Identifying molecular differences using ISSR markers on three quinoa genotypes (Chenopodiumm quinoa) under Sinai conditions, Egypt

Abstract

This study aimed to evaluate the performance of three quinoa genotypes (Giza1, Danish KVL3704, and Misr1) under three irrigation intervals (every three, six, and nine days). Genetic diversity among nine quinoa accessions was assessed using eight ISSR primers, yielding robust amplification products and polymorphic fingerprint patterns. A total of 102 bands were generated, averaging 12.75 bands per primer. Among these, 52 fragments were polymorphic, resulting in an average of 6.5 polymorphic bands per primer and an overall polymorphism level of 40.9%. Primer ISSR-8 exhibited the highest polymorphic capacity with 17 polymorphic bands, while primer ISSR-15 displayed the highest frequency (0.9). In contrast, primers ISSR-8 and ISSR-10 exhibited the lowest frequency (0.5). The polymorphism percentage ranged from 20% for primer ISSR-15 to 88% for primer ISSR-10. The similarity index revealed a minimum value of 78% between treatments, clustering the nine accessions into eight groups across four similarity levels, from 80% to a maximum of 93%. Dendrogram analysis underscored the utility of ISSR-PCR in detecting genetic relationships among quinoa accessions. These findings highlight the potential of ISSR-PCR as a reliable tool for genetic diversity studies and its applicability in quinoa breeding programs.

Full text

Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202401
Research Article
Open Access
1
Plant Production Department, Faculty of
Environmental Agricultural Sciences, Arish
University, North Sinai, Egypt.
2
Department of Agronomy, Faculty of Agricul-
ture, Suez Canal University, Ismailia 41522,
Egypt.
3
Agricultural Genetic Engineering Research
Institute, Agricultural Research Center, Giza,
Egypt.
4
Faculty of Environmental Agricultural Sciences,
Arish University, Egypt.
5
Department of Agronomy, Faculty of Agricul-
ture, Cairo University, Giza, Egypt.
6
Department of Biological Sciences,Environ-
mental Studies Institution, Arish University,
Egypt.
*To whom correspondence should be ad-
dressed: mobark.mohamed99@yahoo.com
Editor: Morad Mokhtar, University Mohammed VI
Polytechnic, BenGuerir, Morocco.
Reviewer(s):
Suleiman Aminu, Department of Biochemistry,
Ahmadu Bello University, Zaria, Nigeria.
Fida Alo, International Center for Agricultural
Research in the Dry Areas (ICARDA)
Received: February 19, 2024
Accepted: September 9, 2024
Published: December 24, 2024
Citation: Mubarak MH, Emam MA, Shafik
DI, Moatz AM, Abd El-Wahab MMH, Ibrahim
AAH. Identifying molecular differences using
ISSR markers on three quinoa genotypes
(Chenopodiumm quinoa) under Sinai conditions,
Egypt . 2024 Dec. 24;7:bs202401
Copyright: ©2024 Mubarak MH et al.. This is
an open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and supplementary materials.
Funding: The authors have no support or funding to
report.
Competing interests: The authors declare that they
have no competing interests.
Identifying molecular differences using ISSR markers on three quinoa
genotypes (Chenopodiumm quinoa) under Sinai conditions, Egypt
Mohamed H. Mubarak
1>< �
, Mohamed A. Emam
2>< �
, Shafik D. Ibrahim
3>< �
,
Moatz A. Mohamed
4>< �
, Mustafa M. H. Abd El-Wahab
5>< �
, Ahmed A.
Hegazy6>< �
Abstract
This study aimed to evaluate the performance of three quinoa genotypes (Giza1, Danish
KVL3704, and Misr1) under three irrigation intervals (every three, six, and nine days).
