Journal of Natural Fibers

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/wjnf20

Characterization and Genetic Diversity of

Photoperiodic among Mutant Kenaf (Hibiscus
Cannabinus L.) Using EST-SSR Markers

Md Al-Mamun, Mohd Y. Rafii, Yusuff Oladosu, Azizah Binti Misran, Zulkarami

Berahim, Zaiton Ahmad, Fatai Arolu & Md Mahmudul Hasan Khan

To cite this article: Md Al-Mamun, Mohd Y. Rafii, Yusuff Oladosu, Azizah Binti Misran, Zulkarami
Berahim, Zaiton Ahmad, Fatai Arolu & Md Mahmudul Hasan Khan (2021): Characterization and
Genetic Diversity of Photoperiodic among Mutant Kenaf (Hibiscus�Cannabinus L.) Using EST-SSR
Markers, Journal of Natural Fibers, DOI: 10.1080/15440478.2021.2002762

To link to this article: https://doi.org/10.1080/15440478.2021.2002762

Published online: 29 Dec 2021.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=wjnf20
JOURNAL OF NATURAL FIBERS
https://doi.org/10.1080/15440478.2021.2002762

Characterization and Genetic Diversity of Photoperiodic among

Mutant Kenaf (Hibiscus Cannabinus L.) Using EST-SSR Markers
Md Al-Mamuna,b, Mohd Y. Rafii a,c, Yusuff Oladosu a, Azizah Binti Misranc,
Zulkarami Berahima, Zaiton Ahmadd, Fatai Arolua, and Md Mahmudul Hasan Khana,e
a
Laboratory of Climate- Universiti Putra Malaysia, Serdang, Malaysia; bBreeding Division, Bangladesh Jute Research
Institute (BJRI), Dhaka, Bangladesh; cDepartment of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia,
Serdang, Malaysia; dAgrotechnology and Bioscience Division, Malaysian Nuclear Agency, Bangi, Kajang, Malaysia;
e
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh

ABSTRACT 关键词
Information on molecular genetic diversity among Hibiscus cannabinus 红麻; 特征化; 关联分析; 突
L. lines is crucial for developing a new variety from the existing 变育种; 遗传多样性
germplasms based on consumer demand. Based on this premise, the KEYWORDS
allelic diversity of thirty-one kenaf genotypes were assessed using Kenaf; characterization;
polymorphic EST-SSR. From the 72 EST-SSR primers tested, 10 showed association analysis;
sharp and clear polymorphic bands ranging from 4 (137 to 225 bp) to 7 mutation breeding; genetic
(182 to 382 bp) alleles with a mean value of 5.2 per primer. The diversity; EST-SSR
Polymorphism Information Content (PIC) value ranged from 0.531 to
0.737, with a mean of 0.610, indicating that the genotypes are geneti­
cally diverse. Shannon index estimation varied from 0.982 to 1.515,
whereas 0.123 to 0.405 was recorded within the groups. Genetic differ­
entiation ranged from 0.67 to 1 alongside the average gene flow (Nm)
of 0.024. The AMOVA revealed 76% variation within the population,
while the population gain recorded was 24%. UPGMA dendrogram
indicated the relationship and categorized the evaluated kenaf mutants
into five major groups. This study revealed the existence of significant
variation in kenaf mutants based on EST-microsatellites. These results
could be used for future breeding programs and conservation of germ­
plasms to enhance Hibiscus cannabinus L. genotypes.
摘要
木槿品种间的分子遗传多样性信息对于根据消费者需求从现有种质中开
发新品种至关重要. 基于这一前提, 利用多态性EST-SSR对31个红麻基因
型的等位基因多样性进行了评估. 从72个EST-SSR引物中, 10个表现出清
晰的多态性带, 范围从4个 (137-225 bp) 到7个 (182-382 bp) 等位基因,
平均值为5.2个引物. 多态性信息含量 (PIC) 值在0.531到0.737之间, 平均
值为0.610, 表明基因型具有遗传多样性. Shannon指数估计值在0.982到
1.515之间变化, 而在各组内记录到0.123到0.405. 遗传分化范围为0.67至
1, 平均基因流 (Nm) 为0.024. AMOVA显示种群内有76%的变异, 而记录
的种群增长率为24%. UPGMA树状图表明了这种关系, 并将评估的红麻突
变体分为五大类. 本研究揭示了基于EST微卫星的红麻突变体存在显著变
异. 这些结果可用于今后的育种计划和种质保护, 以提高木槿基因型.

CONTACT Mohd Y. Rafii [email protected] Institute of Tropical Agriculture and Food Security , Universiti Putra Malaysia,
Serdang 43400, Malaysia
Characterization and genetic diversity of photoperiodic among mutant kenaf (Hibiscus cannabinus L.) using EST-SSR Markers
© 2021 Taylor & Francis
2 M. AL-MAMUN ET AL.

