated from 208 soybean
MDP lines.

Source Est. Var. Percentage of variation (%)

Inter-mutant pops 11.320 20.6
Intra 94Seori pops 0.820 1.5
Intra KAS360-22 pops 0.231 0.4
Intra BangSa pops 0.866 1.6
Intra PalDal pops 2.765 5.0
Intra DanBaek pops 14.324 26.1
Intra DaePung pops 14.252 26.0
Intra HwangKeum pops 10.342 18.8
Control 94Seori KAS360-22 BangSa PalDal DanBaek DaePung HwangKeum
94Seori –
KAS360-22 0.065 –
BangSa 0.273 0.243 –
PalDal 0.312 0.270 0.285 –
DanBaek 0.239 0.229 0.222 0.268 –
DaePung 0.276 0.245 0.258 0.226 0.129 –
HwangKeum 0.351 0.343 0.322 0.270 0.254 0.126 –
Significance (p < 0.001) was determined based on 1000 iterations.

3.5. Association Analysis

Associations between 329 polymorphic fragments (16 combinations of TRAP markers) and 11
phenotypic traits of 208 soybean MDP lines were analyzed by three methods: (1) SFA; (2) a structure
association analysis using a general linear model where population membership served as covariates
(Q GLM); and (3) a composite approach in which the average relationship was estimated by kinship
(K) and implemented under a mixed linear model (Q + K MLM). The average level of significance of
each marker–trait association was based on the three different analyses are shown in Table 6. A total of
27 significant (p < 0.001) marker–trait associations (SMTAs) were detected using the three methods.
The 157B + Sa4 primer combination was significantly associated with four phenotypic traits (GT, SA,
PH, and NN), while 156A + Sa12, 156A + Sa4, 157B + Ga5, 157B + Ga3, 159A + Sa12, 159A + Ga5, and
159A + Ga3 were associated with one trait each: FC, PH, FC, PH, SHC, SI, and RN, respectively. The
lowest calculated P-value using SFA was for the association of 157B + Sa12_6 with the FC trait (p = 1.44
× 10−12 , R2 = 0.216). Under the Q GLM model, the lowest P-value of a SMTA was that of B14 + Sa4_18
with PH (P = 2.46 × 10−8 , R2 = 0.119). Under the Q+K MLM model, the lowest SMTA P-value was
observed for the association of B14+Sa4_18 with PH (P = 9.81 × 10−7 , R2 = 0.107) (Table 6). Among
the three different analytical approaches (SFA, Q GLM, and Q + K MLM), the highest total number of
SMTAs (626) was detected using SFA, followed by the Q GLM approach (178). The lowest number of
SMTAs (143) was detected using the Q+K MLM approach; this number corresponded to 22.8% and
80.3% of the total number of SMTAs detected with SFA and Q GLM, respectively (Table S2). Six SMTAs
Agronomy 2020, 10, 253 11 of 16

at p < 0.0001—two for GT (156A + Ga5_16 and 157B + Sa4_4), one for FC (157B + Sa12_6), one for SCC
(157B + Sa12_20), one for PH (B14 + Sa4_18), and one for NN (B14 + Sa4_18)—were revealed by all
three approaches when kinship and/or population structure was considered in this collection.

Table 6. Selection of 27 significant marker–trait associations (SMTA) markers based on three association
analysis approaches.

