G C A T

T A C G
G C A T
genes
Article
Uncovering Phenotypic Diversity and DArTseq
Marker Loci Associated with Antioxidant Activity in
Common Bean
Muhammad Azhar Nadeem 1 , Müttalip Gündoğdu 2 , Sezai Ercişli 3 , Tolga Karaköy 4 ,
Onur Saracoğlu 5 , Ephrem Habyarimana 6 , Xiao Lin 7 , Ruştu Hatipoğlu 8 ,
Muhammad Amjad Nawaz 9 , Muhammad Sameeullah 1 , Fiaz Ahmad 10 , Bok-Mi Jung 11 ,
Gyuhwa Chung 12, * and Faheem Shehzad Baloch 1, *
1 Department of Field Crops, Faculty of Agricultural and Natural Science, Bolu Abant Izzet Baysal University,
Bolu 14030, Turkey; [email protected] (M.A.N.); [email protected] (M.S.)
2 Department of Horticulture, Faculty of Agriculture and Natural Sciences, Bolu Abant Izzet Baysal University,
Bolu 14000, Turkey; [email protected]
3 Department of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum 25240, Turkey;
[email protected]
4 Faculty of Agricultural Sciences and Technologies, Sivas University of Science and Technology,
Sivas 58140, Turkey; [email protected]
5 Department of Horticulture, Faculty of Agricultural, Tokat Gaziosmanpasa University, Tokat 60010, Turkey;
[email protected]
6 CREA Research Center for Cereal and Industrial Crops, 40128 Bologna, Italy;
[email protected]
7 School of life Sciences, The Chinese university of Hong Kong, Shatin, 999077, N.T., Hong Kong, China;
[email protected]
8 Department of Field Crops, Faculty of Agricultural, University of Cukurova, Adana 1000, Turkey;
[email protected]
9 Education and Scientific Center of Nanotechnology, Far Eastern Federal University,
Vladivostok 690950, Russian; [email protected]
10 Jamil-ur-Rahman Center for Genome Research, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan; [email protected]
11 Division of Food and Nutrition, Chonnam National University, Gwangju 61186, Korea; [email protected]
12 Department of Biotechnology, Chonnam National University, Chonnam 59626, Korea
* Correspondence: [email protected] (G.C.); [email protected] (F.S.B.);
Tel.: +90-545-540-4239 (F.S.B.)




Received: 11 November 2019; Accepted: 24 December 2019; Published: 28 December 2019


Abstract: Antioxidants play an important role in animal and plant life owing to their involvement
in complex metabolic and signaling mechanisms, hence uncovering the genetic basis associated
with antioxidant activity is very important for the development of improved varieties. Here, a total
of 182 common bean (Phaseolus vulgaris) landraces and six commercial cultivars collected from
19 provinces of Turkey were evaluated for seed antioxidant activity under four environments and
two locations. Antioxidant activity was measured using ABTS radical scavenging capacity and mean
antioxidant activity in common bean landraces was 20.03 µmol TE/g. Analysis of variance reflected
that genotype by environment interaction was statistically non-significant and heritability analysis
showed higher heritability of antioxidant activity. Variations in seed color were observed, and a
higher antioxidant activity was present in seeds having colored seed as compared to those having
white seeds. A negative correlation was found between white-colored seeds and antioxidant activity.
A total of 7900 DArTseq markers were used to explore the population structure that grouped the
studied germplasm into two sub-populations on the basis of their geographical origins and trolox
equivalent antioxidant capacity contents. Mean linkage disequilibrium (LD) was 54%, and mean

Genes 2020, 11, 36; doi:10.3390/genes11010036 www.mdpi.com/journal/genes

Genes 2020, 11, 36 2 of 17

LD decay was 1.15 Mb. Mixed linear model i.e., the Q + K model demonstrated that four DArTseq
markers had significant association (p < 0.01) for antioxidant activity. Three of these markers were
present on chromosome Pv07, while the fourth marker was located on chromosome Pv03. Among
the identified markers, DArT-3369938 marker showed maximum (14.61%) variation. A total of four
putative candidate genes were predicted from sequences reflecting homology to identified DArTseq
markers. This is a pioneering study involving the identification of association for antioxidant activity
in common bean seeds. We envisage that this study will be very helpful for global common bean
breeding community in order to develop cultivars with higher antioxidant activity.

Keywords: Phaseolus vulgaris; germplasm characterization; GWAS; genetic basis; genotype by

environment interaction; mixed linear model

1. Introduction
The world is facing a great threat of climate change, and extreme events throughout the world
are being observed. Besides climate change, rapid growth in the world population is a global issue,
and concerns have been raised to produce a sufficient quantity of food in terms of calories and
nutrients [1]. A report issued by the food and agriculture organization (FAO) [2] stated that the daily
average per capita calories available to the world population were 2789 kcal in the year 2000, but it is
estimated that 3130 kcal will be required on a daily basis during the year 2050. In this context, grain
legumes are key commodities having great potential to mitigate world food shortages and provide
well balanced and cheap food [3].
The common bean (Phaseolus vulgaris L.) is one of the most important proteinaceous crops of Latin
America. The common bean serves a source of food for more than 300 million people [4]. Although
the common bean is an underrated crop, the presence of higher quantity of proteins, carbohydrates,
vitamins, and minerals makes it a rational food of choice, particularly for poor people all around the
world [5]. Global common bean production was 23 million tons in 2010 and 26 million tons in 2016 [6].
The common bean reflects a great level of genotypic and phenotypic diversity, and domestication of
this crop resulted in two distinct gene pools—the Mesoamerican gene pool from Central America and
the Andean gene pool from the Andes Mountains in South America [7]. Turkey is not the center of
origin of the common bean, and it is known that this crop was introduced to Turkey from Europe in the
17th century [8]. However, hundreds of landraces, especially those having bigger seed size due to local
consumer preference, are found in Turkey. The common bean is a very popular food commodity for
Turkish people and is an important part of their daily diet [9]. An increase in the yield of the common
bean has been observed in Turkey during the last two decades; 212,758 tons were produced in the year
2010, and 651,094 tons in the year 2016 [6], making up a share of about 2.75% of the world production.
To date, 200 fresh bean and 39 dry bean cultivars have been registered in Turkey (Variety Registration
and Seed Certification Center; www.tarimorman.gov.tr). According to Ozturk et al. [10], the observed
gradual rise in common bean production is due to the development of improved cultivars reflecting
better adaptation to environmental stresses.
Advancements in the molecular markers and sequencing technologies have boosted breeding
activities [11]. Among major techniques, genome-wide association studies (GWAS) and quantitative
trait loci (QTL) mapping are the two main approaches used to elucidate the genetic basis for traits
of interest. QTL mapping or bi-parental mapping is mainly used for the investigation of QTLs
associated with traits of interest. However, in addition to its advantages, QTL mapping also has
several drawbacks, like low recombination, being time-consuming, and the population specificity of the
identified QTLs [11]. GWAS overcomes all the drawbacks of QTL mapping, and the identified markers
through this approach can be used for any population [12]. GWAS has been successfully carried out
for the common bean in order to elucidate marker-trait association for agronomic traits [13], seed
Genes 2020, 11, 36 3 of 17