Introduction
Kenaf (Hibiscus cannabinus L.), a jute substitute belonging to Malvaceae, recently received great
attention due to its status as a multi-purposes fiber crop. It is presently cultivated in over 20 countries
with several uses such as thermal insulation boards, pulp, energy sources and building materials (Li
et al. 2016). Kenaf is used as an alternative raw material to wood in the textile and paper industries (Al-
Mamun et al. 2020). The economically important product of kenaf is the stem which consists of the
bast (outer bark), which produces a high-quality pulp that is suitable for both technical and textile uses
(for production of carpets, canvases, sacs, cordages, ropes etc.). Furthermore, the core (inner part)
with high hemicellulose and cellulose content is used as an adsorbent in animal bedding and as
a source of bio-ethanol production (Patanè and Sortino 2010). Despite its vast abundance in tropical
countries, kenaf productivity in Malaysia is on a decline owing to the unavailability of high yielding
varieties due to photoperiodic sensitivity. Kenaf has numerous economic importance and offers a wide
range of utility to man; however, efforts are currently lacking on its cultivation expansion by local
producers as the crop offers low economic return due to low production.
The projections of kenaf as an alternative crop in paper and textile industries in Malaysia depend
largely on the possibility of obtaining large quantities of harvest index biomass from the cultivated
kenaf (Al-Mamun et al. 2020). To accomplish this, cultivation and agronomical parameters such as
nitrogen and water use efficiencies, appropriate sowing conditions, planting density and cultivar
selection are among the main determinant factors that must be given due attention. Aside from
agricultural inputs, good management practices are also very essential in ensuring high yield and
adaptability. Due to its tropical origin, kenaf is considered a photoperiod sensitive crop, where the
plant continues its vegetative phase until day length falls below 12 hours or 12 hours and 45 minutes as
reported by Carberry et al. (1992) and Alexopoulou et al. (2013), respectively. In Malaysia, the days to
flowering are categorized as early flowering (31–60 days), intermediate flowering (61–90 days), and
mid-late flowering (91–120 days) (Jeong et al. 2017). Therefore, the understanding and selection of
late-flowering or the less sensitive cultivars is crucial for maximizing yield and productivity in
Malaysia. In addition, the presence of genetic variability is a desirable prerequisite in any successful
breeding program. Classical genetic study (generation means analysis) revealed essential information
that leads to a better understanding of important agronomic traits in kenaf, and selection in the
segregating generations would lead to significant improvement in fiber yield (Al-Mamun et al. 2020).
Modern DNA marker technology is currently the major technique in analyzing plant genetic
resources since morphological markers are greatly affected by environmental factors. Molecular
markers are easily scored, time-efficient, and are not subject to environmental variation. It also
plays a significant role in gene mapping analysis, plant genealogy, genetic map construction, sorting
of characters, and genetic diversity analysis (Chukwu et al. 2019). Different molecular markers,
including ISSR, AFLP and RAPD have been reported in fiber crops. Simple sequence repeat (SSR)
or Microsatellites has shown their unambiguous supremacy, which results from high reproducibility
and abundance, easy scoring, co-dominant nature and extensive coverage. Aside from these, SSR
markers have been widely employed in molecular breeding studies and genetic mapping in plants (Li
et al. 2016). Based on their source, SSRs have been classified into genomic SSRs (gSSRs) and expressed
sequence tag SSRs (EST-SSRs). Genomic SSRs are usually related to non-coding parts, but EST-SSRs
are derived from the genome expressed parts (Li et al. 2004). Germplasm evaluation using SSRs
derived from ESTs may improve the role of genetic markers by analyzing variations in transcribed and
known function genes (Eujayl et al. 2002). EST-SSRs are closely linked to functional genes, which may
control certain important genetic traits (Li et al. 2016). Therefore, EST-SSRs are among the important
genetic markers for marker-assisted breeding, high-density genetic mapping and analysis of genetic
diversity (Li et al. 2016).
The transcribed region of the genome contains EST-SSRs, which may be relatively well conserved
and represent a better relationship between species or varieties. Therefore, this study was conceptua­
lized to enable the development of additional DNA fingerprinting markers for kenaf cultivars. This
 JOURNAL OF NATURAL FIBERS 3

would enable even more precise distinction among cultivars and aid in furthering efforts on Kenaf’s
varieties protection. The study aims to analyze the phylogenetic relationship and genetic diversity
among mutant kenaf accessions using polymorphic markers EST-SSR to find suitable parents for
heterotic hybridization in subsequent breeding programs.

Materials and methods

Genetic materials
The accessions used in this study consist of thirty-one kenaf genotypes, which formed three base
populations: early flowering (31–60 days), intermediate flowering (61–90 days), and mid-late flower­
ing (91–120 days). Among these genotypes, 28 were mutant lines derived from V-36 through acute
and chronic gamma irradiation, 2 commercial varieties from Bangladesh and one commercial variety
from Malaysia (Table 1). In this study, 71 EST-SSR primers reported by Jeong et al. (2017) and Li et al.
(2016) were screened to select suitable markers for the assessment of genetic diversity among the 31
kenaf genotypes (Supplementary Table 1). Following the completion of the PCR, the characteristics of
the polymorphisms and alleles were observed after removing the markers with no clear bands, low
repeatability, or dominant form. Following the screening, only 10 of the primer pairs were deemed
suitable. The 10 EST-SSR primers that showed co-dominance, high repeatability, and high poly­
morphism rates were selected to assess the genetic diversity among the 31 kenaf genotypes as shown in
Table 2.

DNA extraction and quantification

In this study, 0.5 g of two-week-old kenaf leaves consisting of five representatives from each genotype
were bulk harvested for DNA extraction using the CTAB (cetyltrimethylammonium bromide) method
with slight modifications of the method described by Zheng et al. (1995). The DNA pellet formed
during the extraction process was washed with 75% ethanol, before it was dissolved in 50 mL TE buffer
(Tris–ethylenediaminetetraacetic acid) and treated with RNase to remove the impurities. The integrity
of the extracted DNA was assessed using a spectrophotometer (ND1000 USA, NanoDrop
Technologies Inc., Wilmington, DE), where individual samples were measured at the absorbance
ratio from 1.95 to 2.0 of 260 nm divided by 280 nm. The DNA quality was assessed using 1.5% agarose
gel. Lastly, the DNA stock solutions were diluted with 1× TE buffer containing 10 mmol/L Tris–HCl,
1 mmol/L EDTA (ethylenediaminetetraacetic acid), and pH of 8.0 to achieve 70 ng/lL and stored at
−20°C until PCR amplification.

PCR amplification
The DNA thermal cycler (Eppendorf 5331 96 Well PCR Mastercycler Gradient) was used for the PCR
following the modified touchdown protocol. The PCR reaction containing all required components
was prepared in 0.2-mL microcentrifuge tubes. The final volume of 15 μl PCR amplification cocktail
containing; – master mix (7.5 μl), nucleus free water (4.5 μl), forward primer (1 μl), reverse primer
(1 μl) and DNA template (1 μl) were run on the PCR machine. The following conditions were used for
DNA amplification in a programmable Biometra thermocycler (Bio Red, USA): Initial denaturation at
94°C for 5 minutes, followed by 40 cycles of 45 seconds denaturation at 94°C, 45 seconds annealing
(temperature varies with primer), and 90 seconds extension at 72°C. The final extension was con­
ducted for 7 minutes (Ryu et al. 2017).
4 M. AL-MAMUN ET AL.

Table 1. List of H. cannabinus genotypes used in this study.

