Molecular Diversity Analysis of Some Chilli (Capsicum spp.

)
Genotypes Using SSR Markers

Abstract

Chilli belongs to the genus Capsicum which possesses enormous wealth of genetic diversity.
Extent of genetic diversity determines the success level of crop improvement programme.
Simple sequence repeats (SSRs) are the most widely used marker system for molecular diversity
analysis especially in cultivated species. The aim of our present study was to assess the molecular
genet-ic diversity of 20 local chilli genotypes of Bangladesh using SSR markers. Ge-nomic DNA
was extracted from young leaves and PCR reactions were per-formed. Eleven SSR primers were
used in PCR amplification. Total 10 alleles were detected for the five polymorphic SSR loci,
with a mean of 2.00 alleles per primer. Gene diversity ranged from 0.333 to 1.00 with an average
of 0.567. Polymorphic Information Content (PIC) values of the SSR primers ranged from 0.255
to 0.500 with an average value of 0.371. The similarity index ma-trix ranged from 0.00 to 1.000.
It was highest in several germplasms viz. Pop-2 vs Pop-18; Pop-3 vs Pop-5 vs Pop-19 vs Pop-
20 and the lowest in the germplasm Pop-8 vs Pop-18. Dendrogram based on Nei’s genetic
distance using Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated the
segregation of 20 chilli genotypes into two main clusters. The SSR markers showed genetic
variability in the studied pepper genotypes and they are powerful tools for estimating molecular
diversity of chilli. The findings of the present study have potential applications in future breeding
programme for the genetic improvement of chilli.

Keywords
Capsicum, Molecular Diversity, Genotypes, SSR Markers, Polymorphism

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Contents
Introduction .............................................................................................................................................. 3
Literature Review ..................................................................................................................................... 5
Molecular markers ............................................................................................................................... 5
Ideal Desirable Properties of Molecular Markers ............................................................................... 6
Types of molecular markers ................................................................................................................. 7
Microsatellites (SSR) ............................................................................................................................. 8
Advantages of SSRs markers ................................................................................................................ 9
Polyamines improves germination and early seeds growth ............................................................... 9
Genetic diversity and crop germplasm of chillies ............................................................................. 10
Materials and Methods .......................................................................................................................... 11
Experimental Site and Time Duration................................................................................................ 11
Plant Materials ................................................................................................................................... 11
Experimental material and the SSR markers ..................................................................................... 12
Genomic DNA extraction and SSR analysis........................................................................................ 12
Analysis of SSRs and Primer Design ................................................................................................... 13
DNA Isolation ...................................................................................................................................... 14
SSR Analysis ........................................................................................................................................ 14
Electrophoretic Separation of the Amplified Products ..................................................................... 15
Results ..................................................................................................................................................... 15
Gene Diversity and Polymorphism Information Content (PIC) ......................................................... 17
Discussion ............................................................................................................................................... 20
Conclusion............................................................................................................................................... 21
References .............................................................................................................................................. 21

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Introduction
Chilli pepper (Capsicum annuum L.) (Solanaceae) has a chromosome number 2n=2x=24. It is
indigenous to South America and was first introduced in India from Brazil by Portuguese
towards the end of 15"" century (Basu and Krishna, 2003). Pepper is an often-cross pollinated
crop and, therefore, exhibits wide variability for different qualitative and quantitative traits
(Tanksley, 1984). There are five cultivated species of peppers including Capsicum annuum L,
C. frutescens, C. chinense, C. pubescens and C. baccatum (Heiser and Smith, 1957). Bangladesh
is considered to be the secondary centre of diversity for chilli (IBPGR, 1983), especially of C.
annuum, the most important cultivated species. North-Eastern parts are home to the genetic
variability where several interspecific hybrids/derivatives were originated, among which Naga
King is one of the world's hottest peppers. Over the years, chilli has become an important
commercial crop of Bangladesh and the country is currently one of the competitive producer,
consumer and exporter of peppers in the world.

Chilli has numerous chemicals including steam-volatile oil, fatty oils, carotene-ids, vitamins,
protein, fibre and mineral elements and is used for different purposes because of their nutritional
value, flavour, aroma, texture, pungency and colour. It also has antifungal property against
fungal species belonging to Aspergillus and Fusarium. It is source of Vitamin, A, B, C and E
with minerals like molybdenum, manganese, folate, potassium, thiamine, and copper. Capsaicin
has significant physiological action which is used in many pharmaceutical preparations and
ointments for cold, sore throat, chest congestion etc. It is also used in cosmetics like prickly heat
powders and skin ointments.
The chilli landraces of different district in Bangladesh are heterogeneous and a wide variability
in respect of fruit morphology, pungency, bearing habit and crop duration is found throughout
country. Bangladeshi chilli varieties have been developed traditionally by selection,
hybridization and back crossing with locally adapted cultivars. An important source for the
introduction of new traits is the existence of a genetically diverse pool of chilli germplasm
available in the country but they are mostly lying unexplored. There is a strong need to collect
this germplasm and their proper characterization and classification. Subjective classification
based on morphological data has been reported to create confusion and difficulty in classifying
the genus capsicum. In addition, the level of polymorphism for morphological characteristics in
genotypes is sometimes so limited and inadequate to allow variety/genotype discrimination.