weight [14], disease resistance [15], cooking properties [16], anthracnose resistance [17], and drought
tolerance [18]. These studies resulted in the identification of some candidate genes that can be helpful
in the development of new genetic stocks for breeding perspectives in the common bean.
Antioxidants are an important class of chemicals present in our food and play an essential role
in reducing the oxidative stress of the physiological system [19]. Antioxidants play a significant
role against various disorders like cancer, aging, cardiovascular disease, cataracts, immune system
decline, and brain dysfunction. Antioxidants minimize the chances of these diseases by preventing the
production of free radicals [20]. Sudhir et al. [21] stated that higher antioxidants in food minimize the
incidence of cardio and cerebrovascular diseases. Oxygen under various conditions may have serious
deleterious effects on the body [22]. Oxygen is a highly reactive atom with the ability to become part of
potentially damaging molecules known as “free radicals.” These free radicals have the ability to attack
healthy body cells, leading to various diseases like cardiovascular disease, cancer, brain dysfunction,
and a decline in the immune system [20–23]. Antioxidants can electrons to stabilize these free radicals
and prevent their detrimental effects. Antioxidants protect the body from both endogenous and
exogenous molecules [24]. Sikora et al. [25] stated that food containing a higher concentration of
antioxidants can significantly affect the increase of the reactive antioxidant potential of the organism
and decrease the risk of some diseases caused by the production of free radicals. Antioxidants can also
be endogenous or exogenous; exogenous antioxidants enter the body when we eat antioxidant-rich
food, while endogenous antioxidants are solely produced by the body. Exogenous antioxidants are
found in plant and animal sources, and those of plant origin are of great interest because of their
presence in relatively higher quantities [26,27].
The role of antioxidants against various stress conditions in plants has been well documented [28].
Plant species containing a higher level of antioxidant contents, either constitutive or induced, reflect
a higher resistance to environmental stresses [29]. According to Silvana et al. [30], the extent of
oxidative damage to plant cells when exposed to abiotic stresses is mainly governed by the capacity
of their antioxidant defense system. As antioxidant activity is a key trait, its manipulation can be
a relevant target for common bean breeding community to develop new varieties having improved
antioxidant activity. This in turn will be helpful for improving human health. GWAS has been used
previously for the identification of genes associated with antioxidant activity and total flavonoid
contents in barley [31] and rice [32] in order to investigate polyphenol contents and antioxidant activity.
Despite a good understanding of the role of antioxidants for human health, the genetic basis for
antioxidant activity has not been elucidated for most of crops. The common bean is a good source of
antioxidants [29], and Cardador-Martiänez et al. [33] stated that bean extract from the hull contains
a significant level of antioxidants. Weidner et al. [34] explored the antioxidant levels in different colored
common bean genotypes and observed a good amount of antioxidant capacity. The common bean
serves as a source of food for a large population of the world, so variations in the common bean for
antioxidant activities can be used to develop cultivars with better antioxidant content. Therefore, an
effort is made through this study to explore the variations for antioxidant activity and antioxidant
activity–associated markers in Turkish common bean germplasm.

2. Materials and Methods

2.1. Plant Material

A total of 182 common bean landraces and six commercial cultivars (Akman, Goynuk, Karacaşehir,
Onceler, Goksun, and Akdag) were used as plant material in this study. Germplasm was collected
from 19 geographical regions of Turkey (Table S1; Figure 1) and maintained at Bolu Abant Izzet Baysal
University (BAIBU). Commercial cultivars used in this study were developed through a single plant
selection and have been used as standard cultivars in previous studies [35,36].
Genes 2020, 11, 36 4 of 17
Genes 2020, 11, x FOR PEER REVIEW 4 of 17

Figure 1.
Figure 1. Collection
Collection provinces
provinces of
of Turkish
Turkish common
common bean
bean germplasm.
germplasm.