code Accession Mode of development Generation Group

G1 ML1 Acute (200), MNA M6 Early
G2 ML2 Acute (200), MNA M6 Early
G3 ML3 Acute (300), MNA M6 Early
G4 ML4 Acute (300), MNA M6 Early
G5 ML5 Acute (300), MNA M6 Intermediate
G6 ML6 Acute (300), MNA M6 Early
G7 ML7 Acute (300), MNA M6 Early
G8 ML8 Acute (300), MNA M6 Early
G9 ML9 Acute (300), MNA M6 Early
G10 ML10 Acute (300), MNA M6 Early
G11 ML36-3 Acute (300), MNA M5 Early
G12 ML36-8 Acute (300), MNA M5 Early
G13 ML36-10 Acute (300), MNA M5 Intermediate
G14 ML36-11 Acute (300), MNA M5 Intermediate
G15 ML36-16 Acute (800), MNA M5 Intermediate
G16 ML36-18 Acute (800), MNA M5 Early
G17 ML36-19 Acute (800), MNA M5 Early
G18 ML36-20 Acute (800), MNA M5 Intermediate
G19 ML36-21 Acute (800), MNA M5 Early
G20 ML36-22 Acute (800), MNA M5 Early
G21 ML36-24 Acute (1300), MNA M5 Mid-late
G22 ML36-25 Acute (1300), MNA M5 Early
G23 ML36-26 Acute (1300), MNA M5 Intermediate
G24 ML36-27 Acute (1300), MNA M5 Early
G25 ML36-29 Acute (1300), MNA M5 Early
G26 V-36 Conventional method, Malaysia Check Intermediate
G27 HC-95 Conventional method, Bangladesh Check Mid-late
G28 BJRI KENAF4 Conventional method, Bangladesh Check Early
G29 MLRing4 P1 Chronic, MNA M5 Intermediate
G30 MLRing4 P2 Chronic, MNA M6 Mid-late
G31 ML36-21(2) Acute (800), MNA M6 Early
Legend: MNA – Malaysian Nuclear Agency, Bangi, Selangor

Gel electrophoresis
The amplified products were isolated on a 3% MetaPhorTM agarose gel (Lonza Rockland, Inc., USA)
with 1× TBE buffer using the Bio-Rad Horizontal Electrophoresis System (Bio-Rad, CA, USA) at 60 V
for 80 minutes. GelDoc2000 doc was used to visualize and obtain the gels under UV light.

Band scoring
All the genotypes were scored for the presence and absence of the SSR bands, and the data were
entered into a binary matrix as discrete variables, representing present as “1” and absent as “0” inside
an Excel software file. The scoring was performed using UVIDoc version 99.02 to analyze the image.
The scale of a 100 bp DNA ladder (SMOBiO) was used to score the amplified bands. The only bands
that were scored were reproducible and are greater or equal to 100 bp in length. However, bands that
appeared similar were assumed to be homologous.
Table 2. Shows the characteristics of 10 EST-SSR markers used to identify 31 kenaf genotypes.

Marker GenBank ID Sequence of Primers (5΄-3΄) Repeat Motif Allele sizes (bp) Allele frequency Ann. Temp. (0C) No. of Allele
RBRC_Hc_ES_1 KU896377 F: CCGAAGCTCCTGCTTTTATC (AG)6 140–320 0.048–0.387 60.188 5
R: GTCTCAGATGAAGCCACCAC
RBRC_Hc_ES_35 KU896411 F: GTTCCTATGAAGAATCCGGC (CTC)8 135–375 0.032–0.419 59.725 6
R: ACTTTGAGAGGTTGCAAGGG
RBRC_Hc_ES_43 KU896419 F: GGTAAACTGTTGAAGCGGGT (GAA)10 170–325 0.032–0.387 60.321 5
R: GCAGAGCATTTCAACCAG
RBRC_Hc_ES_51 KU896427 F: GGTATGGCAGACGAGATGTT (GAG)4 137–225 0.032–0.645 60.969 4
R: GTGTTAGTAGGCACTGGTGAAG
RBRC_Hc_ES_62 KU896438 F: ATTCAGAAACCGATGCCC (GCT)6 182–382 0.032–0.452 59.621 7
R: GGAATGTCACTGGTCCGAG
RBRC_Hc_ES_67 KU896443 F: GTACACAAAGTGCAACCTCTCC (GGT)6 163–325 0.032–0.419 60.188 5
R: TTCTCCCCTAATTCCTCACC
RBRC_Hc_ES_79 KU896455 F: GGAGTGTCTTGTAATAGCCCAC (TGC)5 142–320 0.032–0.548 60.969 5
R: CTCCAACCTCCCATTGTTC
RBRC_Hc_ES_80 KU896456 F: GAAACCGTGTTGGTCTTGTC (TGC)4 125–296 0.016–0.484 60.969 5
R: AAAGGCCCGATCCAAATC
RBRC_Hc_ES_86 KU896462 F: CAAGGCTTAGGTCGTAGGTATC (TGT)5 110–284 0.032–0.355 59.621 5
R: AAGAGAAGCCAAGATCAGCC
CL1973.Contig3_All_1379_2 Kenaf-856 F: TAAGAGTAAGGGACAGGAGAGG GGGCTA (6*4) 187–312 0.016–0.500 60.151 5
R: TAACAATCCTCCCAGCTCAAGT
Source: (Li et al. 2016; Jeong et al. 2017)
JOURNAL OF NATURAL FIBERS
5
6 M. AL-MAMUN ET AL.

Statistical analysis
POPGENE software version 1.32 was used to calculate genetic variation parameters such as average
observed allele number, percentage of polymorphic loci, number of alleles, number of polymorphic
loci, observed heterozygosity, and expected heterozygosity. The polymorphism information content
(PIC) was computed using the formula given below:

X
n
PIC ¼ 1 P2ij
j¼1

where Pij is the frequency of the jth allele for the ith marker and is summed over n alleles.

Multivariate analysis
Jaccard’s similarity coefficient was computed for the cluster analysis, and a dendrogram was con­
structed using the unweighted pair group method (UPGMA) algorithm and the SAHN with the aid of
NTSYS-pc software (version 2.1). Partitioning of variation between and within the base population in
this study was achieved via analysis of molecular variance (AMOVA) using the GENALEX 6.41
package in the excel software (Peakall and Smouse 2006).

Results
Analysis of polymorphisms in the EST-SSR markers
A total of ten polymorphic EST-SSR markers employed in this study amplified 52 alleles, and the
number of alleles for each primer ranged from 4 to 7 with an average of 5.2 alleles (Table 2). The results
reported in this study are in accordance with the report of Akter et al. (2008) in self-pollinating fiber
plant species, where the researcher observed 2 to 9 alleles per locus with an average 4.61. In this study,
KU896438 had the highest number of alleles (7) with sizes ranging from 182 to 382 bp, while
KU896427 had the lowest alleles (4) with sizes ranging from 137 to 225 bp. Among the ten assessed
markers, seven viz KU896377, KU896419, KU896443, KU896455, KU896456, KU896462 and Kenaf-
5856 which produced five alleles were detected at the loci. In the current study, allele frequency ranged
from 0.016 to 0.645. Figure 1 presents the primer pairs’ DNA banding patterns among the 31 kenaf
genotypes.
The EST-SSR markers with their genetic diversity parameter and PIC values among the genotypes
were not uniform, as shown in Table 3. The effective number of alleles ranged from 2.131 (KU896427)
to 3.798 (KU896427), with an average of 3.145. (KU896377). The highest allele efficiencies were
observed in the KU896377(75.96%) and KU896419 (74.94%), respectively. In contrast, the lowest
allele efficiency (49.30%) was observed in marker KU896438. The estimation of genetic diversity
among the kenaf mutants ranged from 0.982 to 1.515, with the mean value of 1.304 for Shannon’s
Information index (SI). The largest number of alleles (7), with sizes ranging from 182 to 382 bp, were
found with KU896438, which had the highest SI values. The PIC value ranges from 0.531 (KU896427)
to 0.737 (KU896377), with a mean of 0.610, indicating that the EST-SSR markers used in this study
were effective and polymorphic. Genetic differentiation (Fst) values varied from 0.670 to 1.000, with
a mean of 0.914 and Gene flow (Nm) values ranged from zero to 0.123, with an average value of 0.024.
The observed heterozygosity for four markers is zero to 0.484 (KU896419), with a mean value of 0.116
(Table 3). The expected heterozygosity ranged from 0.539 to 0.749, with a mean value of 0.683. The
mean value of Nei’s index was 0.672. Across 31 kenaf genotypes, the highest average heterozygosity
(0.242) was observed in KU896419, followed by KU896438 (0.113), KU896455 (0.113), KU896377
(0.065), KU896456 (0.032) and Kenaf-585 (0.016) in descending order.
 JOURNAL OF NATURAL FIBERS 7