Genetic resources are the most valuable and essential basic raw material to meet the current and
future needs for genetic improvement of any crop. Characterization of the germplasm is

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important for its identification and registration with the competent authority for plant variety
protection. Conventionally, morphological markers called descriptors were used for varietal
identification and genetic diversity analysis in plants that demands collection of extensive data
at different locations. However, the level of polymorphism for morphological characteristics in
elite germplasm is sometimes too limited and inadequate to allow for variety/genotype
discrimination (Geleta et aI., 2005). The traditional method of distinctness, uniformity and
stability (DUS) testing is time-consuming and expensive, requiring large areas of land and
skilled personnel, and is often subjective due to environmental influences (Singh et aI., 2004).
Further, taxonomy of the genus Capsicum is confusing and sometimes it is difficult to identify
an accession using only subjective morpho-agronomic data (Costa et aI., 2006).

Molecular markers are important tool for genotype identification and studying the organization
and evaluation of plant genome. With the advent of molecular biology technique, large number
of highly useful DNA markers has been developed for the identification of genetic
polymorphism. Molecular markers define differences in nucleotide sequences, which are
unaffected by growth stage, season, location, and agronomic practice. Different molecular
markers such as restriction fragment length polymorphisms (RFLPs), random amplified
polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and simple
sequence repeats (SSRs) have been developed for pepper. SSR markers represent highly
polymorphic, reproducible, co-dominant, and multi allelic types of variation. They are used in
genome mapping, gene tagging, and estimation of genetic diversity, variety identification, and
marker-assisted selection (Jang et aI., 2004; Kang et aI., 1997, 2001; Lee et aI., 2004; Lefebvre
et al., 2001; Moon et aI., 2003; Paran et aI., 1998; Prince et aI., 1992). These markers have
proven to be very useful in assessing genetic diversity and phylogeny, characterization of
germplasm and detection of duplicates, parental verification in crosses, gene tagging in marker
assisted breeding and gene cloning in genetic transformation (Costa et aI., 2006). The application
of RFLPs for genetic diversity is limited because it requires the use of radioactivity and is labour
intensive (Nahm et aI., 1997). RAPDs and AFLPs identify only dominant alleles and are
sensitive to PCR amplification. The working group on biochemical and molecular techniques of
UPOV has identified SSR markers as the most widely used marker system for plant variety
characterization (UPOV-BMT, 2002). The SSR markers are especially suitable for diversity
analysis in cultivated species which have low levels of variation as detected by other types of
markers and have also been used in successful prediction of heterosis and performance of F1
hybrids from morphological similarity of their parents (Geleta et aI., 2004). The present

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investigation was undertaken to characterize and give robust genetic diversity estimates in
cultivated chilli peppers using SSR markers.

The main objective of this study is to capture the potential genetic diversity among chilli
genotypes grown in Bangladesh and selection of suitable genotypes for future chilli
hybridization programme. Hence, the present study was carried out for following objectives:

1) Molecular diversity analysis of different chilli genotypes.

2) Polymorphism study among chilli germplasm.

3) Dendrogram establishment in some local chilli genotypes.

Literature Review

Molecular markers

Molecular marker is a sequence of DNA, which are located with a known position on the
chromosome (Kumar 1999), or a gene whose phenotypic expression is frequently easily
discerned and used to detect an individual, or as a probe to mark a chromosomes, nucleus, or
locus (King and Stansfield 1990; Schulmann 2007). Markers show polymorphism, which may
arise due to alteration of nucleotide or mutation in the genome loci (Hartl and Clark 1997) and
make it possible to identify genetic differences between individual organisms or species (Collard
et al. 2005). Molecular markers are used in many different areas such as genetic mapping,
paternal tests, detect mutant genes which are connected to hereditary diseases, cultivars
identification, marker assisted breeding of crops, population history, epidemiology and food
safety, population studies (Hartl and Jones 2005). Collard et al. (2005) studies that genetic
marker used to identify genetic variation among individual species or organisms. Schhulmann
(2007) studies that genetic marker used to construct linkage maps and genetic diversity.
Molecular marker also used in many different areas that are including detecting mutant genes,
genetic mapping, epidemiology and for population studies (Hartl and Jones 2005).