2.2.
2.2. Field
Field Experiment
Experiment and
and Phenotypic
Phenotypic Evaluation
Evaluation
Field
Field experiments
experiments were were conducted
conducted during
during 2017
2017 and
and 2018
2018 at
at two
two experimental
experimental sites—BAIBU
sites—BAIBU
(40 ◦ 440 45” N/31◦ 370 44” E, altitude; 752 m) and Cumhuriyet University Sivas (39◦ 420 16.69” K/37◦ 010 57.48”
(40°44′45″ N/31°37′44″ E, altitude; 752 m) and Cumhuriyet University Sivas (39°42′16.69″
D, altitude; 1293D,m).
K/37°01′57.48″ The experiment
altitude; 1293 m). wasThe laid out according
experiment was laidto augmented
out according blockto design, whichblock
augmented has
been found
design, to be
which hasmore
been effective,
found to precise,
be moretime-saving, and trustworthy
effective, precise, compared
time-saving, to other experimental
and trustworthy compared
designs
to other [37]. Augmented
experimental block
designs design
[37]. contains
Augmented more
block than contains
design one control
morecultivar
than one that are taken
control as
cultivar
replicated treatments,
that are taken and these
as replicated cultivarsand
treatments, are repeated in eachare
these cultivars block. Thesein
repeated control cultivars
each block. are used
These to
control
standardize the data to calculate adjusted means. Evaluated means are later used for
cultivars are used to standardize the data to calculate adjusted means. Evaluated means are later used various statistical
analyses
for various[37,38]. In thisanalyses
statistical study, seeds were
[37,38]. In planted in elementary
this study, seeds wereplots,
plantedeach
inconsisting
elementary ofplots,
a 2 m each
row
with a 50 cm
consisting of ainter-row and 10
2 m row with a 50cmcmintra-row
inter-rowspacing.
and 10 cm Studied germplasm
intra-row was sown
spacing. Studied accordingwas
germplasm to
augmented block design in eight blocks. Six commercial cultivars (Akman,
sown according to augmented block design in eight blocks. Six commercial cultivars (Akman, Goynuk, Karacaşehir,
Onceler,
Goynuk,Goksun, and Akdag)
Karacaşehir, Onceler, were included
Goksun, andin each block
Akdag) wereas a controlingroup.
included Sowing
each block as awas donegroup.
control on 27
April
Sowing 2017 and
was 16 April
done on 272018,
Aprilrespectivelyat
2017 and 16 the BAIBU,
April 2018, while the trial in
respectivelyat Sivas
the location
BAIBU, was
while thesown
trialon
in
15 May 2017 and 19 May 32018. Di-ammonium phosphate (DAP) (130 kg/ha)
Sivas location was sown on 15 May 2017 and 19 May 32018. Di-ammonium phosphate (DAP) (130 and ammonium sulfate
(51 kg/ha)
kg/ha) andwere used as a source
ammonium sulfate of(51
fertilizer,
kg/ha) while
were standard
used as agriculture
a source ofpractices were
fertilizer, performed
while standardat
both locations. Harvesting was done at the time of 90% pods per plant maturity.
agriculture practices were performed at both locations. Harvesting was done at the time of 90% pods
per plant maturity.
2.3. DNA Extraction and Genotyping for DArTseq Markers
2.3. DNA
DNAExtraction and Genotyping
was extracted for DArTseq Markers
from two-week-old seedlings according to CTAB protocol of Doyle
and Doyle [39] with some modifications [40].
DNA was extracted from two-week-old seedlings Agarose gel electrophoresis
according (0.8%) of
to CTAB protocol was usedand
Doyle to
check the purity of isolated DNA and quantification was performed through DS-11
Doyle [39] with some modifications [40]. Agarose gel electrophoresis (0.8%) was used to check the FX series
spectrophotometer/fluorometer
purity of isolated DNA and (Denovix, Wilmington,
quantification was DE, USA). High-quality
performed DNA was
through DS-11 FX further
series
diluted and a final concentration of 50 ng µL-1 was prepared. For genotyping-by-sequencing
spectrophotometer/fluorometer (Denovix, Wilmington, DE, USA). High-quality DNA was further (GBS),
the prepared
diluted and aDNA
final samples were processed
concentration at Diversity
of 50 ng µL-1 Array Technology
was prepared. Pty, Ltd., Bruce, Australia
For genotyping-by-sequencing (GBS),
(http://www.diversityarrays.com/).
the prepared DNA samples were processed at Diversity Array Technology Pty, Ltd.,described
DArTseq analysis was carried out as previously [9].
Bruce,Australia
(http://www.diversityarrays.com/). DArTseq analysis was carried out as previously described [9].
2.4. Extraction and Determination of Total Antioxidant Activity
Antioxidant
2.4. Extraction andactivity was measured
Determination of Total using ABTS radical
Antioxidant Activityscavenging capacity. For the investigation
of antioxidant activity, TEAC extract was firstly prepared to calculate trolox equivalent antioxidant
Antioxidant activity was measured using ABTS radical scavenging capacity. For the
capacity (TEAC). For this purpose, dry samples were extracted with acetone, water, and acetic acid
investigation of antioxidant activity, TEAC extract was firstly prepared to calculate trolox0 equivalent
solution (70/29.5/0.5), respectively for ten days. For TEAC analysis, 7 mM ABTS (2,2 -Azino-bis
antioxidant capacity (TEAC). For this purpose, dry samples were extracted with acetone, water, and
3-ethylbenzothiazoline-6-sulfonic acid) was mixed with 2.45 mM potassium sulfate for 12–16 h as
acetic acid solution (70/29.5/0.5), respectively for ten days. For TEAC analysis, 7 mM ABTS (2,2′-
suggested by Saracoglu et al. [41]. This solution was then mixed with 20 mM sodium acetate (pH 4.5)
Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) was mixed with 2.45 mM potassium sulfate for 12–
buffer solution. Finally, 30 µL of extract was mixed with 2.97 mL of prepared buffer and incubated for
16 h as suggested by Saracoglu et al. [41]. This solution was then mixed with 20 mM sodium acetate
(pH 4.5) buffer solution. Finally, 30 µL of extract was mixed with 2.97 mL of prepared buffer and
incubated for 10 min and measured on a spectrophotometer at a wavelength of 734 nm to 0.700 ± 0.01.
Genes 2020, 11, 36 5 of 17

10 min and measured on a spectrophotometer at a wavelength of 734 nm to 0.700 ± 0.01. Absorbance

values obtained were calculated with Trolox (10–100 µmol/L) and presented as µmol TE/g.

2.5. Statistical Analysis

2.5.1. Phenotypic Data Analysis

An online software for augmented block design developed by Rathore et al. [42] was used to
derive statistical inferences. The analysis was performed in two steps. First, analysis of variance
(ANOVA) was investigated within the environments, and adjusted means were derived. Second,
these evaluated means were used to run ANOVA across the environments, solving the appropriate
mixed model equation to contemporarily account for both genotype and genotype × environment
interaction effects. The Fligner–Killeen test was also computed before combined analysis to evaluate
the homogeneity of variance. Environmental effects were taken as random, and genotypic effects were
taken fixed in the computed mixed model equation, as suggested by Gomez and Gomez [43]. For the
calculation of heritability, appropriate variance components were derived from the linear mixed model
equation fitted attributing genotype and environment random effects as reported by Habyarimana [44].
The model was fitted with a restricted maximum likelihood using the R software [45]. The boxplot and
frequency distribution of antioxidant activity were investigated using GenStat software [46]. Pearson’s
correlation coefficient among various colored seeds and antioxidant activity was investigated using
the statistical software XLSTAT (www.xlstat.com).

2.5.2. Population Structure and GWAS Analysis

Population structure was carried as described by Nadeem et al. [9]. A mixed linear model (MLM,
Q + K) approach was used to investigate the marker-trait association. Q-metrics (Q) and kinship (K)
were used to correct the population and family structure during association analysis. The kinship
matrix was investigated by following the methodology reported by Bradbury et al. [47] through
TASSEL 5.0.5 (https://tassel.bitbucket.io) software. Linkage disequilibrium (LD) was estimated for all
DArTseq markers with r2 based on allele frequencies [48,49]. Pairwise LD values of polymorphic sites
were plotted on both the X- and Y-axis to generate LD heat map. To calculate LD decay, the pairwise
correlations among linked markers in significant LD (p < 0.001) were used. The threshold for LD decay
was considered below r2 = 0.1. Results of association analysis reflected that p value (marker) shows
whether a marker is associated with a trait, and R2 (marker) defines the proportion of phenotypic
variation accounted for by a significant marker [49,50]. Both Bonferroni and FDR thresholds were
used, while DArTseq markers having FDR and Bonferroni p = 0.01 thresholds were taken significantly
associated with antioxidant activity. Statistically significant markers for antioxidant activity were
visualized through the Manhattan plot in R 3.4.1 statistical software (http://www.r-project.org/) by
using qq-man R Package [51]. Quantile-Quantile (q-q) plot was drawn to visualized important
p-value distributions (expected vs. observed p-values on a –log10 scale) through TASSEL 5.2.50
(https://tassel.bitbucket.io) software.

