plants
Article
Genetic and Morphological Diversity Assessment of Five
Kalanchoe Genotypes by SCoT, ISSR and RAPD-PCR Markers
Jameel M. Al-Khayri 1, * , Ehab M. B. Mahdy 2 , Heba S. A. Taha 3 , Ahmed S. Eldomiaty 3 ,
Mohamed A. Abd-Elfattah 4 , Arafat Abdel Hamed Abdel Latef 5, * , Adel A. Rezk 1 , Wael F. Shehata 1 ,
Mustafa I. Almaghasla 6,7 , Tarek A. Shalaby 6,8 , Muhammad N. Sattar 9 , Hesham S. Ghazzawy 10 ,
Mohamed F. Awad 11 , Khalid M. Alali 1 , Shri Mohan Jain 12 and Abdallah A. Hassanin 3, *
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Citation: Al-Khayri, J.M.; Mahdy,
10
E.M.B.; Taha, H.S.A.; Eldomiaty, A.S.;
Abd-Elfattah, M.A.; Abdel Latef,
A.A.H.; Rezk, A.A.; Shehata, W.F.;
Almaghasla, M.I.; Shalaby, T.A.; et al.
Genetic and Morphological Diversity
Assessment of Five Kalanchoe
Genotypes by SCoT, ISSR and
RAPD-PCR Markers. Plants 2022, 11,
1722. https://doi.org/10.3390/
plants11131722
Academic Editor: Andreas W. Ebert
Received: 6 June 2022
Accepted: 25 June 2022
Published: 29 June 2022
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4.0/).
11
12
*
Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; arazk@kfu.edu.sa (A.A.R.); wshehata@kfu.edu.sa (W.F.S.);
kalali@kfu.edu.sa (K.M.A.)
National Gene Bank (NGB), Agricultural Research Centre (ARC), Giza 12613, Egypt; ehab.mahdy@arc.sci.eg
Genetics Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt;
hebasayedtaha@gmail.com (H.S.A.T.); a.salah8373@gmail.com (A.S.E.)
Pomology Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt;
dr.mohamed.alaa@cu.edu.eg
Department of Botany and Microbiology, Faculty of Science, South Valley University, Qena 83523, Egypt
Department of Arid Land Agriculture, College of Agriculture and Food Sciences, King Faisal University,
P.O. Box 420, Al-Ahsa 31982, Saudi Arabia; malmghaslah@kfu.edu.sa (M.I.A.); tshalaby@kfu.edu.sa (T.A.S.)
Plant Pests, and Diseases Unit, College of Agriculture and Food Sciences, King Faisal University, P.O. Box 420,
Al-Ahsa 31982, Saudi Arabia
Horticulture Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt
Central Laboratories, King Faisal University, P.O. Box 420, Al-Ahsa 31982, Saudi Arabia; mnsattar@kfu.edu.sa
Date Palm Research Center of Excellence, King Faisal University, Al-Ahsa 31982, Saudi Arabia;
hghazzawy@kfu.edu.sa
Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia;
m.fadl@tu.edu.sa
Department of Agricultural Sciences, University of Helsinki, 00014 Helsinki, Finland;
jain.mohan70@gmail.com
Correspondence: jkhayri@kfu.edu.sa (J.M.A.-K.); moawad76@gmail.com (A.A.H.A.L.);
asafan@zu.edu.eg (A.A.H.)