Genetic diversity among nine quinoa accessions was assessed using eight ISSR primers,
yielding robust amplification products and polymorphic fingerprint patterns. A total
of 102 bands were generated, averaging 12.75 bands per primer. Among these, 52
fragments were polymorphic, resulting in an average of 6.5 polymorphic bands per
primer and an overall polymorphism level of 40.9%. Primer ISSR-8 exhibited the
highest polymorphic capacity with 17 polymorphic bands, while primer ISSR-15
displayed the highest frequency (0.9). In contrast, primers ISSR-8 and ISSR-10
exhibited the lowest frequency (0.5). The polymorphism percentage ranged from
20% for primer ISSR-15 to 88% for primer ISSR-10. The similarity index revealed a
minimum value of 78% between treatments, clustering the nine accessions into eight
groups across four similarity levels, from 80% to a maximum of 93%. Dendrogram
analysis underscored the utility of ISSR-PCR in detecting genetic relationships among
quinoa accessions. These findings highlight the potential of ISSR-PCR as a reliable
tool for genetic diversity studies and its applicability in quinoa breeding programs.
Keywords: Quinoa, genotypes, ISSR primers, Similarity index, relationship.
Introduction
Quinoa (Chenopodium quinoa) is a pseudo-grain that produces seed-like grains, commonly used
as whole grains in bread and soups and as a versatile ingredient for people with celiac disease. It is
considered one of the most nutritious crops, offering a rich source of protein, essential amino acids,
and fiber, making it an excellent alternative for gluten-free diets. Quinoa demonstrates significant
potential as a sustainable crop for cultivation in salt-affected areas [
1
]. Water scarcity, climate change,
and rapid population growth are significant challenges in Egypt, putting significant pressure on the
country's agricultural productivity and food security. Egypt has extensive marginal lands unsuitable
for agriculture due to high soil salinity or low-quality groundwater. However, these lands are viewed
as an opportunity to enhance agricultural production and ensure food security by cultivating salt-
tolerant crops like quinoa, which is considered one of the strategic solutions to cope with the high
food demand of the growing population. With quinoa's adaptability to harsh conditions, it could
play a critical role in improving agricultural sustainability and helping meet the nutritional needs of
the population [
2
;
3
]. Molecular markers are powerful tools for cultivar identification, germplasm
conservation, and detecting genetic variability in various species. ISSR (inter-simple sequence
repeat) markers are inexpensive and are easily scored manually, and prior knowledge of the flanking
sequences is not required, making them ideal for large-scale genetic studies. The development of
polymerase chain reaction (PCR) has made PCR-based marker systems key tools in genetic analyses.
ISSR-PCR generates reproducible, abundant, and polymorphic multilocus patterns in plant genomes
[4;5].
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Mubarak MH et al., 2024 Quinoa molecular differences using ISSR markers under Sinai conditions
This study aims to evaluate the performance of various quinoa
(Chenopodium quinoa) genotypes under different irrigation inter-
vals and stress conditions. With a focus on identifying drought
and salinity tolerant genotypes suitable for marginal lands in
Egypt. Additionally, to assess the genetic diversity of these
quinoa genotypes using ISSR markers to inform breeding pro-
grams aimed at developing high-yielding, salt-tolerant quinoa
varieties [6;7].
Materials and methods
Field Experiment
The planting and harvesting activities were conducted during
the 2023/2024 agricultural season, and following the DNeasy
kit technique (Qiagen) [
8
], DNA was extracted and processed
to ensure high-quality DNA for further analysis. To maintain
integrity and avoid freezing, fresh quinoa leaf tissue was crushed
using a mortar and pestle in liquid nitrogen. After transferring
the powdered tissue to a tube, up to 100 mg of wet tissue tissue
were mixed with 400
µ
l of Buffer AP1 and 4
µ
l of RNase A (100
mg/ml). The mixture was vortexed thoroughly, it was incubated
at 65
o
C for 10 minutes to lyse the cells, and then cooled on ice
for 5 minutes with the addition of 130
µ
l buffer AP2 to precipitate
contaminants. The lysate was transferred to a spin column of
a QIA shredder and centrifuged at high speed to get rid of cell
debris [9].
