vv

Clinical Group

Scientific Journal of Genetics and Gene

Therapy
DOI CC By

Nidaf khan and Sunita Singh Dhawan*

Review Article
CSIR- Central Institute of Medicinal and Aromatic
Plants, P.O CIMAP, Lucknow, Uttar Pradesh, India

Dates: Received: 23 December, 2016; Accepted: 29

Role of Molecular Markers in
December, 2016; Published: 30 December, 2016
Assessing Genetic Diversity in Mentha:
*Corresponding author: Sunita Singh Dhawan, CSIR-
Central Institute of Medicinal and Aromatic Plants,
P.O CIMAP, Lucknow , Uttar Pradesh, India, E-mail:
A Review
[email protected];

Keywords: Genetic diversity; ISSR markers; SCoT

markers; Polymorphism
Abstract
https://www.peertechz.com
Morphological, phytochemical and genetic differences were studied to evaluate the level and
distribution of diversity among thirteen genotypes of Mentha using both agro-morphological traits and
ISSR markers. Our findings indicated that there were significant differences for agro-morphological
traits and essential oil variations in mint species. Cluster analysis based on these traits grouped the
genotypes into 8 separate clusters. Highest and lowest polymorphism was shown by M. piperita and M.
cardiaca respectively. Plants have evolved various strategies for survival and reproduction during the long
evolutionary course, one of which is the production of an array of small molecular weight compounds,
also known as secondary metabolites or natural products. The potent biological activity of secondary
metabolites has also led to their exploitation as pharmaceuticals and many other functions. Although
much progress has been made, a lot more still need to be learned about the mechanisms responsible
for the formation of most of the important secondary metabolites. SCoT analysis also revealed high
polymorphism with the primers generating polymorphic profiles.

Introduction linalool in Mentha citrata and Piperitone oxide in Mentha

longifolia. Both nutraceuticals of Mentha citrata and Mentha
Mints are herbaceous plants and perennial aromatic herbs longifolia possess antioxidant and anticancer effect that
that are cultivated for their essential oils used both for medicinal could be attributed to the presence of phytosterosl, phenolic
and culinary purposes. The essential oil from M. arvensis contains compounds, unsaturated fatty acids and specific volatile
menthol, M.spicata- carvone, d-limonene, dihydrocarvone, M. constituents [1]. Mentha is one of the most common herb
piperita - menthol, menthone, methyl esters, menthylacetate. which has been known for its medicinal and aromotherapeutic
Mint species are not clearly distinct as hybridization between properties since ancient times and in the last few decades, the
some of them occurs naturally. Plants belonging to the genus insecticidal potential of leaf extracts has also been investigated.
Mentha L. (Lamiaceae) have evolved in nature through natural The insecticidal activity of Mentha against various stored grain
hybridization and selection showing substantial variation in pests and vectors. Insecticidal properties of different Mentha
terms of their natural habitats, growth characteristics, and species are commonly inherent in its essential oils or plant
aromas. Morphological, molecular and biochemical markers extracts which is correlated with their chemical composition.
are complementary in determining the genetic similarity of Mentha species produce valuable secondary metabolites that
inter- and intra-species and the relationship between the scavenge toxic free radicals. The free radical scavenging
populations. Genetic variation is fundamentally involved in the potential (1,1diphenyl2picrylhydrazyl scavenging activity) in
ability of a species to adapt to biotic and abiotic changes and Mentha species were investigated to evaluate and explore new
in its evolution. Recognition of the levels and distribution of potential sources for natural antioxidants [2].
genetic variation within and among populations of a species
is the base for development and selection of plant genotypes Applications of molecular markers in plant genome ana-
in breeding programs and increases the understanding of lysis and breeding
the historical processes underlying the genetic diversity
Molecular markers have been looked upon as tools for
providing information for the management and preservation
a large number of applications ranging from localization
of endangered and geographically restricted species.
of a gene to improvement of plant varieties by marker-
The major volatile components were Linalyl acetate and assisted selection. They have also become extremely popular