The practice in agriculture has since the introduction of molecular makers in the 1980s broaden
and simplified both for commercial and scientific uses to acknowledge new information such as
large number of agronomic and disease resistance traits are available in major crop species

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(Phillips and Vasil 2001; Jain et al. 2002; Gupta and Varshney 2004). A conventional breeding
programme thus involves crossing whole genomes, followed by selection of the superior
recombinants from among the several segregation products. Indeed, such a procedure is time
consuming and laborious, involving several crosses, several generations, and careful phenotypic
selection, and the linkage drag (tight linkage of the undesired loci with the desired loci) may
make it further difficult to achieve the desired objective. Today many plant breeders utilize
molecular markers to proof and identify desirable traits of important plants. Various plant
researchers are used molecular markers to study genetic polymorphism and linkage maps
construct (Schulmann 2007). There are many advantages of molecular markers compared with
morphological and biochemical markers. For instance, when comparing morphological markers
with molecular markers, expression of such markers is influenced both by dominant-recessive
relationship and epistatic-pleiotropic interaction. The use of such markers has been criticized
since the study of genetic variation among germplasm using morphological characteristics is
labors and time consuming (Cooke 1984). When comparing with biochemical markers, their use
is now limited due to large scale analysis and low level of genetic variation in a given species
and subspecies (Rafinski and Babik 2000).

Ideal Desirable Properties of Molecular Markers

The following ideal properties are best described for the molecular markers:

• Easily available

• Assay is rapid and easy

• Reproducible and highly polymorphic

• Co-dominant inheritance and recurrent occurrence in genome

Selectively neutral to environmental conditions

• Data exchange between different laboratories should be easy.

It is really complicated to obtain molecular marker of above criteria. Depending on the type of
study undertaken, a marker system can be recognized that would fulfil the above characteristics.
Diverse types of molecular markers are used to estimate DNA polymorphism and are classified
as hybridization-based markers and polymerase chain reaction (PCR)-based markers. In
hybridization-based markers DNA profiles are visualized by hybridizing the restriction
endonuclease digested DNA fragment, to a labelled probe, which is a DNA fragment of known

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sequence while PCR based markers involve in vitro amplification of particular DNA sequences
with the help of specifically or arbitrarily chosen oligonucleotide sequences (primers) and a
thermostable DNA polymerase enzyme. Electrophoresis and banding patterns separate the
amplified DNA fragments are detected by different methods such as staining (using ethidium
bromide dye) and autoradiography. With the advent of thermostable DNA polymerase the use
of PCR in research and clinical laboratories has increased tremendously. PCR is extremely
sensitive and operates at a very high speed. Its application for diverse purposes has opened up a
multitude of new possibilities in the field of molecular biology (Saiki et al. 1985; Saiki et al.
1988).

Types of molecular markers

Various types of molecular markers are available and new methods are frequently being
developed. Today, no method is ideal for all applications so scientist teams must weigh both
pros and cons of methods when starting a new project (Harlt and Jones 2005).

Molecular markers are grouped after their different abilities of showing homozygosity (dominant
marker) or heterozygosity (co-dominant marker) (Hartl 1988). The most commonly used
dominant DNA marker for genetic diversity in plants are: Random Amplified Polymorphic DNA
(RAPD; Williams et al. 1990), DNA amplification fingerprinting (DAF; Caetano-Anolles et al.
1991), Arbitrarily primed polymerase chain reaction (AP-PCR; Welsh and McClelland 1990),
Inter-simple sequence repeat (ISSR; Zietkiewicz et al. 1994) and Amplified Fragment Length
Polymorphisms (AFLP; Vos et al. 1995), whereas the most common used co-dominant markers
are: Restriction Fragment Length Polymorphisms (RFLP; Botstein et al. 1980), Microsatellites
(SSR; Akkaya et al. 1992); Sequence characterised amplified regions (SCAR; Paran and
Michelmore 1993), Cleaved amplified polymorphic sequence (CAPS; Konieczny and Ausubel
1993), Expressed sequence tag (EST; Adams et al. 1991) and Single Nucleotide Polymorphism
(SNP; Jordan and Humphries, 1994) and sequence tagged sites (STS; Olsen et al. 1989). Both
dominant and co-dominant markers can be used to detect DNA polymorphism, which further
used to assess the level of genetic variation in diverse populations and can indicate for instance
population history, patterns of migration, and breeding structure (Hartl and Jones 2005).

