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Intraspecific variation in Solanum xanthocarpum Schard. and Wendl.
revealed by ISSR marker
Keywords:
Genetic diversity, Solanum xanthocarpum, ISSR.
ABSTRACT:
Inter-simple sequence repeat (ISSR) analysis was performed in seven
accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the
applicability of this analysis for assessing the intraspecific variation. The value
of similarity indices ranged from 0.375 to 0.125. The similarity result
indicates the presence of high level of genetic diversity among the accessions of
Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear
grouping among the populations. The primers showed abilities in detecting genetic
diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus,
ISSR-PCR technology can be used to study genetic variation and genetic relationships
in the genus Solanum xanthocarpum Schard. and Wendl.
146-152 | JRPS | 2012 | Vol 1 | No 2
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and
reproduction in all medium, provided the original work is properly cited.
www.plantsciences.info
Journal of Research in
Plant Sciences
An International Scientific
Research Journal
Authors:
Ajoy Kumar Das1
,
Sailendra Prasad Borah2
.
Institution:
1. Department of
Biotechnology, Indian
Institute of Technology
Guwahati, Assam, India,
781 039.
2. Department of Botany,
Gauhati University,
Guwahati, Assam,
India, 781 014.
Corresponding author:
Ajoy Kumar Das.
Email:
ajoykdas@iitg.ernet.in
Phone No:
+91 99579 96983.
Web Address:
http://www.plantsciences.info
documents/PS0035.pdf.
Dates:
Received: 15 Aug 2012 Accepted: 15 Sep 2012 Published: 13 Dec 2012
Article Citation:
Ajoy Kumar Das, Sailendra Prasad Borah.
Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR
marker.
Journal of Research in Plant Sciences (2012) 1(2): 146-152
Original Research
Journal of Research in Plant Sciences
JournalofResearchinPlantSciences An International Scientific Research Journal
INTRODUCTION
The genus Solanum xanthocarpum Schard. and
Wendl. is an important medicinal perennial herb
belonging to the family Solanaceae. This prickly herb
is a good source of important alkaloids such as
Solasodine, solasonine, solamargine and diosgenin.
Solasodine extracted from this plant has
antispermatogenic activity. Solamargine found in this
herb has anticancer properties. Diosgenin extracted from
Solanum xanthocarpum Schard. and Wendl. is found to
be effective in suppressing FAS expression in HER 2
over expressing breast cancer cells (Singh and
Singh, 2010). Glucoalkaloid solanocarpine, sterol
carpesterol, solanocarpidine and traces of isochlorogenic,
neochronogenic, chronogenic and caffeic acids are also
found in the fruits of this plant. In India, this plant is
used for curing various ailments since ancient times.
Powder made from this herb is very useful in asthma,
breathlessness and allergies of respiratory tracts. It is also
used as carminative, digestive, alternate and astringent.
As the herb is a stimulant to the heart and is a blood
purifier, it is extremely beneficial in the treatment of
cardiac diseases associated with edema. The fruit is
useful as an aphrodisiac in males and the seeds, in
women for irregular menstruation and dysmenorrheal. It
also promotes conception in females. The roots of this
plant are largely used in catarrhal and febrile affection,
cough, chest pain, flatulence, sore throat, toothache
and constipation and in bronchial diseases. Leaves of
this plant are also used in rheumatic pain.
Solanum xanthocarpum Schard. and Wendl. is non toxic
and safe for human use and is regarded as a valuable
plant in both ayurvedic and modern drug development
areas for its versatile medicinal uses.
The taxonomic description of the genus Solanum,
the largest and hyper diverse genus among the members
of the family Solanaceae accumulated so far is mainly
base on morphometrical characters and has left many
issues unresolved. The nomenclature of S. xanthocarpum
had remained controversial in the past. Phenotypically
this species is highly polymorphic. Morphological
character such as presence of hairs on the stem and
petiole, point of origin of spines, arrangement of spines,
length of filaments, flower colour etc. of this plant shows
polymorphism. Even, many taxonomists confused this
species with other Solanum species. The status of this
critical taxa can be justified by using molecular tools.
Again, the existence of genetic variability among
the species is very important for any plant improvement
program. The analysis of genotypes derived from
different geographical areas is important to study genetic
diversity. Though morphological characters are used for
distinguishing the plants but it is often restricted as
Das and Borah, 2012
147 Journal of Research in Plant Sciences (2012) 1(2): 146-152
Table 1 Plant materials used in the study
Name of the species Acc. No. Locality Geographical location(Altitude/latitude)
Solanum xanthocarpum
Schard. And Wendl.