2.5.3. Putative Candidate Gene Analysis

Sequences of DArTseq markers associated with the antioxidant activity were used to BLAST-search
against common bean genome in Phytozome V.12.1 (http://phytozome.jgi.doe.gov/pz/portal.html),
the legume information system (LIS: https://legumeinfo.org/), and the National Center for Biotechnology
Information (NCBI) (http://www.ncbi.nlm.nih.gov/) databases to detect putative genes homologous to
these sequences.
Genes 2020, 11, 36 6 of 17

3. Results

3.1. Phenotypic Diversity Evaluation

Analysis of variance (ANOVA) was performed for four environments to understand the effects
of genotype, environment, and the interaction of both (GEI) for antioxidant activity in Turkish
Genes 2020, 11, x FOR PEER REVIEW 6 of 17
common bean germplasm (Table 1). Genotypic effects were found to be statistically significant at (p <
0.05; F value 11.9), of
Analysis while GEI(ANOVA)
variance was statistically non-significant
was performed for antioxidants
for four environments activity the
to understand in this study.
effects
Heritability analysis
of genotype, revealed theand
environment, existence of higher
the interaction of heritability
both (GEI) for (0.92) (Table 1).activity
antioxidant Antioxidant activity
in Turkish
common as
was evaluated bean
TEAC germplasm
for each(Table 1). Genotypic
environment effects
for both were found
locations (Table to S2).
be statistically
At BAIBUsignificant
in 2017, the at (p
mean
< 0.05; F value 11.9), while GEI was statistically non-significant for antioxidants
TEAC for common bean landraces was 19.78 ranging 1.8 (Hakkari-65) to 67.2 (Balikesir-19) (Table S3). activity in this study.
At the Heritability
same location analysis revealed
in 2018, 20.25the existence
was the mean of higher
TEAC, heritability
while 1.9(0.92) (Table 1). Antioxidant
(Hakkari-65) activity
and 69.9 (Balikesir-19)
was evaluated as TEAC for each environment for both locations (Table S2). At BAIBU in 2017, the
were the minimum and maximum TEAC, respectively. At the Sivas location in 2017, mean TEAC
mean TEAC for common bean landraces was 19.78 ranging 1.8 (Hakkari-65) to 67.2 (Balikesir-19)
was 19.65, while minimum and maximum TEAC reflected by Hakkari-65 and Bitlis-22 landraces were
(Table S3). At the same location in 2018, 20.25 was the mean TEAC, while 1.9 (Hakkari-65) and 69.9
2.4 and 64.3, respectively.
(Balikesir-19) Mean TEAC
were the minimum was 20.43TEAC,
and maximum at Sivas location during
respectively. 2018,
At the Sivas while in
location minimum
2017,
and maximum
mean TEAC TEACwas reflected
19.65, whilebyminimum
Hakkari-65 andand Bitlis-22
maximum TEAClandraces
reflected were 2.5 and 66.8,
by Hakkari-65 andrespectively.
Bitlis-22
Overalllandraces
mean TEAC were 2.4in Turkish
and 64.3,common bean
respectively. landraces
Mean TEAC waswas 20.43
20.03,atranging from 32.15
Sivas location during(Hakkari-65)
2018, while to
60.48 (Balikesir-19). Mean TEAC in common bean cultivars at Bolu in 2017 and 2018 were
minimum and maximum TEAC reflected by Hakkari-65 and Bitlis-22 landraces were 2.5 and13.9
66.8,and
respectively.(Table
14.3, respectively Overall mean
S3). Mean TEAC
TEACin Turkish
in commoncommon beanbean landraces
cultivars was 20.03,
at Sivas in 2017ranging fromwere
and 2018 32.1515.7
(Hakkari-65)
and 16.3, respectively, to 60.48
while(Balikesir-19).
overall mean Mean
TEACTEAC in common
in common beancultivars
bean cultivarsduring
at Bolu the
in 2017
whole andperiod
2018 of
study was 15.1. Boxplot analysis also confirmed the results of ANOVA by revealing that there2017
were 13.9 and 14.3, respectively (Table S3). Mean TEAC in common bean cultivars at Sivas in are no
and 2018 were 15.7 and 16.3, respectively, while overall mean TEAC in common bean cultivars during
significant environmental effects on the seed antioxidant contents (Figure 2). Frequency distributions
the whole period of study was 15.1. Boxplot analysis also confirmed the results of ANOVA by
for TEAC in common bean are presented in Figure 3, which shows that most of the landraces have
revealing that there are no significant environmental effects on the seed antioxidant contents (Figure
less than 10 µmol TE/g
2). Frequency TEAC. Mean
distributions for TEACantioxidant
in common activity
bean arein presented
the colored seeds and
in Figure white
3, which seeds
shows were
that
30.04 and
most of the landraces have less than 10 µmol TE/g TEAC. Mean antioxidant activity in the coloredand
16.22 µmol TE/g TEAC, respectively. Correlation analysis between antioxidant activity
seed caot colors
seeds and was
white also performed,
seeds were 30.04 which revealed
and 16.22 µmola negative
TE/g TEAC, correlation of antioxidant
respectively. Correlationactivity
analysiswith
white seeds
between (Table 2).
antioxidant activity and seed caot colors was also performed, which revealed a negative
correlation of antioxidant activity with white seeds (Table 2).
Table 1. Summary of analysis of variance and estimates of heritability in Turkish common
Table 1. Summary of analysis of variance and estimates of heritability in Turkish common bean
bean germplasm.
germplasm.
Variables Df Sum Sq Mean Sq F Value Pr (>F)
Variables Df Sum Sq Mean Sq F Value Pr (>F)
LINE
LINE 187
187 199487
199487 1066.8
1066.8 11.937
11.937 <2<2 −16
× ×1010 −16
ENV
ENV 33 115
115 38.2
38.2 0.427
0.427 0.734
0.734
Residuals 561 50133 89.4
Residuals 561 50133 89.4
Heritability 0.92
Heritability 0.92

2. Boxplot
FigureFigure analysis
2. Boxplot analysisofofantioxidant activityin in
antioxidant activity Turkish
Turkish common
common bean bean germplasm
germplasm under
under four
four environments.
environments.
Genes 2020, 11, 36 7 of 17
Genes 2020, 11, x FOR PEER REVIEW 7 of 17

FigureFigure 3. Frequency
3. Frequency distribution
distribution ofof antioxidantactivity
antioxidant activity in
in Turkish
Turkishcommon
commonbean germplasm
bean under
germplasm under
four environments.
four environments.

2. Pearson’s
TableTable correlation
2. Pearson’s coefficient
correlation coefficientbetween
between various seedscolors
various seeds colors and
and antioxidant
antioxidant activity.
activity.