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Microsatellites (SSR)

Microsatellites, also known as simple sequence repeats (SSR), variable number tandem repeats
(VNTR) and short tandem repeats (STR) are tandem repeats motifs of 1-6 nucleotides found at
high frequency in the nuclear genomes of most taxa (Beckmann and Weber, 1992). For example,
(A)11, (GT)12, (ATT)9, (ATCG)8, (TAATC)6 and (TGTGCA)5 represent mono-, di-, tri-, tetra-
, penta- and hexa-nucleotide repeats, respectively. A microsatellite locus typically varies in
length between 5 and 40 repeats, but longer strings of repeats are possible. Dinucleotide,
trinucleotide and tetranucleotide repeats are the most common choices for molecular genetic
studies. Dinucleotides are the dominant type of microsatellite repeats in most vertebrates
characterized so far, although trinucleotide repeats are most abundant in plants (Beckmann and
Weber, 1992; Kantety et al. 2002; Chen et al. 2006). Despite the fact, the mechanism of
microsatellite evolution and function remains unclear, SSRs were being widely employed in
many fields soon after their first description (Litt and Luty 1989; Tautz 1989; Weber and May
1989) because of the high variability which makes them very powerful genetic markers.
Microsatellites have proven to be an extremely valuable tool for genome mapping in many
organisms (Knapiket al. 1998), but their applications span over different areas ranging from
kinship analysis, to population genetics and conservation/management of biological resources
(Jarne and Lagoda 1996). Microsatellites can be amplified for identification by the polymerase
chain reaction (PCR), using two unique sequences which are complementary to the flanking
regions as primers. This process results in production of enough DNA to be visible on agarose
or polyacrylamide gels; only small amounts of DNA are needed for amplification as
thermocycling in this manner creates an exponential increase in the replicated segment.

With the abundance of PCR technology, primers that flank microsatellite loci are simple and
quick to use, but the development of correctly functioning primers is often a tedious and costly
process. However, once they are developed and characterized in an organism, microsatellites are
powerful for a variety of applications because of their reproducibility, multiallelic nature,
codominant inheritance, relative abundance and good genome coverage (Liu and Cordes 2004).

Unlike conserved flanking regions, microsatellite repeat sequences mutate frequently by

slippage and proofreading errors during DNA replication that primarily change the number of
repeats and thus the length of the repeat string (Eisen 1999). Because alleles differ in length,
they can be distinguished by high-resolution gel electrophoresis, which allows rapid genotyping
of many individuals at many loci for a fraction of the price of sequencing DNA. Many

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microsatellites have high-mutation rates (between 10-2 and 10-6 mutations per locus per
generation, and on average 5×10-4) that generate the high levels of allelic diversity necessary
for genetic studies of processes acting on ecological time scales.

Advantages of SSRs markers

(i) SSR are used for plant breeding, conservation biology

and population genetics as forensics, paternity analysis and gene mapping (Coates and Byrne
2005).

(ii) Require little amount of DNA, which does not have to be of high quality.

(iii) the simple interpretation of results (de Vicente and Fulton 2003).

Disadvantage of SSRs markers

(i) the requirement of a known sequence to be amplified (Weising et al. 2005).

(ii) Developing new microsatellites are expensive and time consuming (Coates and Byrne 2005).

(iii) the phenomena null-alleles, which are nonamplifying alleles and appears frequently. Null-
alleles leave no bands when having homozygosity, but when having heterozygosity it leaves one
band visual. This will interfere and complicate reading of data, since it will be registered as a
homozygote individual when actually being a heterozygote. To reduce error due to null alleles,
population studies should contain many diverse SSR primers so that different multiple
microsatellite loci are investigated (Weising et al. 2005).

Polyamines improves germination and early seeds growth

Improvement in stand establishment can be obtained by advancements in seed quality and seed
enhancements, genetic improvement, as well as improved seeding techniques. Seed priming is a
technique of seed enhancement that improves seed performance by rapid and uniform
germination, normal and vigorous seedlings, which resulted in faster and higher rate of
germination and emergence in different crops (Farooq et al., 2007; 2008), which also helps
seedlings to grow in biotic or abiotic stress conditions (Ashraf and Foolad, 2005; Khan et al.,
2009a; 2009b). Such seed treatments result in synchronized emergence and uniform stand
establishment leading to improved yield.
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Seeds performance of many vegetables can be improved by inclusion of plant growth regulators
during priming and other pre-sowing treatments (Ashraf and Foolad, 2005; Khan et al., 2009b).
Polyamines (PAs) are small polycationic molecules found ubiquitously in all organisms. Their
function has been reported in a variety of biological processes including transcription, RNA
modification, protein synthesis and the modulation of enzyme activities (Takahashi and Kakehi,
2010). PAs are essential for growth, development, and cell differentiation in plants (Kusano et
al., 2008). Moreover, PAs are also accumulated in plants and perform their function in plants
under normal as well as stress conditions. Exogenous PAs may influence germination of isolated
embryos depending on the type of polyamine, its concentration, and the state of the embryo
dormancy (Farooq et al., 2011). Some studies have shown the positive effects of PAs on seed
germination, seedling vigour, growth and development of different crops when seeds were
primed with PAs e.g. wheat (Iqbal and Ashraf, 2005; Farooq et al., 2011), sunflower (Farooq et
al., 2007), rice (Farooq et al., 2008) and tomato (Afzal et al., 2009). Nonetheless, no study has
been reported to evaluate the effects of polyamine seed priming on hot pepper. Therefore, the
current study was proposed to assess benefits (if any) linked to seed priming with PAs on hot
pepper through exploring responses at seed germination and early growth stages.