Figure 1. Microsatellite profiles of 31 Kenaf genotypes at locus KU89646262 (A) and KU896456 (B); M: molecular wt. marker (100
bp DNA ladder), Lane 1: ML1, Lane 2: ML2, Lane 3: ML3, Lane 4: ML4, Lane 5: ML5, Lane 6: ML6, Lane 7: ML7, Lane 8: ML8, Lane 9: ML9,
Lane 10: ML10, Lane 11: ML36-3, Lane 12: ML36-8, Lane 13: ML36-10, Lane 14: ML36-11, Lane 15: ML36-16, Lane 16: ML36-18, Lane
17: ML36-19, Lane 18: ML36-20, Lane 19: ML36-21, Lane 20: ML36-22, Lane 21: ML36-24, Lane 22: ML36-25, Lane 23: ML36-26, Lane
24: ML36-27, Lane 25: ML36-29, Lane 26: V-36, Lane 27: HC-95, Lane 28: BJRI KENAF4, Lane 29: MLRing4 P1, Lane 30: MLRing4 P2,
Lane 30: ML36-21(2).

Table 3. The amplified EST-SSR markers’ genetic diversity parameter in genotypes of H. cannabinus.

Locus Na Ne SI PIC Fst Nm* Ho He Nei** A.He

KU896377 5 3.798 1.444 0.737 0.912 0.024 0.129 0.749 0.737 0.065
KU896411 6 3.495 1.452 0.714 1.000 0.000 0 0.726 0.714 0
KU896419 5 3.747 1.423 0.733 0.670 0.123 0.484 0.745 0.733 0.242
KU896427 4 2.131 0.982 0.531 1.000 0.000 0 0.539 0.531 0
KU896438 7 3.451 1.515 0.710 0.841 0.047 0.226 0.722 0.710 0.113
KU896443 5 3.372 1.355 0.703 1.000 0.000 0 0.715 0.703 0
KU896455 5 2.673 1.209 0.626 0.820 0.055 0.226 0.636 0.626 0.113
KU896456 5 2.626 1.135 0.619 0.948 0.014 0.065 0.629 0.619 0.032
KU896462 5 3.280 1.293 0.695 1.000 0.000 0 0.707 0.695 0
Kenaf-585 5 2.882 1.234 0.653 0.975 0.006 0.032 0.664 0.653 0.016
Mean 5.200 3.145 1.304 0.610 0.914 0.024 0.116 0.683 0.672 0.058
Legend: Number of Different Alleles = Na, Number of Effective Alleles = Ne, Shannon’s Information Index = SI, polymorphic
information conten = PIC, Genetic differentiation = (Fst), Gene flow = Nm*, Observed Heterozygosity = Ho, Expected
heterozygosity* = He, Average heterozygosity = A.He, Fst = 0.25(1 – Fst) /Fst is used to estimate gene flow (Nm*)

Genetic diversity in H. cannabinus populations

The percentage of polymorphic loci based on grouping of genotypes into Early, Intermediate and Mid-late
populations was 91.30, 63.04 and 23.91%, respectively, with an average of 59.42% (Table 4). Among populations,
Early had the highest genetic diversity, at 91.30%, followed by Intermediate with 63.04%, while the Mid-late had
the lowest value at 23.91 %. Furthermore, the observed number of alleles ranged from 0.696 (mid-late) to 1.826
(early) in these populations, with a mean value of 1.275. The average effective number of alleles (ne) was 1.3, with
values ranging from 1.125 (mid-late) to 1.430 (early). Shannon’s information index ranged from 0.123 (mid-late)
to 0.405 (early), with a mean of 0.281. The expected heterozygosity varied from 0.080 (mid-late) to 0.262 (early),
with a mean value of 0.183
8 M. AL-MAMUN ET AL.

Table 4. Estimation of genetic diversity among the genotypes of H. cannabinus population.

Group N Na Ne I He uHe %P
Early Mean 21.000 1.826 1.430 0.405 0.262 0.268 91.30%
SE 0 0.084 0.051 0.033 0.025 0.026
Intermediate Mean 7.000 1.304 1.343 0.316 0.208 0.224 63.04%
SE 0 0.139 0.052 0.040 0.028 0.030
Mid-late Mean 3.000 0.696 1.125 0.123 0.080 0.096 23.91%
SE 0 0.124 0.036 0.033 0.022 0.026
Average Mean 10.333 1.275 1.300 0.281 0.183 0.196 59.42%
SE 0.659 0.078 0.029 0.023 0.016 0.017 19.54%
Legend: Number of Different Alleles = Na, Number of Effective Alleles = Ne, Shannon’s Information Index = I, Expected
Heterozygosity = He,Unbiased Expected Heterozygosity = uHe, Percentage of Polymorphic Loci = %P

Band patterns across populations

The number of bands for Early, Intermediate and Mid-late populations was presented in Figure 2. Among these,
Early exhibited the highest number of band frequencies i.e. 37, while Intermediate and Mid-late showed 31 and 21
band frequencies, respectively. Among these populations, the number of private bands was 11 (early), 2
(intermediate) and 1 (mid-late), respectively. There were no locally common bands occurring among the
population of the DTF group. The standard error range for mean of expected heterozygosity (he) varied from
0.022 (mid-late) to 0.028 (intermediate), with the average of expected heterozygosity (he) ranging from 0.080
(mid-late) to 0.262 (early). The mean of unexpected heterozygosity (uHe) ranged from 0.096 (mid-late) to 0.268
(early), with a standard error range of 0.026 (mid-late and early) to 0.030 (intermediate).