Variables
Variables WhiteWhite
PurplePurpleBrownBrown BeigeBeige Yellow
Yellow Dark
Darkredred Black
Black Antioxidant
Antioxidant
White 1 −0.448 ** −0.328 ** −0.427 ** −0.236 ** −0.253 ** −0.177 −0.422 **
White 1 −0.448 ** −0.328 ** −0.427 ** −0.236 ** −0.253 ** −0.177 −0.422 **
Purple 1 −0.105 −0.137 −0.076 −0.081 −0.057 0.108
Purple 1 −0.105 −0.137 −0.076 −0.081 −0.057 0.108
Brown Brown 1 1 −0.100
−0.100 −0.055
−0.055 −0.059
−0.059 −0.042
−0.042 0.214 ** **
0.214
BeigeBeige 1 1 −0.072 −0.077
−0.072 −0.077 −0.054
−0.054 0.309 ** **
0.309
Yellow
Yellow 1 1 −0.043
−0.043 −0.030
−0.030 −0.014
−0.014
DarkDark
red red 11 −0.032
−0.032 0.060
0.060
BlackBlack 1 0.080
0.080
Antioxidant
Antioxidant 1 1
Statistically significant****(p(p<<0.01).
Statisticallysignificant 0.01).

3.2. Population Structure and LD

3.2. Population Structure and LD
Whole-genome DArTseq profiling of Turkish common bean germplasm resulted in a total of
Whole-genome DArTseq
15,608 DArTseq markers. profiling
This marker of Turkish
dataset was common bean germplasm
filtered to retain resulted
7900 high-quality in awith
markers total of
15,608less
DArTseq
than 5%markers. ThisPIC
missing data, marker
value dataset was
> 0.10, call filtered
rate > 0.60, to
andretain
100%7900 high-quality
reproducibility. Themarkers
Bayesianwith
clustering
less than model data,
5% missing implemented
PIC value in >STRUCTURE
0.10, call rate > 0.60, grouped
software and 100% studied germplasm The
reproducibility. intoBayesian
two
groups; population A (red) containing 117 landraces (59.57%) and population
clustering model implemented in STRUCTURE software grouped studied germplasm into two groups; B (green) 71 landraces
(37.76%)
population A (Figure S1). The 7900
(red) containing 117highly informative
landraces (59.57%)DArTseq markers were
and population used for
B (green) 71 LD and GWAS
landraces (37.76%)
analysis. Based on each chromosome, the number of mapped markers ranged from 931 (Chr. 2) to
(Figure S1). The 7900 highly informative DArTseq markers were used for LD and GWAS analysis.
591 (Chr. 6) (Table 3). The mean distance between markers on whole-genome was 15.54 Mb with a
Based on each chromosome, the number of mapped 2markers ranged from 931 (Chr. 2) to 591 (Chr. 6)
range of 13.15 (Chr 7) to 18.99 Mbps (Chr. 2). Mean r was 0.47, and 54% was the mean significant LD
(Table(Table
3). The mean distance between markers on whole-genome was 15.54 Mb with a range of 13.15
3). Mean LD decay for whole-genome was 1.15 Mbps which ranged from 1.05 Mbps for
(Chr 7) to 18.99
chromosome Mbps
9 to (Chr.
1.3 for2). Mean r2 was
chromosome 0.47, and trend
5. Nonlinear 54% was theLD
line of mean significant
measure LDphysical
r2 vs. the (Table 3).
mapMean
LD decay for whole-genome was 1.15 Mbps which ranged from 1.05 Mbps for chromosome
distance decayed with almost 1.3 Mbs to r value of 0.5 (Figure S2). Linkage disequilibrium measured
2 9 to 1.3 for
chromosome 2
r vs.
r plotted5.
2 vs.Nonlinear trendpairs
p-value between line of DArTseq
LD measure markers the physical
revealed LD decay map distance
on LG6 (Figuredecayed
S3). with
almost 1.3 Mbs to r2 value of 0.5 (Figure S2). Linkage disequilibrium measured r2 plotted vs. p-value
between pairsTableof3. DArTseq
Distributionmarkers
and linkage disequilibrium
revealed of DArTseq
LD decay on LG6markers
(FigureonS3).
different chromosome of
common bean.
 Genes
Genes 2020,
2020, 11,11,
36x FOR PEER REVIEW 88ofof1717

Number of Mean Distance Significant LD LD Decay

Chr.
Table 3. Distribution
r2
Markers and linkage disequilibrium
(Mbs) of DArTseq markers on (%)different chromosome
(Mb) of
1common bean.658 12.61 0.52 53.76 1.2
2 931Number of 18.99
Mean Distance
0.48 56.47
Significant LD LD Decay
1.2
3 Chr. 863 Markers 16.53 r2 0.51 53.61 1.15
(Mbs) (%) (Mb)
4 1 632 658 13.80
12.61 0.520.41 53.7654.58 1.2 1.18
5 2 659 931 16.38
18.99 0.480.39 56.4759.16 1.2 1.3
6 3 591 863 18.48
16.53 0.510.50 53.6155.16 1.15 1.15
7 4 680 632 13.80
13.15 0.410.45 54.5849.55 1.18 1.08
5 659 16.38 0.39 59.16 1.3
8 863 14.47 0.46 55.4 1.15
6 591 18.48 0.50 55.16 1.15
9 7 637 680 17.03
13.15 0.450.59 49.5545.89 1.08 1.05
10 8 599 863 13.86
14.47 0.460.45 55.450.62 1.15 1.1
11 9 787 637 15.68
17.03 0.590.46 45.89 59.8 1.05 1.2
10 7900 599 13.86
15.45 0.450.47 50.62 54.0 1.1 1.15
11 787 15.68 0.46 59.8 1.2
7900 for Antioxidant
3.3. Marker Trait Association 15.45
Activity 0.47 54.0 1.15