Genetic diversity and crop germplasm of chillies

Genetic diversity assessment and estimation of relationship among accessions are very helpful
for germplasm improvement. So, analysis of genetic diversity is important to examine
differences between individuals of the same species and is also used to compare the genetic
composition of members of different species over a wider taxonomic range (Dale and Schatz,
2002). According to Peeraullee and Sanmukhiya (2013) description establishing the
morphological and agronomic traits has been used to characterization and evaluation of crop
germplasm. However, the expression of qualitative traits appeared less influenced by
environment and so they are considered more important for characterizing plants (Berg et al.,
These variations are governed by allelic differences at one or few numbers of interacting gene
loci and the genotypic differences could be easily identified from the trait’s expressions. Usually
the classification using qualitative traits could be applied for any year, location or environment
(Briggs and Knowles, 1967). But, quantitative traits are highly influenced by environment and
that is why quantitative descriptors are impediment to their use for genetic diversity assessment
(Dagnoko et al., 2013). In Bangladesh, generating information on the degree and pattern of

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genetic diversity of the hot pepper genotypes using morphological qualitative characters were
so far limited evaluation scientifically.

Genetic diversity studies carried out on chilli germplasm from Bangladesh are not
comprehensive (Gogoi and Gautam 2002; Borgohain et al. 2005; Bosland and Baral 2007; Thul
et al. 2009; Sanatombi et al. 2010; Patel et al. 2011). Most of these studies have only used
phenotypic traits such as fruit diameter, number of fruits per plant and leaf diameter for diversity
analysis (Gogoi and Gautam 2002; Borgohain et al. 2005; Thul et al. 2009). Although phenotypic
traits are important for diversity studies, they need to be supported by molecular markers to give
robust genetic diversity estimates. Studies using molecular markers have either taken limited
samples from one geographical location (Sanatombi et al. 2010) or have focused only on
cultivated accessions (Patel et al. 2011). Therefore, a need exists for proper documentation and
analysis of chilli germplasm from different geographic locations in Bangladesh.

Materials and Methods

Experimental Site and Time Duration

The experiment was conducted in Biotechnology Laboratory, Department of Biotechnology,

Sher-e-Bangla Agricultural University (SAU), Dhaka-1207 and Regional Spices Research
Centre, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur-1701,
Bangladesh.

Plant Materials

Twenty different chilli genotypes were used as plant materials for the study and were collected
from different places of Bangladesh (Table 1). The genotypes were grown in poly bags placed
in a shady place under the regular agronomic practices.

Table 1. List of chilli germplasm with their origin and characteristics.

Sl. No. Genotypes Origin Characteristics

1 Pop-1(G1) Rangpur, Large fruit size
Bangladesh
2 Pop-2(G2) Thakurgaon, Small fruit size
Bangladesh
3 Pop-3(G3) Panchagarh, Small fruit size
Bangladesh
4 Pop-4(G4) Panchagarh, Medium fruit size
Bangladesh
5 Pop-5(G5) Bogra, Bangladesh Large fruit size
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6 Pop-6(G6) Rangpur, Medium fruit size
Bangladesh
7 Pop-7(G7) Bogra, Bangladesh Large fruit size
8 Pop-8(G8) Bogra, Bangladesh Large fruit size
9 Pop-9(G9) Dinajpur, Small fruit size
Bangladesh
10 Pop-11(G10) Saidpur, Medium fruit size
Bangladesh
11 Pop-12(G11) Saidpur, Medium fruit size
Bangladesh
12 Pop-13(G12) Rangpur, Large fruit size
Bangladesh
13 Pop-14(G13) Bogra, Bangladesh Small fruit size
14 Pop-15(G14) Thakurgaon, Small fruit size
Bangladesh
15 Pop-16(G15) Panchagarh, Small fruit size
Bangladesh
16 Pop-17(G16) Bogra, Bangladesh Medium fruit size
17 Pop-18(G17) Dinajpur, Small fruit size
Bangladesh
18 Pop-19(G18) Saidpur, Medium fruit size
Bangladesh
19 Pop-20(G19) Bogra, Bangladesh Small fruit size
20 Pop-21(G20) Bogra, Bangladesh Medium fruit size