Q+K Average
Trait Marker SFA a R2 Q GLM b R2 R2
MLM c p-value
Maturity days B14 + Ga5_28 ** 0.073 ** 0.044 * 0.044 *
156A + Ga5_16 ** 0.116 ** 0.101 ** 0.111 **
Growth type
157B + Sa4_4 ** 0.125 ** 0.102 ** 0.102 **
157B + Sa12_6 ** 0.216 ** 0.074 ** 0.072 **
Flower color 156A + Sa12_17 ** 0.159 * 0.057 * 0.057 *
157B + Ga5_9 ** 0.125 * 0.046 * 0.046 *
Seed coat color 157B + Sa12_20 ** 0.083 ** 0.083 ** 0.086 **
159A + Sa12_28 * 0.055 * 0.058 * 0.063 *
Seed hilum color 159A + Sa4_33 ** 0.111 * 0.058 * 0.057 *
157B + Sa12_20 * 0.066 * 0.053 * 0.056 *
156A + Ga3_7 ** 0.082 ** 0.054 * 0.054 **
159A + Sa4_32 ** 0.096 * 0.050 * 0.050 *
Seed index
156A + Ga3_1 * 0.065 * 0.046 * 0.046 *
159A + Ga5_2 ** 0.139 * 0.044 * 0.044 *
Stem
157B + Sa4_7 ** 0.079 ** 0.047 * 0.044 **
anthocyanin
B14 + Sa4_18 ** 0.152 ** 0.119 ** 0.107 **
B14 + Ga5_30 ** 0.173 ** 0.069 * 0.059 **
157B + Ga3_10 ** 0.135 ** 0.062 * 0.052 *
Plant height
157B + Sa4_4 ** 0.072 * 0.053 * 0.051 *
156A + Sa4_6 * 0.056 * 0.048 * 0.049 *
156A + Ga5_13 ** 0.100 ** 0.063 * 0.049 *
B14 + Sa4_18 ** 0.121 ** 0.097 ** 0.087 **
B14 + Ga5_30 ** 0.162 ** 0.078 * 0.066 **
Node number
157B + Sa4_4 ** 0.083 * 0.062 * 0.055 *
156A + Ga5_16 ** 0.074 * 0.046 * 0.054 *
Ramification 159A + Ga3_24 * 0.058 * 0.054 * 0.061 *
number 159A + Sa4_32 ** 0.080 ** 0.072 * 0.058 *
a SFA: single factor analysis of variance. b Q GLM: general linear model using a Q population structure matrix. c Q

+ K MLM: mixed linear model using Q population structure and K kinship matrixes. * p ≤ 0.001, ** p ≤ 0.0001.

4. Discussion
In this study, we constructed an MDP from populations of 1695 gamma-irradiated mutants in two
selection phases over M1 to M12 generations; first, in the M5 generation, we selected 523 mutant lines
exhibiting at least 30% superior agricultural characteristics, and, second, we eliminated redundant
morphological phenotypes in the M12 generation (Figure 1). Finally, we constructed 208 MDP lines
and investigated 11 agronomic traits. Our collection strategy for selecting MDP lines differed in some
respects from the general core-collection method. With the latter approach, a core collection assembled
from an existing collection is chosen to represent the genetic and phenotypic diversity of the larger
collection without overlapping phenotypes [30]. Such an approach has become accepted as an efficient
tool for improving the conservation of many crops [31,32]. In our study, we similarly eliminated
overlapping phenotypes from our collected MDP lines in the second selection phase, but we considered
specific changed agronomic characteristics of individual mutants rather than their representation of
the original populations.
Our examination of agronomic traits in the MDP lines revealed a variety of DF, MD, GT, FC, SA,
PH, NN, and RN phenotypes as well as those related to seed traits, such as SCC, SHC, and SI (Table 2,
Figure 2, Table S1). We also observed changes in phenotypes between MDP lines and their wild types
(Figure 3, Table S3, Figure S1). The FAO/IAEA mutant variety database (MVD, http://mvd.iaea.org)
Agronomy 2020, 10, 253 12 of 16