The flow-through was collected and combined with 1.5 ml of
buffer AP3/E. After centrifuging the resultant solution and dis-
carding the flow-through, 650
µ
l pieces of it were put to a DNeasy
mini spin column. Washing steps were performed using 500
µ
l
of buffer AW, It was centrifuged once for one minute and then
again for two minutes to dry it. Lastly, 100
µ
l of preheated (65
o
C)
buffer AE was applied to the DNeasy membrane, incubating for
5 minutes at room temperature, and centrifuging. The elution
buffer was 50 µl for higher concentration DNA [10;11;12].
Agarose gel electrophoresis was used to estimate the isolated
DNA concentration. A small portion of the DNA sample was
loaded onto a 1% agarose gel alongside a DNA ladder with
known fragment sizes. The concentration of the extracted DNA
can be estimated by comparing the intensity of its bands to those
of the DNA ladder [13].
ISSR-PCR Reactions
A total of 8 ISSR primers
(Table 1)
were used to detect
polymorphisms. The amplification reaction was conducted in a
25
µ
l reaction volume, consisting of 1X PCR buffer, 1.5
mM
MgCl2
, 0.2
mM
dNTPs, 1
µ
M primer, 1 U Taq DNA polymerase,
and 30 ng template DNA [14].
A Perkin-Elmer/
GeneAmp®
PCR System 9700 (PE Applied
Biosystems) was used for the PCR amplification. It was set up
to complete 35 cycles following a 5-minute initial denaturation
cycle. The steps in each cycle were 94
o
C for 1 minute of de-
naturation, 50
o
C for 1 minute of annealing, and 72
o
C for 1.5
minutes of elongation. In the final cycle, the primer extension
was extended to 7 minutes at 72oC [15;16].
Electrophoresis was used to resolve the amplification prod-
ucts in a 1.5% agarose gel with 0.5
µ
g/ww ml of ethidium bro-
mide in 1X TBE buffer at 95 volts. The criterion for molecular
size was a 100 bp DNA ladder. The PCR products were visual-
ized under UV light and photographed using a gel documentation
system (BIO-RAD 2000) [17].
Table 1. ISSR primer names and sequence.
No. Name Primer Sequence 5’-3’
1 ISSR- 1 5’-AGAGAGAGAGAGAGAGYC-3’
2 ISSR- 5 5’-GTGTGTGTGTGTGTGTYG-3’
3 ISSR- 8 5’-AGACAGACAGACAGACGC-3’
4 ISSR- 10 5’-GACAGACAGACAGACAAT-3’
5 ISSR- 13 5’-AGAGAGAGAGAGAGAGYT-3’
6 ISSR- 14 5’-CTCCTCCTCCTCCTCTT-3’
7 ISSR- 15 5’-CTCTCTCTCTCTCTCTRG-3’
8 ISSR- 16 5’-TCTCTCTCTCTCTCTCA-3’
Data Analysis
The genetic relatedness of the samples under investigation
was ascertained by comparing the banding patterns produced by
ISSR-PCR marker tests. Clear and distinct amplification products
were scored as 1 for the presence and 0 for the absence of bands.
Bands with the same mobility were considered identical. The
genetic similarity coefficient (GS) between two genotypes was
calculated using the dice coefficient [18;19].
GS i j =2a
2a+b+c
The genetic similarity coefficient
GS i j
represents the genetic
similarity between individuals
i
and
j
,
a
is the number of bands
shared by both
i
and
j
,
b
is the number of bands present in
i
and
absent in
j
, and
c
is the number of bands present in
j
and absent
in i.
Cluster analysis was performed on the similarity matrix us-
ing the unweighted pair group method with arithmetic mean
(UPGMA) [20].