1 = Sx01 Jalukbari, Assam 26°8'38.8914''N and 90°39'41.3809''E
2 = Sx02 Goalpara, Assam 26°10'0''N and 90°37'0''E
3 = Sx03 Golaghat, Assam 26°30'36''N and 93°85'0''E
4 = Sx04 Rani, Assam 26°3'43.84872''N and 91°36'3.90852''E
5 = Sx05 Khetri, Assam 26°10'00''N and 92°04'00''E
6 = Sx06 Shillong, Meghalaya 25°34'0''N and 91°53'0''E
7 = Sx07 Karbi Anglong, Assam 25°33' N and 92°10' E
Figure 1 Ethidium bromide stained agarose gel
showing genomic DNA of seven accessions of
Solanum xanthocarpum Schard. and Wendl.
M = Marker.
theses character may not be evident at all stages of
development. Now days, variety of genetic markers are
used to assess the genetic variation and identification of
species. Molecular tools provide valuable data in
exploiting the genetic variability that exist among
different genotypes through their ability to detect
variation at DNA level. The use of molecular markers
has been developed as powerful tools, for diversity
analysis in establishing relationship between cultivars, by
many workers all over the world (Awasthi et al., 2004,
Rangan et al., 2008). Among other molecular markers,
random amplified polymorphic DNA (RAPD) markers
and inter - simple sequence repeats (ISSR) markers are
widely used to exploit the variation existed among the
plants species. But though, RAPD is technically simple
and less expensive, ISSR is highly reproducible, more
reliable and very useful for population genetics, variety
identification, gene tagging, germplasm evaluation and
phylogenetic studies, conservation genetics. Due to these
advantages, it might provide valuable data in exploring
Das and Borah, 2012
Journal of Research in Plant Sciences (2012) 1(2): 146-152 148
Table 2 Characters of Primers used in the study
Marker Primer code Sequence (5´- 3´) Annealing Temperature(ºC)
ISSR
UBC 812 GAGAGAGAGAGAGAGAA 50.9
UBC 814 CACACACACACACACACAA 55.0
UBC 840 GAGAGAGAGAGAGAGAAT 58.0
UBC 848 CACACACACACACACAAG 50.9
Figure 2 Polymorphism on the band profiles of genotypes of Solanum xanthocarpum Schard. and Wendl.
revealed by the PCR Amplification generated by UBC 812 (A), UBC 814(B), UBC 840 (C) and UBC 848 (D)
resolved on 2.0% gel. M = Marker.
the genetic variation and distinction among the
genotypes of S. xanthocarpum. In this study, four ISSR
molecular markers were used in the evaluation of the
genetic diversity of seven accessions of S. xanthocarpum
Schard. and Wendl. of Assam and the efficiency of ISSR
markers in the discrimination of accessions was
evaluated.
MATERIALS AND METHODS
Plant material
Seven accessions of Solanum xanthocarpum Schard. and
Wendl. were collected from the different parts of Assam.
Information about the collected plants is included in
Table-1. Fresh, healthy green leaves of these samples
were used for DNA isolation and molecular
fingerprinting analysis.
DNA isolation
Genomic DNA was extracted from 0.5 g
of youn g fr esh leaves usin g CTAB
(Hexadecyltrimethylammoniumbromide) method
(Lassner et al.,1989). The purity of genomic DNA was
evaluated by measuring absorbance (A260 nm/A280 nm
ratio) with a Double Beam UV spectrophotometer. The
Purity and integrity of the DNA isolated were
determined by agarose gel (0.8%) electrophoresis stained
with ethidium bromide and using ladder DNA cleaved as
a size standard.
Primer used in PCRs
For ISSRs, ten primers obtained from UBC
primer set 100/9 (University of British Columbia) were
initially screened for their repeatable amplification with
seven accessions of Solanum xanthocarpum Schard. and
Wendl. After screening, four primers were selected for
further analysis based on their ability to detect distinct,
clearly resolved polymorphic amplified products. To
ensure reproducibility, the primers generating weak
products were discarded. Characters of the selected
primers are given in Table-2.
ISSR amplification
ISSR amplification reactions were carried out in
20 μl volume containing 50 ng template DNA, 0.5 U Taq
DNA polymerase, 10 mM dNTP, 10 μM primer in
1× reaction buffer that contained 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 2.5 mM MgCl2, and 0.01%
gelatin. Amplification was performed PCR amplification
was carried out in a Mini Thermal Cycler (Applied
Biosystems 9700). Amplification conditions were one
cycle at 94°C for 5 min., and 36 cycles each with 94°C
Das and Borah, 2012
149 Journal of Research in Plant Sciences (2012) 1(2): 146-152
Table 3 Degree of polymorphism, percentage of polymorphism (Pol %), polymorphic information content
(PIC) and marker index (MI) for ISSR primers in the seven genotypes of Solanum xanthocarpum Schard.
and Wendl.