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Citation: khan N, Dhawan SS (2016) Role of Molecular Markers in Assessing Genetic Diversity in Mentha: A Review. Scientific J Genet Gene Ther 2(1): 022-026.
khan and Dhawan (2016)

markers for phylogenetic analysis adding new dimensions success of these individuals. Morphological characterization
to the evolutionary theories. If we look at the history of the does not require expensive technology but large tracts of land
development of these markers, it is evident that they have are often required for these experiments making it possibly
been improved over the last two decades to provide easy, fast more expensive than molecular assessment. These traits are
and automated assistance to scientists and breeders. Genome often susceptible to phenotypic plasticity; conversely this
analysis based on molecular markers has generated a vast allows assessment of diversity in the presence of environmental
amount of information and a number of databases are being variation.
generated to preserve and popularize it.
At CSIR-CIMAP, many elite accessions have been developed
Multi locus probes during the last decade which constitutes the major gene pool to
serve as the usable source of genetic variability in the national
A major step forward in genetic identification is the genebank for these commercially important taxa of Mentha.
discovery that about 30–90% of the genome of virtually all the These accessions of the germplasm include introductions,
species is constituted by regions of repetitive DNA, which are clonal selections, mutant selections and selected half-sibs
highly polymorphic in nature. These regions contain genetic from the progenies of superior genotypes. The success of
loci comprising several hundred alleles, differing from each a breeding program depended on the genetic variability
other with respect to length, sequence or both and they are available into the germplasm of the crop. The assessment of
interspersed in tandem arrays ubiquitously. The repetitive genetic diversity at DNA level for these accessions has been
DNA regions play an important role in absorbing mutations considered as the desirable step in the process of developing
in the genome. Of the mutations that occur in the genome, taggable markers to aid genetic improvement in the variety
only inherited mutations play a vital role in evolution or development programme in addition to estimating strength of
polymorphism. Thus repetitive DNA and mutational forces the gene pool [3].
functional in nature together form the basis of a number of
marker systems that are useful for various applications in plant Molecular analysis comprise a large variety of DNA
genome analysis. The markers belonging to this class are both molecular markers which can be employed for analysis of
hybridization-based and PCR-based. variation. Different markers have different genetic qualities.
Molecular markers work by highlighting differences
Microsatellites and minisatellites (polymorphisms) within a nucleic sequence between different
individuals. These differences include insertions deletions
Both are multilocus probes creating complex banding translocations duplications and point mutations. They do not
patterns and are usually non-species specific occurring however encompass the activity of specific genes. Molecular
ubiquitously. They essentially belong to the repetitive DNA marker are widely used to detect and characterize somaclonal
family. Fingerprints generated by these probes are also known variations at the genetic level. Molecular marker are suitable
as oligonucleotide fingerprints. The methodology has been for generating DNA profile have proved to be an effective tool
derived from RFLP and specific fragments are visualized by in assessing the genetic stability of regenerated plants. These
hybridization with a labelled micro- or minisatellite probe. markers are not influenced by environmental factors and
These loci contain tandem repeats that vary in the number generate reliable and reproducible results.
of repeat units between genotypes and are referred to as
variable number of tandem repeats (VNTRs) or hypervariable Genotyping of cultivars
regions (HVRs). Microsatellites and minisatellites thus form
The repetitive and arbitrary DNA markers are markers of
an ideal marker system creating complex banding patterns
choice in genotyping of cultivars. Microsatellites and mini
by simultaneously detecting multiple DNA loci. Some of
satellites have been employed in DNA fingerprinting for the
the prominent features of these markers are that they are
detection of genetic variation, cultivar identification and
dominant fingerprinting markers and codominant STMS
genotyping. This information is useful for quantification
(sequence tagged microsatellites) markers. Many alleles exist
of genetic diversity, characterization of accessions in plant
in a population, the level of heterozygosity is high and they
germplasm collections and taxonomic studies. Microsatellites
follow Mendelian inheritance.
have been useful for generation of STMS markers, revealing
Genetic diversity analysis polymorphisms within closely related cultivars. The first
application of microsatellites in plants has been in cultivar
Genetic diversity refers to the total number identification, wherein microsatellites have been used
of genetic characteristics in the genetic makeup of a species. to genotype unequivocally diverse materials like rice,
It is distinguished from genetic variability, which describes wheat, grapevine (Vitis unifera) [4], soybean [5] etc. This is
the tendency of genetic characteristics to vary. Genetic important especially for protection of proprietary germplasm.
diversity serves as a way for populations to adapt to changing Microsatellite markers have also been advantageous in pedigree
environments. With more variation, it is more likely that some analysis as they represent single locus. The multi allelism
individuals in a population will possess variations of alleles that of these markers facilitates comparative allelic variability
are suited for the environment. Those individuals are more detection reliably across a wide range of germplasm and allows
likely to survive to produce offspring bearing that allele. The individuals to be ubiquitously genotyped, so that gene flow and
population will continue for more generations because of the paternity can be established.