Results and Discussions
Polymorphism Analysis Identified Using ISSR Markers
In this study, eight ISSR primers were used for the analysis
of nine Quinoa accessions, producing amplification products that
resulted in polymorphic fingerprint patterns as shown in
Figure
1
and
Table 2
. The eight PCR primers generated a total of 102
bands, with an average of 12.75 bands per primer. Of these 102
amplified fragments, 52 were polymorphic, yielding an average
of 6.5 polymorphic bands per primer. This corresponds to a
polymorphism rate of 40.9% across the eight primers. Primer
ISSR-8 produced the highest number of polymorphic bands, with
17 polymorphic amplification products detected. In contrast,
primer ISSR-15 yielded the fewest polymorphic fragments, with
only 2 detected. The polymorphism percentage ranged from 20%
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Mubarak MH et al., 2024 Quinoa molecular differences using ISSR markers under Sinai conditions
Figure 1. ISSR profiles of the using primer ((A) ISSR-8, (B) ISSR-13, (C)
ISSR-14 and (D) ISSR- 16). 1 to 9 Samples accession, M: DNA molecular
weight marker (100bp DNA ladder).
(for primer ISSR-15) to 88% (for primer ISSR-10). Primer ISSR-
15 exhibited the highest frequency (0.9), while primers ISSR-8
and ISSR-10 showed the lowest frequency (0.5).
Table 2. Eight ISSR used in this study, the total bands (TB), monmorphic bands
(MB), polymorphic bands (PB), percentage of polymorphic bands (%PB) and
frequency (F).
Primer TB MB PB %PB F
ISSR- 1 9 6 3 33 0.8
ISSR- 5 15 9 6 40 0.8
ISSR- 8 22 5 17 77 0.5
ISSR- 10 8 1 7 88 0.5
ISSR- 13 11 5 6 55 0.6
ISSR- 14 11 6 5 45 0.7
ISSR- 15 10 8 2 20 0.9
ISSR- 16 16 10 6 38 0.8
Total 102 50 52
Average 12.75 6.25 5.5 40.9 0.7
The results of primer ISSR-1 were illustrated in (
Table S1
).
It gave 6 monomorphic and 3 polymorphic bands with different
fragment sizes ranging from 160 to 958 bp for Chenopodium
quinoa. Six common bands were observed across all sites at
fragment sizes of 160, 196, 278, 323, 498, and 747 bp. A band
with a fragment size of 248 bp was exclusively present in Giza
1 with irrigation every 3 days (I3), Giza 1 with irrigation every
6 days (I6), and Misr 1 across all treatments, but was absent in
Giza 1 with irrigation every 9 days (I9) and Danish Kvl3704
in all treatments. A fragment size of 426 bp was exclusively
present in Giza 1 (I3 and I6), Danish Kvl3704 (I3), and Misr 1
under all treatments, but it was absent in Giza 1 under I9 and in
Danish Kvl3704 under I6 and I9. A fragment size of 958 bp was
exclusively present in Danish Kvl3704 with I3 and Misr 1 under
all treatments, but it was absent in Giza 1 under all treatments
and in Danish Kvl3704 under I6 and I9.
The results of primer ISSR-5 were illustrated in (
Table S2
).
It gave 9 monomorphic and 6 polymorphic bands with different
fragment sizes ranging from 132 to 989 bp for Chenopodium
quinoa. Nine common bands were observed across all sites at
fragment sizes of 132, 158, 170, 263, 312, 372, 430, 538, and 637
bp. A band with a fragment size of 190 bp was exclusively present
in Giza 1 across all treatments, but was not found in Danish
Kvl3704 or Misr 1 across all treatments. A fragment size of 214
bp was exclusively present in Misr 1 across all treatments, but
was not found in Giza 1 or Danish Kvl3704 across all treatments.
A fragment size of 235 bp was exclusively present in Giza 1
and Misr 1 across all treatments, but was not found in Danish
Kvl3704 across all treatments. In contrast, a fragment size of 703
bp was exclusively present in Giza 1 and Danish Kvl3704 across
all treatments, but was absent in Misr 1 across all treatments. A
fragment size of 772 bp was exclusively present in Misr 1 across
all treatments, but was not found in Giza 1 or Danish Kvl3704
across all treatments. A fragment size of 989 bp was exclusively
present in Giza 1 (I3), Danish Kvl3704 (I6), and Misr 1 across
all treatments, but was absent in Giza 1 (I6 and I9) as well as
Danish Kvl3704 (I3 and I9).