Abstract: Determining the appropriate parents for breeding programs is the most important decision
that plant breeders must make to maximize the genetic variability and produce excellent recombinant
genotypes. Several methods are used to identify genotypes with desirable phenotypic features for
breeding experiments. In this study, five kalanchoe genotypes were morphologically characterized by
assessing plant height, number of inflorescences, number of flowers, flower length, flower diameter
and number of petals. The analysis showed the distinction of yellow kalanchoe in the plant height
trait, while the orange kalanchoe was distinguished in the number of inflorescences, the number of
flowers and flower length traits, whereas the violet kalanchoe possessed the largest flower diameter
and the highest number of petals. The molecular profiling was performed by random amplified
polymorphism DNA (RAPD), inter-simple sequence repeats (ISSR) and start codon targeted (SCoT)polymerase chain reaction (PCR) tools. Genomic DNA was extracted from young leaves and the PCR
reactions were performed using ten primers for each SCoT, ISSR and RAPD marker. Only four out
of ten primers showed amplicon profiles in all PCR markers. A total of 70 bands were generated
by SCoT, ISSR and RAPD-PCR with 35 polymorphic bands and 35 monomorphic bands. The total
number of bands of RAPD, ISSR and SCoT was 15, 17 and 38, respectively. The polymorphism
percentages achieved by RAPD, ISSR and SCoT were 60.25%, 15% and 57%, respectively. The cluster
analysis based on morphological data revealed two clusters. Cluster I consisted of violet and orange
kalanchoe, and cluster II comprised red, yellow and purple kalanchoe. Whereas the cluster analysis
based on molecular data revealed three clusters. Cluster I included only yellow kalanchoe, cluster II
comprised orange and violet kalanchoe while cluster III comprised red, and purple kalanchoe. The
study concluded that orange, violet and yellow kalanchoe are distinguished parents for breeding
Plants 2022, 11, 1722. https://doi.org/10.3390/plants11131722
https://www.mdpi.com/journal/plants
Plants 2022, 11, 1722
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economically valued traits in kalanchoe. Also, the study concluded that SCoT and RAPD markers
reproduced reliable banding patterns to assess the genetic polymorphism among kalanchoe genotypes
that consider the basis stone for genetic improvements in ornamental plants.
Keywords: genetic polymorphism; diversity assessment; molecular markers; SCoT; ISSR; RAPD
1. Introduction
Kalanchoe is a medicinal plant largely used in folk medicine for the treatment of
kidney stones, gastric ulcer, pulmonary infection and rheumatoid arthritis [1] and is grown
commercially as a flowering potted plant [2]. The kalanchoe genus comprises 125 species
of Crassulaceae succulent plants [3]. The majority of the kalanchoe genotypes are native
to Madagascar and tropical Africa, and many of them are popular due to their growing
indoors [4]. Kalanchoes are very low-maintenance houseplants, however, they require
direct sunlight. They can also endure bright indirect light and only need to be watered
when completely dry. Leaf or stem cuttings can be used to reproduce all species. Kalanchoe
genotypes are mostly perennial herbaceous plants, with a few shrubs and annuals. The
thick leaves are waxy or hairy and come in a variety of shapes. They are frequently borne
on the stems in opposite directions. From the plant’s base or along the leaf margins, several
species develop clonal plantlets [3].
The species of kalanchoe play an important role in scientific research of genetic diversity and evolutionary aspects of plants. Plant diversity has captivated humans throughout
history, owing to the enormous variation in molecular and morphological features found
in nature. The naturally occurring variations among plant species provide a familiar environment for evolution by natural selection, plant taxonomy and phylogeny are based on
genetic diversity [5–7].
In the field of ornamental plant improvement, distant hybridizations are still a common method of promoting genetic diversity in plants. More than 100 plant species have
derived from this variation in breeding and genetic improvement programs [6,8,9] by
applying direct selection for specific traits. Understanding the genetic basis and molecular
profiles of all this naturally occurring diversity is one of the fundamental challenges of
modern biology.
Several studies discussed the genetic diversity of various ornamental plants such
as Allium species [10], ornamental Coffea Arabica plants [11], ornamental pomegranates
(Punica granatum L.) [12], ornamental pepper plants [13] and Dianthus [14].
Conventional DNA markers have numerous applications in determining genetic diversity in plants. These markers include inter simple sequence repeat (ISSR) markers [15],
sequence-related amplified polymorphism (SRAP) markers [16], and simple sequence repeat (SSR) markers [16]. Recently, new promising techniques have emerged. Start Codon
Targeted (SCoT) is a dominant and reproducible marker that is based on the short conserved
region in plant genes surrounding the ATG start codon [17]. To obtain more information
about the morphological and molecular profiles of some kalanchoe genotypes and their
phylogenetic relationship, we have applied phenotypic characterization and molecular profiling; SCoT, ISSR and RAPD-PCR analyses to compare five selected kalanchoe genotypes.
This investigation was performed to assess the genetic diversity among five kalanchoe
genotypes by combining morphological characterization with the RAPD, ISSR and SCoT
molecular markers and phylogeny analyses.
2. Results
2.1. Morphological Polymorphism
Five kalanchoe genotypes were maintained under greenhouse conditions and assessed
for morphological features; number of petals, flower length, number of flowers, flower
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diameter, number of inflorescences and plant height (Figures 1 and 2) which have economic
importance in ornamental plants.