MLM (Q + K) model was applied to investigate marker-trait association for antioxidant activity
3.3. Marker Trait Association for Antioxidant Activity
in studied Turkish common bean germplasm. MLM based on Q + K model showed that out of 7900
MLM (Q
DArTseq + K) model
markers, only was
fourapplied
markers to (DArT-3369938,
investigate marker-trait association
DArT-8668387, for antioxidant
DArT-8207664, andactivity
DArT-
in3371498)
studied reflected
Turkish common bean germplasm. MLM based on Q + K model showed that
statistically significant association for antioxidant activity (Table 4; Figure 4). out of 7900
The
DArTseq markers,
phenotypic only four
variation markers(R(DArT-3369938,
percentage 2 DArT-8668387,
) reflected by each marker ranged DArT-8207664, and DArT-3371498)
between 0.094% to 0.156% for
reflected statistically
DArT-3371498 and significant association
DArT-3369938, for antioxidant
respectively. activity
The p-value of(Table 4; Figurereflecting
each marker 4). The phenotypic
significant
variation 2
level of percentage
association(R ) reflected
between by each
marker andmarker ranged
antioxidant between
activity 0.094% to 0.156%
is presented for DArT-3371498
in the form of Manhattan
and DArT-3369938,
plots respectively.plots
(Figure 4). Manhattan p-value ofthat
The showed eachmarkers
marker associated
reflecting significant level of association
with antioxidant activity are
between marker
present on and antioxidant
chromosomes 3 and 7. activity is presented
Quantile-quantile in plot
(q-q) the form
(Figureof 5)
Manhattan plots
reflected the (Figureof4).
goodness fit
Manhattan
of the bestplots
modelshowed that markersp-value
by implementing associated with antioxidant
distributions (expectedactivity are present
vs. observed on chromosomes
p-values on a -log10
3 scale).
and 7. Quantile-quantile (q-q) plot (Figure 5) reflected the goodness of fit of the best model by
implementing p-value distributions (expected vs. observed p-values on a -log10 scale).
Table 4. Significant marker-trait associations identified for antioxidant activity in Turkish common
Table Significant marker-trait associations identified for antioxidant activity in Turkish common
bean4.germplasm.
bean germplasm.
Marker Chromosome Position p-Value R2 Putative Gene
2
DArT-3369938
Marker 7
Chromosome 40398124 p-Value
Position 7.93 × 10−7 0.15657 Putative
R Phvul.009G180200.2
Gene
DArT-8668387
DArT-3369938 37 47385021 7.93
40398124 7.40 × 10 0.15657
× 10 −7 −5 0.09497 Phvul.009G180200.2
Phvul.003G239700
DArT-8207664
DArT-8668387 73 40398185 7.40
47385021 1.93 ×−5
× 10 10−5 0.09497
0.12493 Phvul.003G239700
Phvul.007G281200.1
DArT-8207664
DArT-3371498 77 40398185
40409459 1.93 ×−5
× 10
1.03 0.09404 Phvul.007G281200.1
10−4 0.12493 Phvul.007G171700.1
DArT-3371498 7 40409459 1.03 × 10−4 0.09404 Phvul.007G171700.1

Figure4.4. Manhattan
Figure plot reflecting
Manhattan plot reflectingmarker
markertrait
traitassociation
association
forfor antioxidant
antioxidant activity
activity in common
in common bean
bean germplasm.
germplasm.
Genes 2020, 11, 36 9 of 17
Genes 2020, 11, x FOR PEER REVIEW 9 of 17

Figure
Figure5.5.Quantile-quantile
Quantile-quantile(q-q)
(q-q)plots
plotsfor
forantioxidant
antioxidantactivity
activityininTurkish
Turkishcommon
commonbean
beangermplasm.
germplasm.

3.4. Putative
3.4. PutativeCandidate
CandidateGenes
Genes for
for Antioxidant
Antioxidant Activity
Activity
Sequences of
Sequences of all
all four
four identified
identified linked
linked markers
markers were
were used
used as
as queries
queries toto BLAST-search
BLAST-search against
against
Phytozome (V.12.1), LIS, and NCBI databases to detect predicted genes homologous
Phytozome (V.12.1), LIS, and NCBI databases to detect predicted genes homologous to these to these sequences.
A zinc-finger
sequences. protein family
A zinc-finger gene
protein (Phvul.009G180200.2)
family located onlocated
gene (Phvul.009G180200.2) Ch Pv07 onwas retrieved
Ch Pv07 was in BLAST
retrieved
using
in BLASTDArT-3369938 marker as
using DArT-3369938 a querry
marker as a(Table
querry4). The 4).
(Table search for thefor
The search homologous candidate
the homologous gene
candidate
against DArT-8207664 marker resulted in the retrieval of Phvul.007G281200.1
gene against DArT-8207664 marker resulted in the retrieval of Phvul.007G281200.1 on chromosome on chromosome Pv07.
The gene
Pv07. The encodes a methylene
gene encodes tetrahydrofolate
a methylene reductase
tetrahydrofolate also known
reductase as theasMTHFR
also known the MTHFRgene,gene,
and folic
and
acid production is predicted function of this gene. A search against the query marker
folic acid production is predicted function of this gene. A search against the query marker DArT- DArT-8668387
resulted resulted
8668387 in the retrieval
in the of Phvul.003G239700
retrieval located on located
of Phvul.003G239700 chromosome Pv03. This gene
on chromosome Pv03.encodes
This genefor
a UDP-glycosyltransferase
encodes superfamily protein.
for a UDP-glycosyltransferase Phvul.007G171700.1
superfamily was found against
protein. Phvul.007G171700.1 DArT-3371498.
was found against
DArT-3371498. The gene located only 20 kb upstream of DArT-3371498 belongs tofamily
The gene located only 20 kb upstream of DArT-3371498 belongs to the Pentatricopeptide repeat the
(PPR_2).
Pentatricopeptide repeat family (PPR_2).
4. Discussion
4. Discussion
In this study, a total of 182 Turkish common bean landraces and six commercial cultivars were used
In this study, a total of 182 Turkish common bean landraces and six commercial cultivars were
to identify marker trait association for antioxidant activity. Results of phenotypic characterization for
used to identify marker trait association for antioxidant activity. Results of phenotypic
antioxidant activity showed a great level of diversity ranging from 2.15 to 60.48 with the mean TEAC
characterization for antioxidant activity showed a great level of diversity ranging from 2.15 to 60.48
of 20.03 µmol TE/g (Table S3). The observed range and average TEAC in the studied germplasm was
with the mean TEAC of 20.03 µmol TE/g (Table S3). The observed range and average TEAC in the
much higher than reported by Huber et al. [52], Weidner et al. [34], and Bojilov et al. [53]. Antioxidant
studied germplasm was much higher than reported by Huber et al. [52], Weidner et al. [34], and
activity during 2018 at both locations was recorded as higher compared to 2017. García-Díaz et al. [54]
Bojilov et al. [53]. Antioxidant activity during 2018 at both locations was recorded as higher compared
also observed higher antioxidant activity in common bean seeds in one year as compared to the other
to 2017. García-Díaz et al. [54] also observed higher antioxidant activity in common bean seeds in one
year of study. These differences in the antioxidant activity of common seeds grown in different years
year as compared to the other year of study. These differences in the antioxidant activity of common
have been previously explained to be influenced by environmental variations, crop management
seeds grown in different years have been previously explained to be influenced by environmental
practices, and genetic differences [55]. Frequency distribution reflected that a good number of landraces
variations, crop management practices, and genetic differences [55]. Frequency distribution reflected
acquired TEAC contents similar to the results of earlier studies [34–53]. The ANOVA was performed
that a good number of landraces acquired TEAC contents similar to the results of earlier studies [34–
within and across the four environments to evaluate the genotypic effect and explore the GEI for
53]. The ANOVA was performed within and across the four environments to evaluate the genotypic
antioxidant capacity. The ANOVA confirmed that statistically significant variations in TEAC are due to
effect and explore the GEI for antioxidant capacity. The ANOVA confirmed that statistically
the genetic background of landraces, while GEI reflected a non-significant effect for antioxidant capacity.
significant variations in TEAC are due to the genetic background of landraces, while GEI reflected a
These results were further strengthened by the results of heritability analysis. Higher heritability
non-significant effect for antioxidant capacity. These results were further strengthened by the results
(0.92) in this study reflected that this trait is mainly under the control of genetic forces, while the
of heritability analysis. Higher heritability (0.92) in this study reflected that this trait is mainly under
environmental forces have very little effect. García-Díaz et al. [54] investigated the effect of cropping
the control of genetic forces, while the environmental forces have very little effect. García-Díaz et al.
season and genotype on antioxidant activity in the common bean and revealed that variations are
[54] investigated the effect of cropping season and genotype on antioxidant activity in the common
mainly due to genotypic effect. Di Silvestro et al. [56] investigated how genotype and environment
bean and revealed that variations are mainly due to genotypic effect. Di Silvestro et al. [56]
investigated how genotype and environment effect antioxidant activity in wheat under multiyear
Genes 2020, 11, 36 10 of 17