Analysis of molecular variance (AMOVA) using EST-SSR markers

The EST-SSR profiles of the kenaf genotypes in this research were analyzed using AMOVA to
determine the inter-population genetic variances. The inter-genetic and intra-genetic variances
revealed that 24% of the total variation occurred between flowering time groups. In comparison,
76% variation occurred within the groups (Table 5). The AMOVA revealed that the majority of the
molecular variation of genetic variability between three groups had an estimated variance of 2.252,
while the variance within groups was 7.204. Furthermore, the AMOVA study revealed significant
(P ≤ 0.05) genetic variations between and within populations (early, intermediate, and mid-late
flowering).

Figure 2. Banding patterns of 31 kenaf genotypes using EST-SSR data set.

 JOURNAL OF NATURAL FIBERS 9

Table 5. Determine the inter-population genetic variances by using AMOVA.

Source df SS MS Est. Var. Percentage (%) P value

Among Populations 2 47.963 23.982 2.252 24 0.003
Within Populations 28 201.714 7.204 7.204 76 0.003
Total 30 249.677 9.456 100
Legend: df stands for degree of freedom; SS stands for number of squared observations; and MS stands for mean of squared
observations.

Genetic relationship among the accessions

To obtain detailed information on the genetic variability among the 31 kenaf accessions, cluster
analysis was performed to construct a phylogenetic tree based on the 10 polymorphic SSR markers.
A significant variation was observed among the evaluated genotypes with a genetic similarity coeffi­
cient ranging from 0.00 to 1.00. Using the dissimilarity coefficient of 0.75 as the standard for the
classification, the 31 genotypes were divided into five major clusters (Figure 3). Group III had the
highest number of (11) genotypes among the five clusters, followed by group IV and V with six
genotypes each. Group I had five genotypes while group II had the lowest number (3) of genotypes.
This grouping implies that the genotypes belonging to different categories would have some agro-
morphological differences among them. Based on population-wise, group I and II are categorized as
early and mid-late flowering genotypes, respectively. Meanwhile, group III, IV and V are mixtures of
early and intermediate flowering genotypes.

Evaluation of genetic relationships among 31 kenaf genotypes

Multidimensional Scaling analyses were used to visualize the genetic distances among the 31 Kenaf
Genotypes, and the relationship between the genetic distances and the genetic identity of the three
base populations. From the combined data of the 10 primers, the values of pairwise comparisons of
genetic distance between germplasms were computed, and the values ranged from 0.00 to 0.983
(Table 6). A greater genetic distance (0.983) was observed between accessions G8 and G7. The lowest
genetic distance (0.00) was found between genotypes pair G2 vs G1, G3 vs G1, G3 vs G2, G4 vs G1, G4
vs G2, G4 vs G3, G7 vs G2, G13 vs G1, G13 vs G3, G13 vs G4, G15 vs G10, G19 vs G7, G22 vs G6, G22
vs G8, G24 vs G1, G24 vs G2, G24 vs G3, G24 vs G4, G24 vs G6, G27 vs G2, G29 vs G7, G18 vs G17,
G20 vs G16, G20 vs G17, G23 vs G17, G27 vs G18, G27 vs G20, G27 vs G21, G27 vs G22, G28 vs G18,
G28 vs G20, G28 vs G23 as shown in Table 7 and 8.

Discussion
Photoperiod insensitivity and late-flowering have been identified as the essential traits required for the
cultivation of kenaf plants in a tropical country such as Malaysia (Hossain et al. 2011). However, in the
absence of diverse traits in the genepool, mutagenesis is often used in crops to create valuable traits
such as plant height, days to flowering, color variation and pathogen tolerance (Kang et al. 2016;
Oladosu et al. 2016). The selection procedure of varieties in the breeding program can be practised
based on polymorphism in DNA level and molecular analysis of genetic relatedness is often used to
select parents for crossing. The transcribed region of the genome contains EST-SSRs, which may be
relatively well conserved and represent a better relationship between species or varieties. As a result,
EST-SSRs are useful genetic markers for studying genetic diversity, high-density genetic mapping, and
marker-assisted breeding (Datta, et al., 2021). The 10 EST-SSR markers successfully identified 52
alleles with an average of 5.2 alleles per locus. However, Jeong et al. (2017) recorded a total of 203
alleles using 70 EST-SSR markers to study the genetic diversity among 45 kenaf accessions. In the
10 M. AL-MAMUN ET AL.

Figure 3. Differentiation between 31 H. cannabinus genotypes according to microsatellite analysis is summarized in a UPGMA
dendrogram based on Nei’s (1972) genetic distance.

present study, the 31 kenaf genotypes showed high genetic diversity and gene flow values were found
to differ, indicating lower differentiation among the genotypes examined, including the mutant lines
and commercial varieties.
The polymorphism information content (PIC) values that reflect allele diversity was used to
estimate the marker’s discriminating power by taking into account both the number of alleles at
a locus and their relative frequencies. EST-SSR primers were extremely informative in nine out of ten
cases (PIC value 0.6 or higher). Lower PIC values could result from genotypes with similar genetic
 JOURNAL OF NATURAL FIBERS 11

Table 6. Distribution of 31 H. cannabinus genotypes under diverse clusters using EST-SSR data.

Population Early Intermediate Mid-late

Cluster I ML1, ML2, ML3, ML4, ML36-27
Cluster II ML36-24, HC-95, MLRing4 P2
Cluster III ML6, ML36-25, ML8, ML9, ML10, ML36-3, ML36-8, ML5, ML36-10, ML36-11, ML36-16,
Cluster IV ML7, ML36-21, ML36-29, ML36-21(2), MLRing4 P1, V-36,
Cluster v ML36-18, ML36-22, ML36-19, BJRI KENAF4 ML36-20, ML36-26