Figure 1. Morphological characterization of five kalanchoe (Kalanchoe blossfeldiana) genotypes.
(A) Different kalanchoe plants with various colors illustrate plant architecture. (B) Flower colors
polymorphism. (C) Difference in leaf shape and size.
The yellow kalanchoe genotype showed the highest plant height (40 cm), while
the orange kalanchoe genotype got the lion’s share due to recording the highest number of inflorescences (9 inflorescences plant−1 ), number of flowers (321 flowers plant−1 )
and flower length (2.1 cm). The violet genotype recorded the highest number of petals
(40 petals flower−1 ).
Statistical analysis revealed significant and non-significant differences among the
five kalanchoe genotypes in morphological characteristics based on the least significant
difference (LSD) values. A significant difference was noticed between the yellow kalanchoe genotype and the remaining genotypes, except for the orange kalanchoe in plant
height character (Figure 2A). The analysis showed a significant difference in the number of
inflorescences between orange kalanchoe genotype and the rest of the genotypes except
the violet genotype, a significant difference also appeared between each of red and yellow genotypes and violet and purple kalanchoe genotypes (Figure 2B). Two insignificant
differences; the first one appeared between purple and violet kalanchoe and the second
one between red and yellow kalanchoe (Figure 2B). The number of flowers showed a
significant difference between orange kalanchoe genotype and the rest of the genotypes
except the violet genotype exactly as the statistical profile appeared in the number of
inflorescences (Figure 2C).
Flower length (cm) was significantly different among both orange and yellow genotypes and the rest genotypes, while violet, purple and red genotypes were insignificantly
different from each other (Figure 2D). Flower diameter showed an insignificant difference
between the orange and the violet kalanchoe genotypes, while they presented significant
differences with the rest of the genotypes (Figure 2E). Orange and red genotypes were
insignificantly different from each other, while they recorded significant differences with
purple and yellow genotypes. The yellow genotype presented a significant difference with
all genotypes in flower diameter character (Figure 2E). Finally, the number of petals showed
a significant difference between the violet genotype and the rest of the genotypes which
presented insignificant differences from each other’s (Figure 2F).
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Figure 2.
Selected characteristics of five kalanchoe genotypes;
each pre≤ 0.05) accord’
sented value is means
± standard error,
and the different letters mean
–
–
that values are significantly different (p ≤ 0.05) according to Fisher’sLSD0.05 .
(C) Number of flowers (D) Flower length
(A) plant height (B) Number of inflorescences.
(E) Flower diameter (F) Number of petals. Note: (A–C) Average of 10 plants (D–F) Average of
50 flowers/plant.
2.2. Molecular Polymorphism Analyses
Genetic polymorphism analysis of kalanchoe genotypes was conducted by RAPD, ISSR
and SCoT-PCR amplifications using 30 arbitrary primers (10 primers for each PCR reaction
type) (Please see materials and methods section) to examine the molecular polymorphism
among kalanchoe genotypes. Finally, only four primers for each reaction produced reliable
polymorphic banding profiles within all the studied genotypes (Table 1 and Figure 3). The
reactions of PCR for the three techniques produced 70 loci, 35 of which were polymorphic,
while 35 were monomorphic. The total number of amplified loci for RAPD, ISSR and
SCoT-PCR was 17, 17 and 38, respectively (Table 1). The polymorphism revealed by RAPDPCR ranged from 50% to 70% while the polymorphism revealed by ISSR-PCR ranged
between zero % to 33% and the polymorphism produced by SCoT-PCR ranged from 44%
to 66% (Table 1).
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Table 1. Numbers of loci; total, monomorphic, polymorphic, and unique, generated by four out
of ten primers of each RAPD, ISSR and SCot-PCR reaction in five kalanchoe genotypes, and the
associated polymorphism.