The results of primer ISSR-8 were illustrated in (
Table S3
).
It gave 5 monomorphic and 17 polymorphic bands with different
fragment sizes ranging from 180 to 1250 bp for Chenopodium
quinoa. Five common bands were observed in all sites at frag-
ment sizes of 350, 460, 500, 830, and 1100 bp. A band with a
fragment size of 180 bp was exclusively present in Giza 1 in (I3),
but it was not found in Giza 1 with (I6 and I9), Misr 1 under all
treatments, and Danish Kvl3704 under all treatments. A frag-
ment size of 200 bp was exclusively present in Giza 1 under all
treatments, but it was not found in Misr 1 under all treatments
and Danish Kvl3704 under all treatments. Respectively, a frag-
ment size of 210 bp was exclusively present in Danish Kvl3704
with (I9), Misr 1 in (I3), as well as Misr 1 with (I6), but it was
not found in Giza 1 under all treatments, Danish Kvl3704 in (I3
and I6), and Misr 1 with (I9). A fragment size of 240 bp was
exclusively present in Danish Kvl3704 under all treatments and
Misr 1 with (I6 and I9), but it was not found in Giza 1 under
all treatments and Misr 1 under (I3). A fragment size of 260
bp was exclusively present in Giza 1 under (I3 and I6), Danish
Kvl3704 in (I3 and I9), as well as Misr 1 with (I3 and I9), but it
was not found in Giza 1 under (I9), Danish Kvl3704 with (I6),
and Misr 1 under (I6). Respectively, a fragment size of 290 bp
was exclusively present in Giza 1 in (I3 and I6), Danish Kvl3704
with (I6), and Misr 1 under (I3), but it was not found in Giza 1
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Mubarak MH et al., 2024 Quinoa molecular differences using ISSR markers under Sinai conditions
with (I9), Danish Kvl3704 under (I3 and I9), as well as Misr 1
with (I6 and I9).
A fragment size of 300 bp was exclusively present in Giza
1 under (I9), Danish Kvl3704 with (I3 and I9), as well as Misr
1 under (I6 and I9), but it was not found in Giza 1 with (I3
and I6), Danish Kvl3704 under (I6), and Misr 1 with (I3). A
fragment size of 320 bp was exclusively present in Giza 1 with all
treatments and Danish Kvl3704 under (I6), but it was not found in
Danish Kvl3704 under (I3 and I9) and Misr 1 with all treatments.
Respectively, a fragment size of 390 bp was exclusively present
in Giza 1 under all treatments and Danish Kvl3704 in (I3), but it
was not found in Danish Kvl3704 with (I6 and I9) as well as Misr
1 under all treatments. A fragment size of 410 bp was exclusively
present in Danish Kvl3704 under (I6 and I9), but it was not found
in Giza 1 with all treatments, Danish Kvl3704 in (I3), and Misr 1
under all treatments. A fragment size of 610 bp was exclusively
present in Giza 1 under (I6 and I9) as well as Misr 1 in (I6), but
it was not found in Giza 1 under (I3), Danish Kvl3704 under all
treatments, and Misr 1 with (I3 and I9). A fragment size of 650
bp was exclusively present in Giza 1 under (I3 and I9) as well as
Misr 1 with (I3), but it was not found in Giza 1 under (I6), Danish
Kvl3704 in all treatments, as well as Misr 1 with (I6 and I9). A
fragment size of 730 bp was exclusively present in Giza 1 with all
treatments, Danish Kvl3704 under (I9), and Misr 1 under (I3 and
I6), but it was not found in Danish Kvl3704 with (I3 and I6) as
well as Misr 1 with (I9). Respectively, a fragment size of 900 bp
was exclusively present in Giza 1 with (I6), but it was not found
in Giza 1 with (I3 and I9), Danish Kvl3704 under all treatments,
and Misr 1 with all treatments. A fragment size of 1000 bp was
exclusively present in Giza 1 with (I6), but it was not found in
Giza 1 under (I3 and I9), Danish Kvl3704 with all treatments,
and Misr 1 under all treatments. A fragment size of 1150 bp
was exclusively present in Giza 1 under all treatments, but it
was not found in Danish Kvl3704 with all treatments and Misr
1 in all treatments. A fragment size of 1250 bp was exclusively
present in Giza 1 with (I9), but it was not found in Giza 1 with
(I3 and I6), Danish Kvl3704 in all treatments, and Misr 1 under
all treatments.