Marker Primer code Total No. of bands
Total No. of
polymorphic bands
POL% PIC MI(Pol% x PIC)
ISSR
UBC 812 9 9 100 0.42 42.0
UBC 814 10 10 100 0.42 42.0
UBC 840 12 11 91.6 0.49 43.9
UBC 848 8 8 100 0.48 49.0
Sx01 Sx02 Sx03 Sx04 Sx05 Sx06 Sx07
Sx01 1.000
Sx02 0.375 1.000
Sx03 0.333 0.227 1.000
Sx04 0.360 0.370 0.308 1.000
Sx05 0.227 0.200 0.125 0.233 1.000
Sx06 0.227 0.304 0.286 0.276 0.364 1.000
Sx07 0.316 0.333 0.250 0.296 0.273 0.333 1.000
Table 4 Similarity matrices of seven genotypes of Solanum xanthocarpum based on ISSR profiling.
for 30 sec., 42°C for 30 sec., and 72°C for 1 min. The
amplified products were loaded on 2% agarose gel and
separated in 0.5× TBE buffer at 75 V and documented
using a gel documentation and image analysis system
(Make: Gel Doc. 2000, Bio Red).
Data scoring and analysis
Duplicate samples from each individual were
tested and, only well resolved and reproducible bands
amplified in both cases, were considered for the scoring
and data analysis. The numbers of polymorphic and
monomorphic amplification products were determined
for each primer for seven accessions of
Solanum xanthocarpum Schard. and Wendl. Scoring was
done as 1 for the presence and 0 for the absence across
the genotypes. The similarity matrix was computed to
estimate all pair wise differences in similarity matrices of
the amplification product, using sequential, hierarchical
clustering option of the SPSS version 11.0 software
package (Information Technology Science Centre,
Lingnan University, 2002). Level of similarity among
species was established as percentage of polymorphic
bands and a matrix of genetic similarity was compiled
using the Dice’s coefficient (Dice, 1945). The program
also generated a dendrogram which grouped the species
on the basis of Nei Genetic distance (Nei, 1978) using
unweighted pair group method with arithmetic average
(UPGMA) cluster analysis (Sneath and Sokal, 1973).
RESULTS
Genomic DNA isolation
The genomic DNA of nine genotypes of Solanum
xanthocarpum Schard. and Wendl. was extracted. The
genomic DNA extracted were pure and intact bands of
each sample was obtained when run in 0.8% agarose gel
(Figure 1).
ISSR amplification
The total number of DNA bands amplified with
the genotypes as well as the number of polymorphic
bands along with percentage of polymorphic bands
(POL%), polymorphic information content (PIC) and
marker Index (MI) is presented in Table 3. A total of 39
mappable ISSR markers were generated by four primers.
Out of these, 38 were polymorphic (97.43%).The
number of amplification products obtained per primer is
in the range of 8 to 12, with the primer UBC 848
producing the minimum number (8) and UBC 840
producing the maximum number (12) of bands. Except
primer UBC 840 all the primers showed 100% POL
percentage. The PIC value ranged from 0.42 to 0.49.
Marker index (MI) value was found to be ranging from
42.0 to 49.0. Primer UBC 848 showed maximum value
of MI i.e. 49.0 (Table 3). The amplification products
obtained by all the primer used in the study are
exhibited in Figure 2, which exemplified the typical
ISSR banding patterns observed.
Gene diversity analysis with ISSR marker
The ISSR data revealed that the genetic
similarity indices ranged from 0.125 to 0.375
(Table No. 4). The minimum similarity (0.125) was
found between Solanum xanthocarpum (Sx03) collected
from Golaghat and Sx05 collected from Khetri.
Maximum level of similarity indices (0.375) was found
between Sx01 and Sx02 collected from Jalukbari and
Goalpara respectively (Table 4). The genetic similarity
value ranged from 0.125 to 0.375 suggesting a wide
genetic base within the genotypes used in the present
investigation. Dendrogram based on the similarity
Das and Borah, 2012
Journal of Research in Plant Sciences (2012) 1(2): 146-152 150
Figure 3 UPGMA dendrogram based on ISSR data
for the studied population
matrices of ISSR-PCR banding patterns clearly
distinguished the samples of Solanum xanthocarpum
Schard. and Wendl. collected from different parts of
Assam into two clusters (Figure 3). Cluster-I has four
accessions comprising Sx01, Sx02, Sx04, and Sx03
collected from Jalukbari, Goalpara, Rani and Golaghat.