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khan and Dhawan (2016)

Mapping and tagging of genes linkage groups in Arabidopsis, while their integration into the
genetic linkage maps is still in progress in rice, soybean, maize,
Plant improvement, either by natural selection or through etc. The most recent microsatellite map has been generated for
the efforts of breeders, has always relied upon creating, potato [10]. Similar to microsatellites, looking at the pattern of
evaluating and selecting the right combination of alleles. The variation, generated by retrotransposons, it is now proposed
manipulation of a large number of genes is often required that apart from genetic variability, these markers are ideal for
for improvement of even the simplest of characteristics [6]. integrating genetic maps [11].
With the use of molecular markers it is now important to trace
valuable alleles in a segregating population and mapping them. Once mapped, these markers are efficiently employed in
tagging several individual traits that are extremely important
Inter simple sequence repeat (ISSR) technique is a PCR for a breeding programme like yield, disease resistance, stress
based method, which involves amplification of DNA segment tolerance, seed quality, etc. A large number of monogenic and
present at an amplifiable distance in between two identical polygenic loci for various traits have been identified in a number
microsatellite repeat regions oriented in opposite direction. of plants, which are currently being exploited by breeders and
The technique uses microsatellites, usually 16–25 bp long, molecular biologists together, so as to make the dream of
as primers in a single primer PCR reaction targeting multiple marker-assisted selection come true. Tagging of useful genes
genomic loci to amplify mainly the inter- SSR sequences of like the ones responsible for conferring resistance to plant
different sizes. pathogen, synthesis of plant hormones, drought tolerance and
a variety of other important developmental pathway genes, is a
A novel marker system called start codon targeted (SCoT)
major target. Such tagged genes can also be used for detecting
polymorphism that is based on the short conserved region
the presence of useful genes in the new genotypes generated in
in plant genes surrounding the ATG translation start (or
a hybrid programme or by other methods like transformation,
initiation) codon that has been well characterized in previous
etc.
studies. SCoT technique is a type of targeted molecular marker
technique with the ATG context as one part of a functional gene, SCoT markers have proved their importance as markers for
markers generated from SCoT marker technique may be mostly gene tagging and are very useful in locating and manipulating
correlated to functional genes and their corresponding traits quantitative trait loci (QTL) in a number of crops. SCoT markers
[7]. SCoT markers are highly reproducible as longer primers as they reveal the genetic diversity at the level of genes thus
are used. ATG translation codon is more advantageous than have the possibility of finding new alleles among a given
the intersimple (ISSR), mini satellte (VNTR) or SSR regions germplasm collection. Allele-specific associated primers have
due to conservation of ATG translation start site and flanking
also exhibited their utility in genotyping of allelic variants of
sequences in plant genes.
loci that result from both size differences and point mutations.
Some of the genuine examples of this are the waxy gene locus
This method uses single 18-mer primers in single primer
polymerase chain reaction (PCR) and an annealing temperature in maize [12] the Glu D1 complex locus associated with bread
of 50°C. PCR amplicons are resolved using standard agarose making quality in wheat [13], the Lr1 leaf rust resistance locus in
gel electrophoresis. This method was validated in rice wheat [14], the Gro1 and H1 alleles conferring resistance to the
using a genetically diverse set of genotypes and a backcross root cyst nematode Globodera rostochiensis in potato [15], and
population. Start codon targeted (SCoT) markers were allele-specific amplification of polymorphic sites for detection
generally reproducible but exceptions indicated that primer of powdery mildew resistance loci in cereals [16]. A number of
length and annealing temperature are not the sole factors other traits have been tagged using ASAPs in tomato, lettuce,
determining reproducibility. SCoT marker PCR amplification etc.. Besides ASAPs, AFLP and SSR markers have been identified
profiles indicates dominant marker like RAPD markers. Gene- to be associated with quantitative resistance to Globodera
targeted markers are preferable for numerous applications pallida (stone) in tetraploid potato, which can be very well
in plant molecular genetics especially QTL mapping since employed in marker-assisted selection [17].
recombination levels between marker and gene/QTL are
STMS markers have displayed a potential use as diagnostic
generally lower compared with ‘indirect random markers’ such
markers for important traits in plant breeding programmes,
as RAPDs, ISSRs, or SSRs.
e.g. (AT) 15 repeat has been located within a soybean heat
These markers once mapped enable dissection of the shock protein gene which is about 0.5 cM from (Rsv) a gene
complex traits into component genetic units more precisely, conferring resistance to soybean mosaic virus (Yu et al 1994).
thus providing breeders with new tools to manage these complex Several resistance genes including peanut mottle virus (Rpv),
units more efficiently in a breeding programme [8]. Genetic phytopthera (Rps3) and Javanese rootknot nematode are
maps have been constructed for several other crops like potato, clustered in this region of the soybean genome. Recently,
barley, banana and members of Brassicaceae family [9]. Once ISSRs, which too belong to the arbitrary marker category, but
the framework maps are generated, a large number of markers are found to be devoid of many of the drawbacks shared by
derived from various techniques are used to saturate the maps RAPD class of markers, have been employed as a reliable tool
as much as possible. Microsatellite markers, especially STMS for gene tagging. An ISSR marker (AG) 8YC has been found to
markers, have been found to be extremely useful in this regard. be linked closely (3.7 ± 1.1 cM) to the rice nuclear restorer gene,
About 30 microsatellites have already been assigned to five RF1 for fertility. RF1 is essential for hybrid rice production and