The results of primer ISSR-10 were illustrated in
(Table S3)
.
It gave 1 monomorphic and 7 polymorphic bands with different
fragment sizes ranging from 265 to 840 bp for Chenopodium
quinoa. One common band was observed in all sites at a frag-
ment size of 316 bp. A band with a fragment size of 265 bp was
exclusively present in Giza 1 under (I6 and I9), Danish Kvl3704
in all treatments, and Misr 1 with all treatments, but it was not
found in Giza 1 with (I3). A fragment size of 378 bp was exclu-
sively present in Giza 1 under (I3 and I9), Danish Kvl3704 with
all treatments, as well as Misr 1 under (I3 and I9), but it was not
found in Giza 1 with (I6) and Misr 1 under (I6). A fragment size
of 426 bp was exclusively present in Danish Kvl3704 under all
treatments and Misr 1 in (I6), but it was not found in Giza 1 under
all treatments as well as Misr 1 with (I3 and I9). A fragment size
of 462 bp was exclusively present in Giza 1 with (I9) and Misr
1 under (I6), but it was not found in Giza 1 under (I3 and I6),
Danish Kvl3704 with all treatments, as well as Misr 1 under (I3
and I9). A fragment size of 570 bp was exclusively present in
Giza 1 under (I9), Danish Kvl3704 with (I6 and I9), as well as
Misr 1 in (I9), but it was not found in Giza 1 under (I3 and I6),
Danish Kvl3704 in (I3), and Misr 1 with (I3 and I6). A fragment
size of 699 bp was exclusively present in Danish Kvl3704 under
(I6 and I9) and Misr 1 with (I9), but it was not found in Giza 1
under all treatments, Danish Kvl3704 with (I3), and Misr 1 under
(I3 and I6). A fragment size of 840 bp was exclusively present
in Giza 1 with (I9), but it was not found in Giza 1 under (I3 and
I6), Danish Kvl3704 with all treatments, and Misr 1 under all
treatments.
The results of primer ISSR-13 were illustrated in (
Table S4
).
It gave 5 monomorphic and 6 polymorphic bands with different
fragment sizes ranging from 177 to 1074 bp for Chenopodium
quinoa. Five common bands were observed in all sites at frag-
ment sizes of 177, 264, 345, 397, and 472 bp. A band with a
fragment size of 214 bp was exclusively present in Giza 1 under
(I6) as well as Misr 1 with (I9), but it was not found in Giza 1
under (I3 and I9), Danish Kvl3704 with all treatments, as well
as Misr 1 under (I3 and I6). A fragment size of 217 bp was
exclusively present in Giza 1 with (I3), Danish Kvl3704 under
(I3 and I9), and Misr 1 with (I6), but it was not found in Giza 1
under (I6 and I9), Danish Kvl3704 with (I6), and Misr 1 in (I3
and I9). A fragment size of 510 bp was exclusively present in
Giza 1 under (I3) and Danish Kvl3704 with (I3 and I6), but it
was not found in Giza 1 with (I6 and I9), Danish Kvl3704 in (I9),
and Misr 1 under all treatments. Respectively, a fragment size of
550 bp was exclusively present in Giza 1 with (I9 and I6), Danish
Kvl3704 in (I9), and Misr 1 under all treatments, but it was not
found in Giza 1 under (I3) as well as Danish Kvl3704 with (I3
and I6). A fragment size of 666 bp was exclusively present in
Giza 1 with (I3 and I6) as well as Danish Kvl3704 under (I9), but
it was not found in Giza 1 under (I9), Danish Kvl3704 with (I3
and I6), and Misr 1 under all treatments. A fragment size of 1074
bp was exclusively present in Danish Kvl3704 with (I9), but it
was not found in Giza 1 under all treatments, Danish Kvl3704
with (I3 and I6), and Misr 1 under all treatments.