effect antioxidant activity in wheat under multiyear experiments. A lesser effect of the environmental
forces was observed in their results supports our observations of the common bean. A similar study
on corn concluded that most of the variations for studied traits are contributed by the genotypes as
compared to environmental forces [57].
We also observed variations in seed color and recorded that white color (58% of the accessions)
was prevalent in our selected population. Seed color in the common bean is associated with bioactive
compounds, such as anthocyanin and tannins which are found in higher quantity in colored seeds
as compared to white colored seeds [58]. Earlier studies explored the relationship between the
seed color and antioxidant activity in common bean and concluded the role of seed color in higher
antioxidant activity [58,59]. In this study, mean antioxidant activity was found much higher in colored
seeds as compared to the white ones. These results are in line with the previous studies stating the
existence of higher antioxidant activity in colored seeds as compared to white seeds [58–60]. Pearson’s
correlation coefficient showed a highly significant correlation between colored seeds (brown, beige)
and antioxidant activity. On the other hand, there was a negative correlation between white seeds and
antioxidant activity. Earlier studies revealed the presence of higher anthocyanin in the seed coat of the
colored seed, which ultimately contributes toward a higher antioxidant activity [58,59].

4.1. DArTseq Markers Analysis and LD

A total of 7900 markers were employed in this study to investigate population structure and
marker-trait association for antioxidant activity. The number of mapped markers ranged from 931
(Chr. 2) to 591 (Chr. 6) (Table 3). The mean distance between markers on the whole nuclear genome
was 15.54 Mb with a range of 13.15 (Chr. 7) to 18.99 Mbps (Chr. 2). The number of markers used in
this study was much higher than earlier studies used for GWAS in various plant [61,62], which in
turn leads toward a more efficient genome-wide screening. Using marker information, the structure
algorithm divided whole germplasm into two heterotic populations on the basis of their 100-seeds
weight, TEAC contents, and collection point. Population A clustered a total 117 landraces, while 71
landraces clustered in population B. Akdag, Onceler, and Goynuk cultivars grouped in population
A and Akman, Goksun, and Karacasehir reflected much similarity with population B. Landraces
clustered in population A mainly contained higher 100-seed weight and relatively more TEAC contents,
while landraces having lower to moderate TEAC contents with lower 100-seed weight clustered into
population B. Geographic locations also played a role in the clustering and landraces from same
provinces were grouped together. Earlier studies have confirmed the clustering of common bean
germplasm based on plant height, 100-seed weight, and collection point. These previous reports
strengthen the findings of our study [63].
Linkage disequilibrium is a nonrandom association of alleles at particular loci in a sampled
population genome [64]. In this study, mean r2 was 0.47 while the mean significant LD was 54%
(Table 3). The maximum and minimum LD was 59.8% and 45.89% reflected by chromosomes 11 and
9, respectively. Mean LD decay was 1.15 Mb for the whole genome and ranged from 1.05 Mbps for
chromosome 7 to 1.3 Mbps for chromosome 5. A nonlinear trend line between r2 vs. distance bp
showed that LD decayed at 1.3 Mbs and r2 value was 0.5 (Figure S2). When an LD decay plot is
constructed, usually the distance point is looked for where the LD value (r2 ) decreases below 0.1 or half
of D’ strength (D’ = 0.5) based on curve of nonlinear logarithmic trend line [65,66]. This shows a rough
estimation of extent of LD, however, highly significant threshold LD values (r2 ≥ 0.2) are used for
more precise LD calculation. Our results are in a disagreement with the findings of Campa et al. [67],
who stated that chromosome 9 shows maximum LD. Linkage disequilibrium measured r2 plotted
vs. p-value between pairs of DArTseq markers the studied germplasm showed a relatively bigger
LD decay on LG6 (Figure S3). Mean r2 found in this study was slightly lower as compared to the
findings by Ates et al. [68]. The higher LD in their study may be due to human selection, which leads
in particular usage, combining distinct variations through a long history of cultivation and multiple
nationwide expeditions.
Genes 2020, 11, 36 11 of 17