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Citation: khan N, Dhawan SS (2016) Role of Molecular Markers in Assessing Genetic Diversity in Mentha: A Review. Scientific J Genet Gene Ther 2(1): 022-026.
khan and Dhawan (2016)

this marker would be useful not only for breeding both restorer sequence repeat (ISSR) markers have been analysed [21].
and maintainer lines, but also for the purity management of Genetic diversity in Mentha cervina was analysed based on
hybrid rice seeds [18]. Similarly ISSR marker (AC)8 YT has been morphological traits, essential oils profile and ISSRs markers
found to be linked to the gene for resistance to fusarium wilt [20]. Characterization of twenty wheat varieties have been
race 4 in repulsion at a distance of 5.2 cM in chickpea. analysed by ISSR Markers [22]. Assessment of genetic diversity
in Mentha using RAPD markers has been done previously [23]
Molecular profiling in Mentha but assessment using ISSR markers in Mentha have not been
The assessment of genetic diversity at DNA level for reported yet.
these accessions has been considered as the desirable step
Genetic improvement of Mentha should be based on
in the process of developing taggable markers to aid genetic
morphological as well as molecular differences. Accessions
improvement in the variety development programme
appearing to be in the same group morphologically, many times
in addition to estimating strength of the gene pool [3].
show different molecular groupings. The molecular diversity
Assessment of molecular variation among different romanian
database can prove to be directly useful as attempted in the
and foreign barley cultivars was done to determine the level of
present study, for Mentha breeders to develop and analyse novel
genetic similarity among them [19]. ISSR markers are highly
intra as well as inter-specific hybrids as the morphological data
polymorphic and are useful in studies on genetic diversity,
alone may be limiting and misleading. In future, analysis with
phylogeny, gene tagging, genome mapping and evolutionary
more markers is required to find out more specific and unique
biology. The technique uses microsatellites, usually 16–25
sequences in Mentha species. This comparative analysis and
bp long, as primers in a single primer PCR reaction targeting
correlation among different genotypes of Mentha species would
multiple genomic loci to amplify mainly the inter- SSR
be helpful as the probes developed would be utilized for genetic
sequences of different sizes. ISSRs segregate mostly as
dominant markers following simple mendelian inheritance. improvement of Mentha to increase the yield of secondary
However, they have also been shown to segregate as co- metabolites by development of new and improved genotypes.
dominant markers in some cases thus enabling distinction
Concluding Remarks
between homozygotes and heterozygotes.
There was a considerable genetic variation among the
Due to the basis of SCoT primer design, SCoT markers is
studied genotypes based on the agro-morphological traits
distributed within gene regions that contain genes on both plus
and generated ISSR and SCoT profiling. To quantify genetic
and minus DNA strands. It is also possible that pseudogenes
diversity among the genotypes based on agro-morphological
and transposable elements may be used as primer binding
traits, cluster analysis was performed using Euclidean distance
sites by SCoT polymorphism technique. An important factor
matrix by UPGMA method and the genotypes were clearly
is the distance in base pairs between primer binding sites of
grouped into 8 main clusters. As it was expected, the inter