includes 174 publicly released soybean mutants. These mutants have various desirable agronomical
and biochemical characteristics, such as an improved maturity date, yield, protein content, fatty acid
content, and changed seed/stem color, with approximately 62% of released mutants mainly selected for
their altered maturity dates and yields. In our phenotypic evaluation of the 208 MDP lines, we detected
a wider variety of changes to the quantitative traits, including SI, PH, NN, and RN (Figure 3), as well
as to the qualitative traits, such as FC, SCC, and SHC (Figure S1). According to our previous study,
in addition, some of DB- and DP-derived mutants in the MDP lines had changed compositions of fatty
acids, including linolenic acid and oleic acid [33]. Given all of these results, our MDP lines may be
useful resources as a genetic diversity pool for soybean breeding.
To investigate genetic relationships among the 208 MDP lines, we evaluated DNA polymorphism
patterns in these lines using TRAP markers. In the rapid, efficient PCR-based TRAP marker
system, expressed sequence tag database information and bioinformatics tools are used to generate
polymorphic markers around targeted candidate gene sequences. Previous studies of lettuce (Lactuca
sativa) [34], sugarcane (Saccharum officinarum) [35], spinach (Spinacia oleracea) [29], geranium (Pelargonium
inquinans) [36], sunflower (Helianthus annuus) [37], and faba beans (Vicia faba) [16] have demonstrated
that TRAP markers are useful for assessing genetic diversity. Using this system in the present study,
we PCR-amplified 551 fragments with 16 primer combinations and observed considerable variation
in the percentage of polymorphic amplicons among primer pairs—from 32.35% to 84.00% (Table 4).
In a study of faba beans, Kwon et al. [16] obtained 221 amplified fragments with 12 TRAP primer
combinations and observed an average polymorphism rate of 55.2%. In the present study, we observed
a polymorphism level of 59.7% among 551 amplified fragments. In contrast, a study of sugarcane
detected a polymorphism rate of 74% from 925 amplified fragments [17], a level much higher than
in the soybeans (Glycine max) and the faba beans. Compared with the results of previous studies of
soybeans based on ISSR and RAPD techniques [38,39], the use of the TRAP system yielded more DNA
fragments per primer combination. A previous AFLP analysis generated an average of 40 to 50 DNA
fragments per primer pair [40,41], similar to the outcome of our TRAP analysis. The present results
demonstrate that the TRAP marker system is a simple yet powerful technique for estimating soybean
genetic diversity.
To reveal relationships among the 208 MDP lines, we constructed a UPGMA-based dendrogram
using the TRAP marker data. On the basis of genetic distances, the 208 MDP lines clustered into four
groups. An analysis of the population structure based on an ad-hoc statistic (∆K) likewise divided
the MDP lines into four groups. These results indicate that four genotype-based sub-populations are
present in the 208 MDP lines (Figure 4) that largely correspond to their wild-type cultivars. As denoted
by different colors, the main membership composition of the four groups and their subgroups is as
follows: Group I including two wild types (94seori, and KAS360-22) possessed 52% red and 46% blue;
Group II including BS and P was 80% blue; Group III-a including DP was 88% yellow; Group III-b
including HK was 71% green; and Group IV including DB was 90% red. In a previous genetic diversity
analysis based on 20 SSR markers, 91 Korean soybean cultivars were divided into seven groups at a
genetic distance of 0.81. In that study, HK and P were clearly separated, but three cultivars (BS, DB,
and DP) grouped together [42]. Using TRAP markers in the present study, we were able to better
resolve groups of wild-type cultivars. In addition, we performed an AMOVA to separate the total
molecular variance of the mutants into inter- and intra-population components (Table 5) and assessed
their significance using permutational testing procedures. Overall, based on the dendrogram and
population structure, 201 mutant lines grouped with their wild type except 29 (14%) mutant lines,
including 22 DP- and 7 HK-mutants. Nevertheless, these mutant lines also possessed their genetic
membership according to population structure. In AMOVA, all intra-mutant population also showed
lower variation than inter-mutant population except for two populations, DB- and DP-, since DB- and
DP- had most large mutant lines, 64 and 60, respectively. A similar result was described by Lee et
al. [20]. Each of the ten wild types was clustered with their M1 generation mutants by gamma radiation
in faba bean. However, the genetic variation of the mutants is not much higher than among cultivars
Agronomy 2020, 10, 253 13 of 16