4.2. Marker Trait Association and Putative Candidate Genes for Antioxidant Activity
We used GWAS for the whole-genome screening of the selected germplasm. This approach has
rapidly gained the attention of breeders to identify genomic regions for various traits of interest that can
be used for marker-assisted breeding [69]. For the investigation of marker–trait association, the MLM
(Q + K) approach was used during GWAS analysis to eliminate possible spurious associations [68].
In this study a total of four markers (DArT-3369938, DArT-8668387, DArT-8207664, and DArT-3371498)
were significantly associated with seed antioxidant contents (Table 4; Figure 4). DArT-3371498
accounted for 15.6% of the total variation and was the most statistically significant marker, while
DArT-3371498 accounted for the least variation among these four identified markers. The Manhattan
plot showed that DArT-3369938, DArT-8207664, and DArT-3371498 are located on chromosome 7,
while DArT-8668387 reflected association with chromosome 3. As these markers reflected significant
association for antioxidant activity in the common bean, these markers can be used for marker-assisted
breeding to develop cultivars with better antioxidant activity.
Located only 100 kb upstream of DArT-3369938, on Pv09, Phvul.009G180200.2 is putative candidate
gene to explain the phenotypic variation associated with this marker. This gene encodes for zinc-finger
domain–containing protein. Zinc-finger proteins (ZFPs) constitute large protein families and have an
important role in various plant developmental stages and to biotic and abiotic stress [70]. Hichri et al. [71]
stated that this protein family contains transcription factors that control various aspects of plant
development and shown a pivotal role in abiotic stress tolerance. Cao et al. [72] found that ZFPs
have resistant to biotic stress such as rice blast fungus infection. Cheuk and Houde [73] explored the
role of ZFPs in abiotic stress and investigated 53 Q-type C2H2 zinc-finger proteins (TaZFPs) from
Triticum aestivum, and stated that these TaZFPs have positive response to high light (44/53), H2 O2
(37/53), drought (37/53), and flooding (31/53). Zang et al. [74] confirmed that this protein is involved
in abiotic stress by investigating abiotic stress in Arabidopsis and concluded that ZFPs increase the
salt and osmotic tolerance in this plant through a series of physiological processes. Zhang et al. [75]
identified ZFPs in rice and concluded that overexpression of ZFPs in their study elevated the activities
of antioxidant enzymes and enhanced the tolerance of rice plants against water and oxidative stresses.
The BLAST search against DArT-8668387 retrieved a gene (Phvul.003G239700) encoding
UDP-Glycosyltransferase superfamily protein; a multifunctional protein family in plants. Glycosylation
is one of the final steps involved in the biosynthesis of triterpenoid for the production of
various plant defensive compounds like phenolic, glucosinolates, salicylates, and anthocyanins [76].
UDP-glycosyltransferases (UGTs) are one of the biggest gene families in the plant kingdom and play
an important role in transferring sugar, controlling various metabolic processes, and play an effective
role against various biotic and abiotic stresses [77,78]. Various studies have explored the biological role
of the putative UGTs against different abiotic stresses. Li et al. [77] comprehensively explained the
role and functioning of a homolog of this protein in enhanced plant tolerance to low temperatures,
drought, and salt stresses in Arabidopsis. Sun et al. [79] found that ectopic expression of UGT85A5
in tobacco resulted in enhanced salt stress tolerance. Rehman et al. [78] performed a comparative
genomic and transcriptomic analyses study for brassica species and Arabidopsis and found the role of
UDP-Glycosyltransferase in biotic and abiotic stresses.
Phvul.007G281200.1 resulted as the putative candidate gene for DArT-8207664 marker that encodes
for the methylene tetrahydrofolate reductase MTHFR gene. MTHFR enzyme is involved in the
metabolism of folate and homocysteine (Hcy). This gene is linked with folate metabolism which
plays a positive effects on oxidative stress by its protecting action against cell death and increased
folic concentrations stabilize MTHFR enzyme [80]. Earlier studies have found the synergetic effect
of folic acid on growth, yield, and yield quality of many plant species. Folic acid can play an
effective role by catching the free radicals or active oxygen produced during the stress conditions
and help the plant defense against environmental stresses [81]. Phvul.007G171700.1 encodes for
PPR containing plant protein (RNA-binding proteins) having a key role in post-transcription via
RNA editing, cleavage, splicing, stability, or translation in plastids or mitochondria [82]. Earlier
Genes 2020, 11, 36 12 of 17

studies revealed that PPR proteins might be target genes of some miRNAs regulating abiotic stress
responses [83]. Zsigmond et al. [84] revealed that PPR proteins are involved in oxidative respiration
that contributes to abiotic stress tolerance in Arabidopsis. Recently, Chen et al. [85] aimed to investigate
the functioning of PPR gene family in rice and explored the role of this family under stress condition.
They investigated the expression pattern of this gene family and concluded that PPR proteins have
crucial roles in response to different abiotic stresses in rice.
As is obvious from the previously discussed evidence, our diverse association panel reflected
a wide range of antioxidant activity in common bean seeds. These genetic variations can be utilized
for various common bean improvement programs. The role of antioxidants in human health has
been universally accepted, and earlier studies revealed that higher antioxidant levels in food can
prevent various disorders [20–22]. Germplasm characterization is an important way to elucidate novel
variations which can be used for the breeding perspective of any crop [86–88]. Therefore, there is
a need to identify the genetic basis for association with antioxidant activity. The common bean serves
as a source of food for millions of people all over the world, and thus, the intake of beans with higher
antioxidant levels will reduce the risk of various disorders. We identified four DArTseq markers
(DArT-3369938, DArT-8668387, DArT-8207664, and DArT-3371498) with significant association for
antioxidant activity in Turkish common bean germplasm. These DArTseq marker can be used for
the marker-assisted breeding of the common bean in order to develop cultivars having improved
antioxidant activity.

5. Conclusions
A wide range of phenotypic variations were observed for antioxidant activity in Turkish common
bean germplasm. Heritability analysis revealed that antioxidant activity is controlled by genotypic
effect. A negative correlation was observed between antioxidant activity and white seeds. This is
a pioneer study revealing the marker trait association for antioxidant activity in Turkish common bean
seeds. A total of four DArTseq markers showed statistically significant association for seed antioxidant
activity, and these markers can be used in the future for the development of common bean cultivars
with better antioxidant contents.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/1/36/s1,

Figure S1: Population structure of Turkish common bean germplasm revealed by DArTseq markers, Figure S2:
Scatter plot reflecting association between linkage disequilibrium (r2 ) and distance bp, Figure S3: Linkage
disequilibrium (LD) measured r2 plotted vs. P-value between pairs of DArTseq markers in Turkish common bean
germplasm Table S1: Passport data of Turkish common bean germplasm used in this study, Table S2: Antioxidant
activity (µmol TE/g fw) in four environments for Turkish common bean germplasm, Table S3: Comparison of
mean, maximum and minimum for antioxidant activity (µmol TE/g fw) between Turkish common bean landraces
and commercial cultivars under four environments.
Author Contributions: Conceptualization, F.S.B.; methodology, F.S.B. and S.E.; software, M.A.N. (Muhammad
Azhar Nadeem), E.H. and X.L.; validation, T.K. and M.A.N. (Muhammad Azhar Nadeem); formal analysis,
M.A.N. (Muhammad Azhar Nadeem), E.H., X.L., and M.S.; investigation, T.K. and M.G.; resources, F.S.B.,
G.C., and B.-M.J.; data curation, M.A.N. (Muhammad Azhar Nadeem), O.S., and T.K.; writing—original draft
preparation, M.A.N. (Muhammad Azhar Nadeem); writing—review and editing, M.A.N. (Muhammad Amjad
Nawaz), R.H., F.A., and S.E.; visualization, F.S.B.; supervision, F.S.B.; project administration, F.S.B; funding
acquisition, F.S.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Scientific and Technological Research Council of Turkey TUBİTAK
under research grant number (TOVAG-2015O630 to FSB).
Conflicts of Interest: The authors declare no conflict of interest.

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