Experimental material and the SSR markers

The experimental material comprised 64 germplasm accessions. Majority of the accessions (49)
belonged to the indigenous sources and the remaining (15) to the exotic sources. Except Naga
King, Tabasco and Punjab Longi, all lines belong to the species C. annuum. Naga King,
popularly grown in Eastern India is believed to be a naturally occurring hybrid between C.
chinense and C. frutescens; and Tabasco belongs to C. frutescens. Phylogeny of Punjab Longi
is not clear but resembles that of C. frutescens. For diversity analysis, the germplasm was
screened using 50 SSR markers of which 27 showed polymorphism (Table 1). Some of the
markers used have been published (Lee et al., 2004) while others developed by AVRDC- The
World Vegetable Center, Taiwan are unpublished.

Genomic DNA extraction and SSR analysis

The genomic DNA was extracted from fresh leaf tissues following CTAB method (Saghai-
Maroof et aI., 1984). Quality and quantity of DNA was checked both by gel electrophoresis and
spectrophotometer. In vitro amplification was performed in a 96 well Eppendorf Master Cyclerm
(Saiki et aI., 1988). The gels were visualized under UV light and photographed using photo
documentation system (UV Transilluminator).

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The number of alleles per locus, major allele frequency, gene diversity and Polymorphism
Information Content (PIC) values were determined using POWER MARKER version 3.25, a
genetic marker da-ta analysis software. The individual fragments were assigned as alleles of the
ap-propriate microsatellite loci. The allele frequency data from POWER MARKER was used to
export the data in binary format (presence of allele as “1” and ab-sence of allele as “0”) for
analysis with NTSYS-PC (Numerical Taxonomy and Multiware Analysis System) Version 2.2
software. Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram was
constructed using a computer programme, POPGENE (Version 1.31) based on Nei’s genetic
distance.

The SSR allele sizes were determined depending on the position of bands relative to the ladder
(Fermantas Gene Ruler 1 KB DNA ladder). Total number of alleles was recorded for each SSR
marker in all the 64 genotypes by assigning allele number as 1, 2, 3, 4 and so on. The allele
amplified in a particular set having highest molecular weight was numbered as allele 1. The
amplified alleles were recorded as 1 (band present) or 0 (band absent) in a binary matrix. The
polymorphic information content (PIC) values for all the primers were estimated using the
formula:

PlC=1- ∑ Pij2

j=1

Where, Pij is the frequency of jth allele in the ith primer and summation extends over ‘n’ patterns.
Genetic diversity and cluster analysis The SSR marker amplification profile of 64 genotypes was
used to estimate genetic similarity based on number of shared amplified.

Analysis of SSRs and Primer Design

Perl script MISA software (MISA http://pgrc.ipk-gatersleben.de/ misa/; RRID:OMICS_00110)

was used to mine microsatellites from the assembled sequences. Sequences with at least nine
repetitions for dinucleotides, six repetitions for trinucleotides, and five repetitions for tetra-,
penta-, and hexa-nucleotides, excluding the polyA and polyT repeat, were identified.
Dinucleotide repeats such as AT/TA and CT/GA were treated as the same type of repeat motif.
Primer Premier 7.0 software (PREMIER Biosoft International, Palo Alto, CA) was used to
design appropriate primers from the flanking sequences, based on the following criteria: primer

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length of 18–22 bp (optimally 20 bp), GC content of 40–60%, annealing temperature (Tm) of
50–60◦C (with the Tm of forward and reverse primers differing by no more than 4◦C), and
expected amplified product size of 100–400 bp. Primers were synthesized by Invitrogen
Biological Engineering Technology & Services Co., Ltd (Shanghai, China).

DNA Isolation

Genomic DNA extractions from fresh young leaf at 3 - 4 leaf stage of seedling was done in SRC
lab using CTAB (cetyl trimethyl ammonium bromide) buffer. CTAB extraction buffer was
prepared by Doyle & Doyle method with some minor modification. Approximately 200 mg of
sterilized young leaves were used for DNA extraction. Extracted DNA was visualized in 1%
agarose gel. Approximate 20 - 25 ng of DNA was used as template in PCR reaction.