PCR Type
Primer
Number
of Loci
RAPD
OPA 2
OPA 7
OPA 9
OPA 10
2
8
3
2
3.75
15
4
3
6
4
4.25
17
9
9
11
9
9.5
38
1
2
1
1
1.25
5
4
3
4
3
3.5
14
3
5
4
4
4
16
1
6
2
1
2.5
10
0
0
2
1
0.75
3
6
4
7
5
5.5
22
0
2
2
0
1
4
0
0
0
0
0
0
3
1
1
0
1.25
5
70
35
35
9
Average
Total
ISSR
Average
Total
SCoT
Average
Total
Total number
of loci
ISSR-3
ISSR-5
ISSR-8
ISSR-10
SCoT 3
SCoT 11
SCoT 13
SCoT 14
Monomorphic Polymorphic
Loci
Loci
Unique Loci
Polymorphism Percentage Fidelity
(%)
of the RAPD/SCoT
50%
75%
66%
50%
60.25%
0%
0%
33%
25%
15%
4/5 × 100− 15/38 ×
100 = 80 − 39.47 =
40.53%
66%
44%
63%
55%
57%
Figure 3. DNA fragment patterns of RAPD, ISSR and SCoT-PCR amplification of five kalanchoe
genotypes. (A) RAPD-PCR amplification using primers OPA 2, OPA 7, OPA 9 and OPA 10, respectively. (B) ISSR-PCR amplification using primers ISSR-3, ISSR-5, ISSR-8 and ISSR-10, respectively.
(C) SCoT-PCR amplification using primers SCoT3, SCoT11, SCoT13 and SCoT14, respectively.
M = 100 bp Plus DNA Ladder. The monomorphic loci are presented in green numbers and the
polymorphic loci are presented in yellow numbers.
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2.3. Phylogeny Analyses
Phylogenetic relationships among the five kalanchoe genotypes were inferred based
on the data recorded from morphological criteria. The clustering analysis grouped the five
kalanchoe genotypes into two groups (I, II) (Figure 4). Cluster I comprised the violet and
the orange kalanchoe genotypes and cluster II included red, yellow and purple kalanchoe
genotypes. On the other hand, the phylogenetic relationship was determined among the
kalanchoe genotypes based on the banding profiles revealed by RAPD, ISSR and SCoT-PCR.
Phylogenetic analysis (Figure 5) divided the five kalanchoe genotypes into three clusters
according to the data scored from the molecular analysis. Yellow kalanchoe independently
formed cluster I. The analysis grouped both violet and orange kalanchoe in cluster II.
independently
formedpurple
cluster and
Ι. The
analysis
grouped
both violetThe
and only
orange
kalanchoebetween
Cluster
III included
Red
kalanchoe
genotypes.
difference
independently
formed
clustergenotypes
Ι. The analysis
grouped
both violetand
andmorphological
orange kalanchoe
the
clustering of
kalanchoe
based
on molecular
attributes
is that the yellow kalanchoe grouped in an independent group based on the molecular
profile (Figure 5).
Kalanchoe genotypes
Red Kalanchoe
II
Yellow Kalanchoe
Purple Kalanchoe
Violet Kalanchoe
I
Orange Kalanchoe
0
20
40
60
80
100
Genetic Distance
Figure 4. Phylogenetic tree of five kalanchoe genotypes revealed by the weighted pair group method
using arithmetic average (WPGMA) method based on morphological features.
Red Kalanchoe
Kalanchoe genotypes
III
Purple Kalanchoe
Violet Kalanchoe
II
Orange Kalanchoe
I
Yellow Kalanchoe
3.8
3.9
4.0
4.1
4.2
4.3
4.4
4.5
Genetic Distance
Figure 5. Phylogenetic tree of five kalanchoe genotypes revealed by the weighted pair group method
using arithmetic average (WPGMA) method based on SCoT, ISSR and RAPD banding patterns.
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3. Discussion
The success of breeding programs is determined by the accurate selection of parents.
At this point, breeders begin their search for a specific plant genotype that will meet market
expectations. Even while recombination may play role in expanding the polymorphism
among segregating populations, the ability of two parents to combine their progeny and
their excellent performance in agronomic variables will identify whether the progeny will
be successful elite lines [18,19]. Given the scarcity of knowledge on combining abilities,
investigations that highlight genotype correlations will be critical sources for investigations,
assisting breeders to select parents for breeding experiments. As a result, morphological
and DNA marker characterizations, as well as multivariate statistical analyses, will be
critical components in improving our capability to select the best parents for crosses [18].