the template. Therefore, a relatively long extension time of the
cluster distances in all cases were more than the intra cluster
thermal cycle is important. For QTL mapping, SCoT markers
distance indicating higher genetic variability between the
could be integrated into existing framework maps to increase
genotypes of different clusters. Genetic variation at the DNA
the marker density or to target specific chromosomal regions.
In another analogy with RAPD, we propose that important level was also considerable and a high level of polymorphism
SCoT markers, such as those identified to be tightly linked to (100%) was detected with ISSR markers set used. Primers
a gene or QTL of interest, could be converted into sequence were synthesized taking into consideration monoterpene
characterized amplified regions markers or sequence tagged biosynthetic pathway genes.
site markers in order to make the marker single locus and
Larger inter than intra cluster distances implies the
improve robustness. The use of ISSR fingerprints could be a
presence of higher genetic variability between the genotypes
powerful tool to assess the genetic diversity in Mentha [20].
of different groups. The generated dendrogram based on ISSR
Morphological, phytochemical and genetic differences were
profiles divided the genotypes into 7 groups. Species specific
studied to evaluate the level and distribution of diversity among
bands could also be recognized from some ISSR primers in
thirteen genotypes of Mentha using both agro-morphological
this study indicating the high potential use of this marker
traits and ISSR markers [20].
for discrimination of genotypes. SCoT analysis also revealed
In recent years, there has been a significant increase in high polymorphism with the primers generating polymorphic
the application of molecular genetics methods for assessing profiles. Species-specific and genotype specific markers can be
the conservation and use of plant genetic resources. Molecular useful for introgression studies where plant breeders want to
techniques have been applied in the analysis of specific transfer some desirable traits from one species into another.
genes, as well as to increase understanding of gene action, Genetically distinct cultivars can be identified that could be
generate genetic maps and assist in the development of gene potentially important sources of germplasm. ISSR and SCoT
transfer technologies. Additionally, they are not confounded markers can then be used for linkage mapping of Quantitative
by environmental, pleiotropic and epistatic effects. Genetic Trait Loci and for indirect selection (Marker assisted selection)
diversity across the natural populations of three montane for genetic improvement in Mentha. Localization of these
plant species in the Western Ghats (India), Symplocos laurina, markers on the chromosomes would be useful for keeping
Gaultheria fragrantissima and Euryanitida using inter simple track of important traits that need to be transferred.

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khan and Dhawan (2016)

Key message 9. Winter P, Kahl G (1995) Molecular marker technologies for plant improvement.
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Copyright: © 2016 khan N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and r eproduction in any medium, provided the original author and source are credited.

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Citation: khan N, Dhawan SS (2016) Role of Molecular Markers in Assessing Genetic Diversity in Mentha: A Review. Scientific J Genet Gene Ther 2(1): 022-026.