The results of primer ISSR-14 were illustrated in
(Table S5)
.
It gave 6 monomorphic and 5 polymorphic bands with different
fragment sizes ranging from 720 to 2000 bp for Chenopodium
quinoa. Six common bands were observed in all sites at fragment
sizes of 720, 790, 860, 950, 1200, and 1500 bp. A band with
a fragment size of 1050 bp was exclusively present in Giza 1
with (I3) and Misr 1 under (I9), but it was not found in Giza 1
with (I6 and I9), Danish Kvl3704 under all treatments, as well
as Misr 1 under (I3 and I6). A fragment size of 1100 bp was
exclusively present in Giza 1 under (I9) and Danish Kvl3704
with (I9), but it was not found in Giza 1 with (I3 and I6), Misr
1 under all treatments, and Danish Kvl3704 in (I3 and I6). A
fragment size of 1350 bp was exclusively present in Giza 1 under
(I9) and Misr 1 with (I9), but it was not found in Giza 1 under (I3
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Mubarak MH et al., 2024 Quinoa molecular differences using ISSR markers under Sinai conditions
Figure 2. Dendogram of similarity index (SI) between the nine studied treat-
ments.
and I6), Danish Kvl3704 in all treatments, as well as Misr 1 with
(I3 and I6). A fragment size of 1650 bp was exclusively present
in Giza 1 with (I3) and Misr 1 under (I9), but it was not found
in Giza 1 under (I6 and I9), Danish Kvl3704 with all treatments,
and Misr 1 under (I3 and I6). A fragment size of 2000 bp was
exclusively present in Giza 1 with (I3), Danish Kvl3704 under
(I9), and Misr 1 in (I9), but it was not found in Giza 1 under (I6
and I9), Danish Kvl3704 in (I3 and I6), and Misr 1 with (I3 and
I6).
The results of primer ISSR-15 were illustrated in (
Table S6
).
It gave 8 monomorphic and 2 polymorphic bands with different
fragment sizes ranging from 310 to 1050 bp for Chenopodium
quinoa. Eight common bands were observed in all sites at frag-
ment sizes of 310, 490, 520, 630, 780, 850, 960, and 1050 bp. A
band with a fragment size of 420 bp was exclusively present in
Giza 1 with (I6), Danish Kvl3704 under all treatments, and Misr
1 with all treatments, but it was not found in Giza 1 under (I3 and
I9). A fragment size of 720 bp was exclusively present in Giza 1
with (I3), but it was not found in Giza 1 with (I6 and I9), Danish
Kvl3704 under all treatments, and Misr 1 with all treatments.
The results of primer ISSR-16 were illustrated in (
Table S7
).
It gave 10 monomorphic and 6 polymorphic bands with differ-
ent fragment sizes ranging from 136 to 944 bp for Chenopodium
quinoa. Ten common bands were observed in all sites at fragment
sizes of 136, 342, 144, 465, 390, 546, 702, 741, 814, and 862 bp.
A band with a fragment size of 324 bp was exclusively present in
Danish Kvl3704 with (I9) and Misr 1 under all treatments, but
it was not found in Giza 1 with all treatments, Danish Kvl3704
under (I3 and I6), as well as Misr 1 in all treatments. A fragment
size of 275 bp was exclusively present in Giza 1 with all treat-
ments and Danish Kvl3704 under all treatments, but it was not
found in Misr 1 under all treatments. A fragment size of 502 bp
was exclusively present in Misr 1 with all treatments, but it was
not found in Misr 1 under all treatments, Giza 1 in all treatments,
and Danish Kvl3704 with all treatments. A fragment size of
591 bp was exclusively present in Giza 1 under all treatments
and Misr 1 with all treatments, but it was not found in Danish
Kvl3704 with all treatments. A fragment size of 908 bp was
exclusively present in Danish Kvl3704 under all treatments and
Misr 1 with all treatments, but it was not found in Giza 1 under
all treatments. A fragment size of 944 bp was exclusively present
in Giza 1 under all treatments, but it was not found in Danish
Kvl3704 under all treatments and Misr 1 with all treatments.