In this investigation, five kalanchoe genotypes were characterized regarding their
morphological and molecular features. The current results of morphological characterization revealed that some kalanchoe genotypes had advantages in some traits, and this can
be considered as a cornerstone in breeding and genetic improvement of kalanchoe. The
orange kalanchoe offered advantages in its number of inflorescences, number of flowers
and flower length, which are important traits in the characterization of ornamental plants.
Hence, orange kalanchoe genotypes may be used as parents in breeding programs for
these parameters.
In the same context, the yellow kalanchoe presented advantages in both plant height
and flower length, also the violet kalanchoe showed the highest score in flower diameter and
number of petals which are considered the most important features in the ornamental field.
For successful Kalanchoe breeding programs, it is important to completely characterize the
morphological traits, especially those that have economic importance such as number of
inflorescences, number of flowers and flower length. Achieving desired breeding results
requires correct parent selection and understanding of the genetic distance either based
on morphological features or molecular profiling and understanding the inheritance of
the desired traits [20]. This study clarified that Kalanchoe genotypes are morphologically
diverse and hence are great candidates for intraspecific hybridization to produce new lines
with favorable features.
The current study emphasized the potential importance of DNA polymorphism detection for plant breeding programs and genetic improvement. Plant genetic diversity can be
studied and detected with the help of molecular markers [21]. For genetic diversity studies
in plants, a variety of molecular markers are used, including RAPD [22,23], ISSR [24],
amplified fragment length polymorphism (AFLP) [25,26] and SCoT [27].
The molecular markers (RAPD and SCoT) produced reliable polymorphic banding
patterns enabling the determination of genetic polymorphism among kalanchoe genotypes.
The ISSR produced a low polymorphism ratio compared to both RAPD and SCoT markers.
The RAPD and SCoT-PCR markers used in this study may be considered as more efficient
in identifying the genetic polymorphism based on their polymorphic banding profiles
than ISSR-PCR which produced a low polymorphism percentage. This may provide
important clues in distinguishing the relationship among kalanchoe genotypes. These
results are similar to those reported by Collard and Mackill [17], they found that results of
amplification yield using SCoT technique were more reproducible than other molecular
markers results in rice. Furthermore, genetic polymorphisms produced by SCoT marker
were better in determining the relationship among mango cultivars than ISSR markers [28].
In the same context, an investigation on potato indicated the greater effectiveness of SCoT
markers in determining somaclonal variation compared to ISSR and RAPD markers [29].
Based on the results obtained from morphological data and molecular profiles of the
kalanchoe genotypes, we generated two phylogenetic trees. Phylogeny and alignment are
important analyses to determine the genetic diversity among different species [6,30–35].
The phylogeny analysis showed similar results in clustering of the genotypes except
the analysis based on molecular banding patterns separated the yellow kalanchoe in an
independent cluster, while the other four genotypes were grouped in two clusters similarly
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in both analyses based on morphological and molecular profiles. It is clear from the cluster
analysis that genotypes of the same cluster may have common ancestors. So we combined
results from both molecular and morphological analyses to precisely interpret and shed
light on the genetic relationship among kalanchoe genotypes that can be considered a
cornerstone for genetic improvement. Based on the genetic relationship, suitable parents
can be selected for hybridization experiments to achieve desired heterosis effects. Generally,
the number of data derived from morphological observations is significantly fewer than
that derived from genetic markers, resulting in a bias toward the molecular analyses’
outcome [18]. When morphological criteria and molecular markers were examined in
wheat, three out of four clusters in the molecular analysis were consistent with the distance
indicated by phenotypic features [36].
Despite the current study may support the conventional breeding methods for genetic improvement of kalanchoe genotypes, however recent genetic approaches such as
genetic engineering [37] or genome editing approaches [38,39] could be efficiently used in
kalanchoe breeding and improvement for different desirable features.
4. Materials and Methods
4.1. Plant Materials
The five genotypes of kalanchoe used in this study were obtained and classified by a
plant taxonomist in the Faculty of Science. The plants were kept in a controlled greenhouse
environment with natural light, at a temperature of 15 ◦ C at night and 25 ◦ C during the day.
4.2. Morphological Polymorphism
Morphological data; plant height, number of inflorescences, number of flowers, flower
length, flower diameter and number of petals were collected. Plant height was recorded
on the day when the first flower bloomed. Flower diameter, floral length, and the number
of petals were recorded at the sticky stage of stigma [40]. On the day that the first wilted
flower was spotted, the number of blooms and inflorescences were counted.