content for a specific locus, whereas higher PIC values could result from genotypes with varied genetic
content. The PIC revealed that the most informative marker was KU896377, which indicated that this
marker is highly useful for differentiating among the accessions whereas; the least informative marker
was KU896427, which separated the least number of accessions. The repeat number and sequence of
SSR markers have an impact on the number of alleles amplified by a primer and its PIC values
(Chukwu et al. 2020). Hence, SSR markers are useful for genetic research and determining the degree
of polymorphism on a specific marker locus (Sarif et al. 2020).EST-SSR primers such as KU896377,
KU896419, KU896411, KU896438, KU896443, and KU896462 showed high polymorphism among the
evaluated accessions with He values exceeding 70%. (0.749, 0.745, 0.726, 0.722, 0.715 and 0.707,
respectively.) (Table 4). Obtaining a heterozygosity value of more than 70% for population studies is
more reliable, insightful, and gives precise information (Myint et al. 2021). The locus KU896377 had
the highest effective number of alleles (3.798), while KU896427 had the lowest effective number of
alleles (2.131).
Furthermore, studies of genetic polymorphism among various populations indicate varying levels
of genetic diversity. The mid-late group population had a significantly lower genetic diversity based on
the Shannon’s Information Index and Expected Heterozygosity values. Meanwhile, Shannon’s
Information Index showed that early flowering (0.405) and intermediate flowering (0.316) populations
had the most genetic heterogeneity. Despite the large distance between the populations, early and
intermediate groups were closer. To assess the extent of population difference among the 31 kenaf
genotypes, EST-SSR markers were also used to calculate the pairwise population matrix for Nei’s
genetic distance among the three days to flowering groups. Early and intermediate groups have a lower
genetic distance than other groups, suggesting that they are genetically identical. This may be due to
distinct characteristics of the Mid-late population, which is lacking in other populations.
The genetic diversity of different kenaf mutants was assessed using the means of genetic distances
between germplasm. It was observed that there were variations among 31 kenaf germplasms based on
the differences between the highest and lowest genetic distance values. Genetic diversity demonstrated
the likelihood of two randomly selected alleles differing from the population (Myint et al. 2021).
According to the observations, the germplasm with the most genetic variance can be used as a parental
source for breeding lines to expand kenaf varieties. Among the 31 kenaf genotypes, the total genetic
variation within populations was 76%, whereas genetic variation between the three base populations
was 24%. Moreover, the AMOVA analysis revealed significant genetic variations (P ≤ 0.05) between
and within populations, indicating that high genetic dissimilarity existed within-population compared
to among the population.
Analysis of the UPGMA cluster divided the 31 kenaf genotypes into five distinct clusters, including
five early flowering genotypes in Cluster I, three mid-late flowering genotypes in Cluster II, and 23
mixed flowering (early and intermediate) genotypes in Cluster III, IV and V. This result is in
agreement with Jeong et al. (2017), who reported that the phylogenetic and population structure of
45 accessions was divided into three classes using EST-SSR markers through de novo RNA sequencing.
Similarly, Korean Kenaf cultivars were divided into three categories; early flowering, mid-late flower­
ing, and late flowering based on their flowering dates (Kang et al. 2016). In addition, Ryu et al. (2017)
 12

Table 7. Genetic identity (above diagonal) and genetic distance (below diagonal) values among 31 H. cannabinus genotypes according to Nei (1972).

Genotypes G16 G17 G18 G19 G20 G21 G22 G23 G24 G25 G26 G27 G28 G29 G30 G31
Genotypes G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15
G1 0.308 0.211 0.053 0.264 0.100 0.105 0.053 0.051 0 0.325 0.300 0.600 0.271 0.211 0.400 0.205
G2 0.564 0.580 0.158 0.527 0.100 0.316 0.105 0.154 0 0.434 0.300 0 0.434 0.474 0.100 0.257
G3 0.308 0.211 0.053 0.264 0.100 0.105 0.053 0.051 0 0.325 0.300 0.600 0.271 0.211 0.400 0.205
G4 0.308 0.211 0.053 0.264 0.100 0.105 0.053 0.051 0 0.325 0.300 0.600 0.271 0.211 0.400 0.205
M. AL-MAMUN ET AL.

G5 0.421 0.324 0.270 0.324 0.205 0.433 0.473 0.368 0.502 0.362 0.410 0.205 0.223 0.270 0.308 0.158
G6 0.421 0.433 0.270 0.379 0.205 0.406 0 0.368 0 0.306 0.257 0.154 0.334 0.324 0.154 0.158
G7 0.105 0.054 0.541 0 0.564 0.352 0.108 0.684 0.115 0.056 0.308 0.154 0.028 0 0.205 0.053
G8 0.526 0.541 0.162 0.487 0.103 0.297 0 0.158 0.057 0.417 0.154 0.051 0.445 0.433 0.051 0.263
G9 0.667 0.474 0.158 0.527 0.100 0.316 0.334 0.257 0.056 0.488 0.200 0.100 0.434 0.474 0.200 0.257
G10 0.579 0.379 0.162 0.433 0.103 0.324 0.054 0.263 0.057 0.445 0.308 0.205 0.334 0.379 0.308 0.263
G11 0.684 0.487 0.162 0.541 0.103 0.324 0.054 0.263 0.057 0.445 0.205 0.103 0.445 0.487 0.205 0.263
G12 0.658 0.460 0.189 0.514 0.154 0.297 0.054 0.316 0.086 0.390 0.257 0.103 0.417 0.460 0.205 0.237
G13 0.579 0.487 0.270 0.541 0.205 0.433 0.108 0.368 0.115 0.445 0.205 0.103 0.445 0.595 0.205 0.263
G14 0.649 0.444 0.167 0.500 0.105 0.306 0.056 0.270 0.059 0.429 0.158 0.158 0.457 0.444 0.158 0.270
G15 0.579 0.379 0.162 0.433 0.103 0.324 0.054 0.263 0.057 0.445 0.308 0.205 0.334 0.379 0.308 0.263
G16 **** 0.579 0.379 0.865 0 0.216 0.108 0.053 0.057 0.779 0.410 0.103 0.779 0.811 0.103 0.579
G17 0.057 **** 0.811 0.833 0 0.222 0.111 0 0.059 0.686 0.422 0.105 0.829 0.861 0.105 0.595
G18 0.788 0 **** 0.056 0.843 0.667 0.278 0.865 0.118 0.114 0.316 0 0 0.111 0.105 0.108
G19 0.039 0.049 0.781 **** 0.053 0.278 0.167 0.054 0.059 0.743 0.474 0.105 0.800 0.833 0.105 0.595
G20 0 0 0.046 0.795 **** 0.791 0.158 0.872 0.112 0.109 0.400 0 0 0.105 0.100 0.103
G21 0.414 0.406 0.110 0.346 0.063 **** 0.111 0.649 0.059 0.314 0.474 0 0.200 0.333 0.158 0.216
G22 0.601 0.594 0.346 0.484 0.498 0.594 **** 0.162 0.030 0.200 0.053 0 0.172 0.222 0.359 0.324
G23 0.796 0 0.039 0.788 0.037 0.117 0.491 **** 0.115 0.111 0.359 0.103 0 0.108 0.205 0.105
G24 0.772 0.765 0.578 0.765 0.592 0.765 0.952 0.585 **** 0.152 0.112 0.112 0.121 0.118 0.112 0.115
G25 0.067 0.102 0.586 0.080 0.600 0.313 0.435 0.593 0.510 **** 0.434 0.109 0.677 0.743 0.054 0.723
G26 0.241 0.233 0.311 0.202 0.248 0.202 0.795 0.277 0.592 0.226 **** 0.200 0.488 0.422 0.400 0.410
G27 0.615 0.608 0 0.608 0 0 0 0.615 0.592 0.600 0.435 **** 0.271 0.105 0.800 0.205
G28 0.067 0.051 0 0.060 0 0.435 0.476 0 0.570 0.106 0.194 0.353 **** 0.858 0.163 0.779
G29 0.057 0.040 0.594 0.049 0.608 0.297 0.406 0.601 0.578 0.080 0.233 0.608 0.042 **** 0.105 0.649
G30 0.615 0.608 0.608 0.608 0.622 0.498 0.441 0.428 0.592 0.788 0.248 0.060 0.491 0.608 **** 0.103
G31 0.148 0.140 0.601 0.140 0.615 0.414 0.304 0.608 0.585 0.088 0.241 0.428 0.067 0.117 0.615 ****
 JOURNAL OF NATURAL FIBERS 13