or accessions. Although TRAP markers have most commonly been used for genetic mapping and
dendrogram studies, they have also recently been applied to detect DNA mutations. Because of their
many advantages, including simplicity, reliability, moderate throughput, and ease of sequencing of
selected bands, TRAP markers have been used widely in plants. For example, the TRAP system has
been used to study genetic variability induced by gamma ray treatments in sugarcane [43] and sorghum
(Sorghum bicolor) [19]. Lee et al. [20] recently exploited a TRAP marker to estimate the frequency of
mutations induced by gamma rays in an M1 generation of faba bean. The 242 amplified fragments
obtained using eight primer combinations had an average polymorphism rate of 66.7%, which is higher
than the percentage in our study because they used early generation. TRAP markers have several
advantages over other types of markers: they are easy to use (like RAPDs), high in polymorphisms
(like AFLPs), and their primers can be readily designed from known sequences of putative genes [44].
In association mapping, false discoveries are a major concern and can be partially attributed to
spurious associations caused by population structure and unequal relatedness among individuals. Two
major approaches, namely, GLM and MLM, are used to study marker–trait associations. The number of
SMTAs detected by GLM is generally much higher than that revealed by MLM [45]. GLM-based studies
of marker–trait associations consider only the Q matrix generated during the study of population
structure. In contrast, MLM simultaneously accounts for both population structure and kinship
(genetic relatedness among individuals) and is hence more reliable. In the present study, the GLM
method (Q) uncovered 178 SMTAs between the 11 phenotypic traits and 27 TRAP markers. Using
the MLM method (Q + K), 143 SMTAs involving 27 TRAP markers were identified (Table 6, Table S2).
These results confirm a previous observation that the number of SMTAs estimated with GLM is higher
than that uncovered with MLM [46,47]. Most interestingly, the three approaches considering kinship
and/or population structure in the MDP collection in this study revealed six SMTAs at p < 0.0001 in all
approach methods. These six SMTAs involved five agronomic traits: GT (2), FC (1), SCC (1), PH (1),
and NN (1).

5. Conclusions
In this study, we successfully constructed soybean MDP lines and compared their agronomic traits.
We also performed the first-ever study of genetic diversity and relationships using the TRAP marker
system in soybean. To examine MDP genetic diversity and relationships, we performed dendrogram,
population structure, and molecular variance analyses based on their TRAP genotypes. Finally, we
uncovered six SMTAs (p < 0.0001) involved with TRAP genotypes and agronomic traits using three
association mapping methods (SFA, Q GLM, and Q + K MLM). Our results can serve as a foundation
for future research on genotype–phenotype interactions in large mutant populations.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/2/253/s1,

Figure S1: Changes in five qualitative-trait phenotypes of 208 MDP lines; Figure S2: ∆K values for different
numbers of populations (K) assumed in the STRUCTURE analysis, Table S1: Agronomic characteristics of the
208 soybean MDP lines used in this study; Table S2: Comparison of the number of SMTAs (p < 0.01) based on
three analytical approaches (SFA, Q GLM, and Q + K MLM); Table S3: Comparison of phenotypes and genotypes
among seven original cultivars.
Author Contributions: Conceptualization, D.-G.K., J.I.L., and S.-J.K.; methodology, D.-G.K., J.I.L., and S.-J.K.;
software, D.-G.K. M.-K.L.; validation, D.-G.K., J.-B.K., C.-H.B., and S.-J.K.; investigation, D.-G.K., J.M.K.;
resources, D.-G.K., M.J.H.; data curation, D.-G.K., M.-K.L., N.N.H.; writing—original draft preparation, D.-G.K.;
writing—review and editing, J.I.L., S.-J.K.; visualization, M.J.H., J.-B.K.; supervision, C.-H.B., S.-J.K.; funding
acquisition, J.-B.K., S.-J.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Radiation Technology R&D Program (NRF-2017M2A2A6A05018538)
through the National Research Foundation of Korea funded by the Ministry of Science and ICT.
Acknowledgments: We thank Edanz Group (www.edanzediting.com/ac) for editing the English text of a draft of
this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Agronomy 2020, 10, 253 14 of 16

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