Table 3. Similarity index (SI) of nine treatments based on ISSR PCR analysis.
1 2 3 4 5 6 7 8 9
1 100
2 89 100
3 85 88 100
4 82 83 81 100
5 81 81 81 91 100
6 78 80 81 89 89 100
7 83 85 81 85 82 83 100
8 78 82 81 87 81 84 93 100
9 80 81 79 85 82 84 91 90 100
The similarity index (SI) based on ISSR–PCR is shown in
Table 3
as a cluster analysis in percentage. Among all studied
treatments, the similarity index recorded the highest value be-
tween treatments (7 and 8), while the lowest value was recorded
(78%) between treatments (6 and 1).
The dendrogram in (
Figure 2
) was based on the results of
the similarity index. It was stratified into eight clusters indicating
the relationship between the nine studied treatments, arranged in
four levels of similarity from (80%) as the lowest level to (93%)
as the highest recorded level.
In this respect, [
21
] have used seven ISSR markers to charac-
terize and genetically compare five quinoa genotypes. The results
revealed 85 loci, of which 37 were polymorphic, accounting for
43.53% of the total. The number of amplified loci varied from
four (UBC-817) to 26 (UBC-845). The size of the amplified
fragments ranged from 139 to 1,111 bp. Additionally, the overall
PIC values ranged from 0.00 to 0.4. Likewise, [
22
] have used
10 ISSR primers to genetically differentiate among five quinoa
genotypes. The results yielded 53 amplicons, 33 of which were
polymorphic, resulting in an average polymorphism percentage
of 61.83%. With an average of 5.3 pieces per primer, the range of
amplicons per primer was three to ten. There were between one
and six polymorphic amplicons, with an average of 3.3 pieces
per primer. Finally, the size of the amplified fragments varied
depending on the ISSR primer employed, ranging from 130 to
1,456 bp. Fifteen ISSR primers were employed in this study,
producing a total of 172 amplified bands, with an average of 11.7
bands per primer. A total number of 90 amplified polymorphic
Highlights in BioScience Page 5 of 7December 2024|Volume 7

Mubarak MH et al., 2024 Quinoa molecular differences using ISSR markers under Sinai conditions
bands were obtained, with an average of 6.0 bands per primer;
the average level of polymorphism was 49.7%. The effectiveness
of ISSR markers in distinguishing the studied genotypes was
assessed by calculating the PIC values, which ranged from 0.21
to 0.84, with an average value of 0.69 [23].
Conclusions
The obtained deference’s of the dendrogram indicated that
ISSR–PCR was useful in detecting the similarity between the
studied treatments. Also, it could be recommended as a suit-
able candidate method between the studied treatment in further
breeding ISSR programs to evaluate some quinoa genotypes to
different irrigation intervals on stress conditions.
Supplementary
Table S1: Survey of olymorphic and monomorphic ISSR
bands in Chenopodium Quinoa using primer ISSR-1. Table
S2: Survey of polymorphic and monomorphic ISSR bands in
Chenopodium Quinoa using primer ISSR-5. Table S3: Survey
of polymorphic and monomorphic ISSR bands in Chenopodium
Quinoa using primer ISSR-8. Table S4: Survey of polymor-
phic and Monomorphic ISSR bands in Chenopodium Quinoa
using primer ISSR-10. Table S5: Survey of polymorphic and
Monomorphic ISSR bands in Chenopodium Quinoa using primer
ISSR-13. Table S6: Survey of polymorphic and Monomorphic
ISSR bands in Chenopodium Quinoa using primer ISSR-14. Ta-
ble S7: Survey of polymorphic and Monomorphic ISSR bands in
Chenopodium Quinoa using primer ISSR-15. Table S8: Survey
of polymorphic and Monomorphic ISSR bands in Chenopodium
Quinoa using primer ISSR-16.
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