Table 8. Based on EST-SSR markers, five of each higher and lower Nei’s (1972) genetic distance (D) between pairs of H. cannabinus
genotypes.

Five Five
higher Genotype lowers
D values combination D values Genotype combination
0.983 G8 x G7 0.000 G2 x G1, G3 x G1, G3 x G2, G4 x G1, G4 x G2, G4 x G3, G7 x G2, G13 x G1, G13 x G3, G13 x G4, G15
x G10, G19 x G7, G22 x G6, G22 x G8, G24 x G1, G24 x G2, G24 x G3, G24 x G4, G24 x G6, G27 x G2,
G29 x G7, G18 x G17, G20 x G16, G20 x G17, G23 x G17, G27 x G18, G27 x G20, G27 x G21, G27 x G22,
G28 x G18, G28 x G20, G28 x G23
0.968 G28 x G7 0.007 G11 x G9, G12 x G11, G14 x G11
0.952 G24 x G22 0.014 G14 x G9
0.803 G6 x G1 0.015 G14 x G12
0.796 G31 x G7 0.022 G12 x G9

reported that the SSR phylogenetic tree divided 32 kenaf genotypes into three flowering groups (early,
mid-late, and non-flowering). Active breeding of kenaf cultivars for yield and economic importance
with adaptation to local conditions have been conducted using these genetic tools (Alexopoulou et al.
2013; Kang et al. 2016). In the kenaf selection breeding process, the introduction of new cultivars is the
most effective method of increasing yield and improving functional quality (Jeong et al. 2017). The
lowest genetic similarity was observed between ML 1 and ML 2, whereas the highest similarity was
found between ML1 and BJRI KENAF4. The difference between the lowest and highest values of
genetic distance showed a wide range of variability among the 31 evaluated genotypes. The cluster
I with five genotypes (ML1, ML2, ML3, ML4, ML36-27) were clustered in close proximity and had
more homology with genetic distance between the five genotypes. The three genotypes ML36-24, HC-
95 and MLRing4 P2 formed the second cluster and they were closely related with a lower genetic
distance. The mid-late mutants ML36-24 and MLRing4 P2 cultivars were derived by acute (800
gamma-ray) and chronic treatment, respectively. Based on the flowering period and phylogenetic
tree, there was an uncertain pattern of division between kenaf mutants (ML36-24 and MLRing4 P2)
and their parent (V-36), with mid-late flowering mutants dominating from the original variety. It is
important to develop hybrids that would continue to grow irrespective of flowering at a critical
daylight period for maximum fiber yield (Olawale and Oluwatoyin 2019). Late maturation, on the
other hand, increases the risk of seed production. As a result, it is crucial to cultivate local mid-late
maturing groups with high biomass and seed yields (Ryu et al. 2013). The result of this study indicated
the presence of high genetic differentiation with desirable molecular traits among the kenaf mutants
discovered using EST-SSR markers, demonstrating the markers’ efficacy in detecting polymorphisms
that are more diverse and can be recommended as a parent in the crossing programme.
Diverse genotypes belonging to different clusters are more likely to produce heterotic offspring.
Considering the allelic diversity levels, genetic structure, and relationships of their codominance
performances, crosses between cluster I and cluster V will lead to high heterosis. Similarly, in future
hybridization programmes, the kenaf mutants ML5, ML9, ML36-10, ML36-25, ML36-21(2), and the
Bangladeshi commercial variety BJRI KENAF4 may be chosen as parents. More diverse parents will
result in more hybrid vigor or heterosis, resulting in a photo-resistant variety with a high fiber yield.

Conclusion
A wide range of genetic variation was observed among the 31 kenaf mutant and inbred lines using
EST-SSR markers which provides accurate and compelling evidence of dissimilarities. The micro­
satellite markers are valuable tools in determining the genetic relationship among kenaf mutants based
on days to flowering – viz; early, intermediate, and mid-late. The dendrogram generated by UPGMA
analysis and the determination of genetic diversity by cluster analysis revealed that kenaf mutants were
14 M. AL-MAMUN ET AL.

divided into three groups. The molecular variance analysis indicate a high polymorphism level within
the population with 76% total genetic variations, whereas a low polymorphism level (24% genetic
variation) was recorded among populations. Following the improvement of kenaf mutants and
widening of their genetic base, the population with the least genetic similarities can be selected as
parental materials. This assessment could be advantageous in developing photo insensitive varieties
with high fiber yield variety in future breeding programs.

Acknowledgments
The authors would like to express their heartfelt gratitude to the Bangladesh Agriculture Research Council (BARC) and
Universiti Putra Malaysia (UPM) for allowing them to conduct this research. The authors are also grateful to the
Bangladesh Jute Research Institute (BJRI) and the Ministry of Agriculture of the People’s Republic of Bangladesh for
referring the lead author to UPM for a PhD programme.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research was funded by a research grant under the BARC (PIU-BARC, NATP-2) programme from the Ministry of
Agriculture, People’s Republic of Bangladesh, and UPM, Malaysia, with the research grant vote number 6282507;
Bangladesh Agriculture Research Council and Universiti Putra Malaysia[Grant vote number 6282507];

ORCID
Mohd Y. Rafii http://orcid.org/0000-0003-4763-6367
Yusuff Oladosu http://orcid.org/0000-0002-2092-971X