4.3. Genomic DNA Extraction
The genomic DNA was extracted from 5 g of sterilized (0.05% Clorox) young leaf
samples using the cetyltrimethylammonium bromide (CTAB) method [41]. A Nano Drop
2000 (Thermo Scientific™, Waltham, MA, USA) was used for measuring extracted DNA
concentrations in the samples; electrophoresis on a 1% agarose gel was performed to verify
the quality and quantity of extracted DNA. The concentrations of DNA were set up to
50 ng·µL–1 and DNA was stored at –20 ◦ C for the next amplification experiments.
4.4. Random Amplified Polymorphism DNA (RAPD-PCR)
PCR amplification was performed by ten primers as shown in Table 2 according to
Williams, Kubelik, Livak, Rafalski and Tingey [22]. The RAPD-PCR amplification reaction
was conducted in a 25 µL reaction mixture including 15 µLof 2x fidelity Taq PCR Master
Mix (USB Corporation, Cleveland, OH), 2 µLdNTPs (200 µM), 1.5 mM MgCl2 (25 mM), 1
µM of each primer (10 pmol) and 2 µL of genomic DNA (20 ng/µL). The final volumes
were adjusted with sterile distilled water up to 25 µL. The RAPD-PCR-based amplification
was performed using a 96 well plate thermal cycler (Applied Biosystem) as the following:
95 ◦ C for 1 min for initial denaturation, followed by 40 cycles of 95 ◦ C for 30 s, 1 min at the
annealing of each primer and 72 ◦ C for 1 min for extension and the final extensions were
done at 72 ◦ C for 5 min.
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Table 2. Codes and sequences of RAPD, ISSR and SCoT primers.
No.
RAPD Primers
Code
1
2
3
4
5
6
7
8
9
10
OPA2
OPA7
OPA9
OPA10
OPA18
OPB5
OPC4
OPC5
OPC8
OPD5
Sequence
ISSR Primers
′ 3–5′
TGCCGAGCTG
GAAACGGGTG
GGGTAACGCC
CTGCTGGGAC
AGGTGACCGT
TGCGCCCTTC
CCGCATCTAC
GATGACCGCC
TGGACCGGTG
TGAGCGGACA
Code
ISSR-1
ISSR-2
ISSR-3
ISSR-4
ISSR-5
ISSR-6
ISSR-7
ISSR-8
ISSR-9
ISSR-10
Sequence
SCoT Primers
′ 3–5′
(ga) 6 gg
(cac)3 gc
(gag) 3 gc
cac (tcc) 5
tgta (ca) 7
tac (ca) 7
(ag) 8 t
cgtc (ac) 7
tcga (ca) 7
(ag) 8 ct
Code
Sequence ′ 3–5′
SCoT 2
SCoT 3
SCoT 4
SCoT 5
SCoT 6
SCoT 9
SCoT 11
SCoT 12
SCoT 13
SCoT 14
ACCATGGCTACCACCGGC
ACGACATGGCGACCCACA
ACCATGGCTACCACCGCA
CAATGGCTACCACTAGCG
CAATGGCTACCACTACAG
ACAATGGCTACCACTGCC
ACAATGGCTACCACTACC
CAACAATGGCTACCACCG
ACCATGGCTACCACGGCA
ACCATGGCTACCAGCGCG
4.5. Inter-Simple Sequence Repeats (ISSR-PCR)
ISSR-PCR-based reaction was performed to detect the polymorphism among kalanchoe genotypes using the primers presented in Table 2. The reaction was conducted
according to procedures described by Moreno, Martín and Ortiz [42]. The ISSR-PCR-based
amplifications were conducted in a 25 µL reaction mixture containing 20 ng/µL of template
DNA, 2 µL 5× buffer; 2 µL MgCl2 (25 mM), 2 µL dNTPs (200 µM), 2 µL Primer (10 pmol)
and 1 Unit Taq DNA polymerase (Promega). The conditions of ISSR-PCR amplifications
were used in an initial denaturation of 94 ◦ C for 5 min followed by 35 cycles of 94 ◦ C
for 1 min, the annealing temperature for the various primers for 1 min, 72 ◦ C for 1 min
(extension) and final extension at 72 ◦ C for 5 min.