SSR Analysis

Eleven SSR primers viz. GPMS-113, CAMS-117, CAMS-142, CAMS-153, GPMS- 161,
GPMS-197, CAMS-327, CAMS-405, EPMS-418, CAMS-806 and CAMS-864 described
previously were selected for PCR reaction on 20 local chilli germplasm for their ability to
produce polymorphic band. DNA amplification was performed in a thermal cycler (Esco
Technologies Swift TM Mini Thermal cyclers, Singapore). The PCR reactions were performed
in 10 μl reaction mixture containing 5.0 μl 2X Taq Master Mix, 1.50 μl primers, 1.0 μl sample
DNA and 2.5 μl de-ionized water (Biolab, UK). SSRs primers were amplified under the
following PCR reaction conditions: Pre-denaturation with 95˚C for 4 min; denaturation with
95˚C for 40 sec, annealing at 50˚C - 61˚C (on the basis of Tm value of primer) for 33 sec,
extension at 72˚C for 40 sec, final extension at 72˚C for 5 min continuing with 31 cycles and
finally stored at 4˚C.

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Electrophoretic Separation of the Amplified Products

PCR products for each sample were confirmed by running it in 2% agarose gel containing 1 μl
ethidium bromide in 1X TBE buffer at 90 V for 1 hour. Five micro litre (5 μl) loading dye was
added to the PCR product and spinned them well. Then it was loaded in the wells. The DNA
ladder (50 & 100 bp) (Promega ther-mal cyclers) was used in both left and right side of the gel.
Under ultra-violet light on a trans-illuminator SSR bands were observed. The PCR product was
saved by gel documentation system and photographed by a Gel Cam Polaroid camera.

Results

Highly polymorphic and repeatable PCR based markers Simple Sequence Re-peats (SSRs) were
used here to assess the polymorphism, diversity and similarity identification within those local
chilli germplasms. Results obtained from the study have been presented below under the
following headings. 3.1. Primer Selection and DNA Amplification through SSR Primer

Eleven SSR primer pairs were screened on twenty chilli genotypes to evaluate their suitability
for amplification of DNA. Among them five primer pairs CAMS-117, CAMS-153, GPMS-161,
EPMS-418 and CAMS-806 showed repro-ducible and distinct polymorphic amplification. A
total 10 alleles were detected for the five polymorphic SSR loci, with an average number of
alleles/locus of 2.00 and a range between 1 to 3 alleles (Table 2). It was observed that one SSR
primer GPMS-161 (Figure 1(c)) gave maximum number of allele (3) and allelic frequency
ranging from 0.675 to 0.125 followed by CAMS-117, EPMS-418 and CAMS-806 SSR primer.
In CAMS 153 SSR primers showed allelic frequency 0.737 for only 1 allele (Table 2).

Table 2. Primer sequence, number of allele, allele size and frequency of alleles and gene diversity
index and PIC value at five SSR loci across 20 chilli genotypes.

Primer code Sequence of Number of Allele DNA Fragment Allele frequency Gene Diversity PIC Value
Primers (5'-3') Size (bp)
CAMS-117 For: 2 220 0.850 0.50 0.255
TTGTGGAGGAA
ACAAGCAAA
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 Rev:
CCTCAGCCCAGG
AGACATAA
190 0.150
CAMS-153 For: 1 200 0.737 1.00 0.388
TGCACAAATATG
AATCCCAAGA
Rev:
AGTCAGCAAACA
CATCTGACAA
GPMS-161 For: 3 240 0.200 0.333 0.489
GAAATCCAATA
AACGAGTGAAG
Rev:
CCTGTGTGAACA
AGTTTTCAGG
200 0.675
140 0.125
EPMS-418 For: 2 200 0.850 0.500 0.225
ATCTTCTTCTCA
TTTCTCCCTTC
Rev:
TGCTCAGCATTA
ACGACGTC
180 0.150
CAMS-806 For: 2 210 0.500 0.500 0.500
TGTCACAAGTGT
CAAGGTAGGAG
Rev:
CCCCAAAAATTT
TCCCTCAT
170 0.500
Total - 10 - 2.833 1.857
Mean - 2.00 - 0.567 0.371

The SSR primer CAMS-117 produced 2 DNA band ranges from 190 bp to 220 bp (Table 2)
where 190 bp was amplified in the genotypes of Pop-1, Pop-3, Pop-17 (Figure 1(a)) which is
polymorphic in nature while primer CAMS-153 produced 1 DNA fragment at 200 bp (Table 2)
where no polymorphic band was found. In case of GPMS-161 primer, 3 DNA fragment was
observed ranges from 140 bp to 240 bp (Table 2). The genotypes Pop-2, Pop-4 and Pop-10 were
amplified at 190 bp DNA fragment which is polymorphic. Further, the SSR primer EPMS-418
and CAMS-806 produced 2 DNA amplification which was ranges from 180 bp to 200 bp and
170 bp to 210 bp (Table 2). The primer EPMS-418 gave 180 bp fragments and it was
polymorphic. It showed amplification in the genotypes Pop-1, Pop-3, Pop-18. The DNA band

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was amplified in the genotypes Pop-1, Pop-2 and Pop-3 showed polymorphism at 170 bp of
DNA band in CAMS-806.