References
Akter, J., M. S. Islam, A. A. Sajib, N. Asraf, S. Haque, and H. Khan. 2008. Microsatellite markers for determining genetic
identities and genetic diversity among jute cultivars. Australian Journal of Crop Science 1 (3):97–107.
Al-Mamun, M., M. Rafii, Y. Oladosu, A. B. Misran, Z. Berahim, Z. Ahmad, F. Arolu, and M. H. Khan. 2020. Genetic
diversity among kenaf mutants as revealed by qualitative and quantitative traits. Journal of Natural Fibres 1–18.
doi:10.1080/15440478.2020.1856268.
Alexopoulou, E., Y. Papatheohari, D. Picco, N. Di Virgilio, and A. Monti. 2013. Kenaf: A multipurpose crop for several
industrial applications, 59–82. London: Springer.
Carberry, P. S., R. C. Muchow, R. Williams, J. D. Sturtz, and R. L. McCown. 1992. A simulation model of kenaf for
assisting fibre industry planning in northern Australia. I. General introduction and phenological model. Australian
Journal of Agricultural Research 43(7): 1501–1513.
Chukwu, S. C., M. Y. Rafii, S. I. Ramlee, S. I. Ismail, Y. Oladosu, E. Okporie, and M. Jalloh. 2019. Marker-assisted
selection and gene pyramiding for resistance to bacterial leaf blight disease of rice (Oryza sativa L.). Biotechnology &
Biotechnological Equipment 33 (1):440–55. doi:10.1080/13102818.2019.1584054.
Chukwu, S. C., M. Y. Rafii, S. I. Ramlee, S. I. Ismail, Y. Oladosu, I. I. Muhammad, and B. R. Yusuf. 2020. Recovery of
recurrent parent genome in a marker-assisted backcrossing against rice blast and blight infections using functional
markers and SSRs. Plants 9 (11):1411. doi:10.3390/plants9111411.
Datta, D. R., M. R. Yusop, A. Misran, M. Jusoh, Y. Oladosu, F. Arolu, A. Haque, and N. M. Sulaiman. 2021. Genetic
diversity in eggplant (Solanum melongena L.) germplasm from three secondary geographical origins of diversity
using SSR markers. Biocell, doi:10.32604/biocell.2021.015321.
Eujayl, I., M. E. Sorrells, M. Baum, P. Wolters, and W. Powell. 2002. Isolation of EST-derived microsatellite markers for
genotyping the A B genomes of wheat. Theoretical and Applied Genetics 104 (2):399–407. doi:10.1007/s001220100738.
Hossain, M. D., M. M. Hanafi, H. Jol, and H. A. Hazandy. 2011. Growth, yield and fibre morphology of kenaf (Hibiscus
cannabinus L.) grown on sandy bris soil as influenced by different levels of carbon. African J. Biotech
10 (50):10087–94.
 JOURNAL OF NATURAL FIBERS 15

Jeong, S. W., S. J. Kwon, J. Ryu, J. B. Kim, J. W. Ahn, S. H. Kim, Y. D. Jo, H. I. Choi, S. B. Im, and S. Y. Kang. 2017.
Development of EST-SSR markers through de novo RNA sequencing and application for biomass productivity in
kenaf (Hibiscus cannabinus L.). Genes & Genomics 39 (10):1139–56. doi:10.1007/s13258-017-0582-z.
Kang, S. Y., S. J. Kwon, S. W. Jeong, J. B. Kim, S. H. Kim, and J. Ryu. 2016. An improved kenaf cultivar ‘Jangdae’ with
seed harvesting in Korea. Korean Journal of Breeding Science 48 (3):349–54. doi:10.9787/KJBS.2016.48.3.349.
Li, H., D. Li, A. Chen, H. Tang, J. Li, and S. Huang. 2016. Characterization of the Kenaf (Hibiscus cannabinus L.) global
transcriptome using illumina paired-end sequencing and development of EST-SSR markers. PLoS ONE 11 (3):
e0150548. doi:10.1371/journal.pone.0150548.
Li, Y. C., A. B. Korol, T. Fahima, and E. Nevo. 2004. Microsatellites within genes: Structure, function, and evolution.
Molecular Biology and Evolution 21 (6):991–1007. doi:10.1093/molbev/msh073.
Myint, K. A., Z. Yaakub, M. Y. Rafii, Y. Oladosu, M. Y. A. Samad, S. I. Ramlee, and M. D. Amiruddin (2021). Genetic
Diversity Assessment of MPOB-Senegal Oil Palm Germplasm Using Microsatellite Markers. BioMed research
international, 2021.
Nei, M. (1972). Genetic distance between populations. The American Naturalist, 106(949), 283–292.
Oladosu, Y., M. Y. Rafii, N. Abdullah, G. Hussin, A. Ramli, H. A. Rahim, and M. Usman. 2016. Principle and application
of plant mutagenesis in crop improvement: A review. Biotechnology & Biotechnological Equipment 30 (1):1‒16.
doi:10.1080/13102818.2015.1087333.
Olawale, A. S., and B. M. Oluwatoyin. 2019. Genetic studies of fibre yield-related traits and days to anthesis in some
kenaf accessions. Journal of Agricultural Sciences 64(1): 9‒19 .
Patanè, C., and O. Sortino. 2010. Seed yield in kenaf (Hibiscus cannabinus L.) as affected by sowing time in South Italy.
Industrial Crops and Products 32 (3):381–88. doi:10.1016/j.indcrop.2010.06.002.
Peakall, R. O. D., and P. E. Smouse. 2006. GENALEX 6: Genetic analysis in Excel. Population genetic software for
teaching and research. Molecular Ecology Notes 6 (1):288–95. doi:10.1111/j.1471-8286.2005.01155.x.
Ryu, J., B. K. Ha, D. S. Kim, J. B. Kim, S. H. Kim, and S. Y. Kang. 2013. Assessment of growth and seed oil composition of
kenaf (Hibiscus cannabinus L.) germplasm. Journal of Crop Science and Biotechnology 16 (4):297–302. doi:10.1007/
s12892-013-0074-x.
Ryu, J., S. J. Kwon, D. G. Kim, M. K. Lee, J. M. Kim, Y. D. Jo, and J. B. Kim. 2017. Morphological characteristics, chemical
and genetic diversity of kenaf (Hibiscus cannabinus L.) genotypes. Journal of Plant Biotechnology 44 (4):416‒430.
doi:10.5010/JPB.2017.44.4.416.
Sarif, H. M., M. Y. Rafii, A. Ramli, Y. Oladosu, H. M. Musa, H. A. Rahim, and S. C. Chukwu. 2020. Genetic diversity and
variability among pigmented rice germplasm using molecular marker and morphological traits. Biotechnology &
Biotechnological Equipment 34 (1):747–62. doi:10.1080/13102818.2020.1804451.
Zheng, L., F. Li, J. Hao, and G. Li. 1995. Fluorescence probe studies of bis (2-ethylhexyl) sodium sulfosuccinate (AOT)
and AOT/cetyltrimethylammonium bromide (CTAB) systems. Colloids and Surfaces. A, Physicochemical and
Engineering Aspects 98 (1–2):11–18. doi:10.1016/0927-7757(95)03100-R.