4.6. Start Codon Targeted (SCoT) Amplification
The SCoT primers (Table 2) were chosen according to [17]. The SCoT-PCR-based
reactions were conducted in a 25 µL reaction mixture containing 25 ng template DNA,
0.2 µM dNTPs, 1.5 µM of each primer, 1.5 mM MgCl2 , 2 µL 5× buffer and 1 Unit of Taq
polymerase (Promega). The PCR amplification was set up for the initial denaturation at
94 ◦ C for 5 min, followed by 35 cycles, each cycle comprised of 94 ◦ C for 1 min, 53 ◦ C for
1 min, then 72 ◦ C for 120s, and the final extensions were done at 72 ◦ C for 7 min.
4.7. Gel Electrophoresis
1.5% agarose gel electrophoresis in TBE buffer was used to separate the results of the
RAPD, ISSR, and SCoT reactions according to [43]. The size of DNA bands on the gel was
calculated using 100 bp DNA ladder (GeneRuler 100 bp Plus DNA Ladder, Thermo Fischer
Scientific, USA). Ethidium bromide (MP Biomedicals, Goddard Irvine, CA, USA) was used
to stain the agarose gel that was further visualized using UV illuminator (VilberLourmat,
France). The frequency of polymorphisms and the number of bands produced by each
primer were calculated individually.
4.8. Data Analysis
Microsoft Excel was used to analyze the morphological data. For the number of petals,
flower length (cm), flower diameter (mm), number of inflorescences, number of flowers,
and plant height, significant differences were evaluated using Student t-tests at p ≤ 0.05.
The SCot, ISSR and RAPD-based PCR loci were scored as present 1 or absent 0, each
of which was treated as independent. Genetic diversity was identified by comparing the
banding patterns of all genotypes. Polymorphism levels were estimated by dividing the
polymorphic loci by the total number of scored loci. Percentage fidelity of the RAPD/SCoT
was calculated from the following equation (number unique bands of RAPD-PCR/number
unique bands ofSCoT-PCR × 100—total number of RAPD loci/total number of SCot
loci ×100). 100 bp ladder (Invitrogen, Waltham, MA, USA) was used to estimate band
size. Genetic similarities among kalanchoe genotypes were calculated according to Dice
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coefficient measurement [44] using IBM SPSS statistics software [45]. The clustering analysis
method was used to generate the phylogeny dendrogram [46] using STATISTICA 8 software
package [47].
5. Conclusions
This work discussed the genetic relationship among five kalanchoe genotypes through
the analysis of morphological and molecular features. The information about molecular and
phenotypic properties has great importance in the future selection of breeding populations,
particularly for traits that possess commercial values. According to the findings, orange,
violet, and yellow kalanchoe are distinct parents for breeding commercially valuable
kalanchoe features. The study also concluded that SCoT and RAPD markers exhibited
accurate banding pattern profiles to analyze the genetic diversity of kalanchoe genotypes
as they gave a high polymorphism percentage among kalanchoe genotypes compared with
ISSR marker that gave a very low polymorphism percentage.
Author Contributions: Conceptualization, A.A.H., A.A.H.A.L., J.M.A.-K. and M.F.A.; methodology,
A.A.H., E.M.B.M. and H.S.A.T.; software, A.S.E. and M.A.A.-E.; validation, A.A.H. and H.S.A.T.;
formal analysis, A.A.H. and E.M.B.M.; investigation, A.A.H., E.M.B.M. and H.S.A.T.; resources,
M.A.A.-E. and S.M.J.; data curation, M.A.A.-E., A.S.E. and H.S.A.T.; writing original draft preparation,
A.A.H., J.M.A.-K. and M.F.A.; writing review and editing, A.A.H., A.A.H.A.L., J.M.A.-K. and M.F.A.;
supervision A.A.H., A.A.H.A.L. and J.M.A.-K.; funding acquisition, J.M.A.-K., A.A.R., W.F.S., M.I.A.,
T.A.S., M.N.S., H.S.G., K.M.A. and M.F.A. All authors have read and agreed to the published version
of the manuscript.
Funding: This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Project No. AN000158],
and the Taif University Researchers Supporting Project number (TURSP—2020/111), Taif University,
Taif, Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors extend their appreciation forthe Deanship of Scientific Research,
Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia
[Project No. AN000158], and the Taif University Researchers Supporting Project number (TURSP—
2020/111), Taif University, Taif, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
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