Gene Diversity and Polymorphism Information Content (PIC)

Gene diversity ranged from 0.333 to 1.00. Primer CAMS-153 showed highest

(a)

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(b)

(c)

(d)

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 (e)

Figure 1. SSR profile of twenty chilli genotypes using primer (a) CAMS-117, (b) CAMS-153,
(c) GPMS-161, (d) EPMS-418 & (e) CAMS-806. M1: 50 bp DNA ladder and M2: 100 bp DNA
ladder. Lane 1 Pop-1; 2: Pop-2; 3: Pop-3; 4: Pop-4; 5: Pop-5; 6: Pop-6; 7: Pop-7; 8: Pop-8; 9:
Pop-9; 10: Pop-11; 11: Pop-12; 12: Pop-13; 13: Pop-14; 14: Pop-15, 15: Pop-16; 16: Pop-17;
17: Pop-18; 18: Pop-19; 19: Pop-20 and 20: Pop-21.

gene diversity (1.00) followed by CAMS-117, EPMS-418, CAMS-806 which was showed same
gene diversity (0.500) (Table 2). Total genetic diversity obtained 2.833 with an average 0.567.
Polymorphic Information Content (PIC) value for the five markers ranged from 0.255 to 0.500
with a mean value of 0.371. The high-est PIC value (0.500) was obtained for CAMS-806
followed by GPMS-161 (0.489). The total PIC was 1.857 with an average 0.370 (Table 2). PIC
value revealed that CAMS-806 was considered as the best marker for 20 chilli germplasm
followed by GPMS-161 and CAMS-153. Primer CAMS-117 and EPMS-418 could be
considered as the least powerful marker.

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Discussion

SSR markers are considered more reliable because of their ability to produce highly consistent
profiles. Eleven SSR primers were screened on 20 chilli genotypes to evaluate their suitability
for amplification of the DNA fragment.

Figure 2. UPGMA dendrogram based on Nei’s (1972) genetic distance, summarizing the data
on differentiation between 20 chilli genotypes according to microsatellite analysis.

The average number of alleles per locus provides complementary information of polymorphism
and more adequate to co-dominant markers. The average numbers of alleles (2.00) per SSR
primer pairs were in agreement with earlier works reported in pepper with the mean value of
2.78 alleles/locus and maxi-mum of four alleles were amplified by the primer AVRDC PP 32.
Our observation were partially supported by Tilahun et al. (2013) where they observed 3.22
average number of alleles per primer, range from 1 to 6.

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The PIC values provide an estimate of discriminating power of a marker by taking into account
not only the number of alleles at a locus but also relative frequencies of these alleles. Lower PIC
values might be result of closely related genotypes and vice versa. Senior et al. opined that
marker loci with an average number of alleles running at equal frequencies will have the highest
PIC value. The PIC value obtained in present study varied from 0.255 to 0.500 with an average
0.371. The highest PIC value (0.500) was obtained for CAMS-806. PIC value revealed that
CAMS-806 was considered as the best marker for 20 chilli germplasm followed by GPMS-161
and CAMS-153. Primer CAMS-117 and EPMS-418 could be considered as the least powerful
marker. Our results were partially consistent with Yumnam et al. (2012) (0.52) and kwon et al.
(0.53). On the contrary, 65 polymorphic markers were validated among a wide collection of 21
Capsicum genotypes with allele number and polymorphic information content value per marker
raging from 2 to 6 and 0.05 to 0.64, respectively. The varying levels of polymorphism in chilli
pepper reported by various research group could be attributed to the differences in genetic
structures of the populations screened and the molecular techniques used.

Conclusion

SSR markers showed genetic variability in the studied chilli genotypes and they are powerful
tools for estimating genetic similarities and diversity. The genetic relationships presented among
the genotypes are helpful for future breeding pro-grams through selection of genetically diverse
parents. The present work was the preliminary study to characterize and detect genetic variation
of chilli varieties of Bangladesh and had some limitations in term of limited number of
individuals and varieties as well as number of primers used. The results indicated that the present
study might be used as a guideline for developing mapping population, marker assisted selection
(MAS) and crop improvement of chilli varieties and consequently enables a genetic conservation
plan in Bangladesh.

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