While three accessions (Sx05, Sx06 and Sx07) that were
collected from Khetri, Shillong and Karbi Anglong
formed cluster-II. A close association was observed
between Sx02 and Sx04 and in between Sx05 and Sx06.
DISCUSSION
Assessment of genetic variability within a
germplasm is of interest, for practical applications such
as, for conservation of genetic resources management
and for breeding purposes, to predict the ability to
combine or to rapidly verify the breeding material.
Again, identification of the species is of fundamental
importance in diversity studies in a variety of ways. For
evaluation of species diversity, it is essential that
individuals can be classified accurately. The
identification of taxonomic units, whose genetic
constitution is distinct from their more abundant
relatives, is important in the development of appropriate
conservation strategies. Most of the important members
of Solanum show high polymorphism in morphological
character possessing taxonomic confusion. ISSR
molecular markers have been used successfully in
germplasm bank characterization (Sudupak, 2004,
Carvalho et al., 2005, Essadki et al., 2006 and
Terzopoulos and Bebeli, 2008) especially in the
assessment of the differences among species or varieties
belonging to the same genus. The present study revealed
the genetic diversity within a collection of
Solanum xanthocarpum Schard. and Wendl. germ-plasm
representing different geographical regions of Assam
using ISSR marker. The ISSR data generated in these
population insights into the existing diversity of the
species. In this study, ISSR markers revealed moderate
levels of polymorphism with an average of nine
polymorphic bands per primer. Similarity indices ranged
from 0.125 to 0.375, also emphasizing the effectiveness
of ISSR markers. Cluster analysis based dendrogram
prediction using ISSR markers clearly distinguished the
experimental species. This indicates that the markers
have high discrimination ability of the groups of
Solanum xanthocarpum species.
Evaluation of diversity would also have immense
significance for in situ conservation of this medicinally
important species especially for further genetic
improvement programmes. Moreover, the results suggest
that the application of molecular fingerprinting enables a
rapid and sensitive method in detecting genetic variations
among the different populations of S.m xanthocarpum.
Our results confirm that DNA analysis by ISSR is an
efficient method for the exploration of genetic diversity
in S. xanthocarpum populations. To our knowledge, this
is the first report by ISSR markers to assess the great
variability that exist among germplasm of members of
Solanum xanthocarpum from Assam.
CONCLUSION
Genetic diversity that refers to the total number
of genetic characteristics in the genetic makeup of a
species is the most fundamental component of biological
diversity. Its analysis is a key element for any kind of
genetic improvement and conservation programme.
Again, maintaining diversity gives the population a
buffer against change, providing the flexibility to adapt.
Moreover, the results suggest that DNA analysis by ISSR
marker can be considered as a valuable method for
discrimination of S. xanthocarpum. And this study might
help in formulating new drugs in the field of
pharmacology. To our knowledge, this is the first report
to assess the variability among germplasm of Solanum
xanthocarpum by using ISSR markers from Assam.
Das and Borah, 2012
151 Journal of Research in Plant Sciences (2012) 1(2): 146-152
REFERENCES
Awasthi AK, Nagaraja GM, Naik GV, Kanginakudru
S, Thangavelu K and Nagaraju J. 2004. Genetic
diversity and relationships in mulberry (genus Morus) as
revealed by RAPD and ISSR marker assays. BMC
Genet., 5: 1-7.
Carvalho A, Matos M, Lima-Brito J, Guedes-Pinto H
and Benito C. 2005. DNA fingerprint of F1 interspecific
hybrids from the Triticeae tribe using ISSRs. Euphytica.,
143:93-99.
Dice LR. 1945. Measures of the amount of ecologic
association between species. Ecol., 26: 297-302.
Essadki M, Ouazzani N, Lumaret R and Moumni M.
2006. ISSR variation in olive-tree cultivars from
Morocco and other western countries of the
Mediterranean Basin. Genet Res Crop Evol., 53:475-482.
Lassner MW, Peterson P and Yoder JI. 1989.
Simultaneous amplification of multiple DNA fragments
by polymerase chain reaction in the analysis of
transgenic plants and their progeny. Plant Mol Biol Rep.,
7:116-128.
Nei M. 1978. Estimation of average heterozygosity and
genetic distance from a small number of individuals.
Genetics., 89:583-590.
Rangan L, Das A, Kesari V, Agarwal S and Sarma
GC. 2008. Use of DNA barcodes to identify Curcuma
species of Northeast India. International conference of
emerging technologies and applications in engineering,
technology and sciences, Rajkot, 2116-2120.
Singh OM and Singh TP. 2010. Phytochemistry of
Solanum xanthocarpum : an amazing traditional healer.
JSIR., 69:732-740.
Sneath PHA and Sokal RR. 1973. Numerical
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230-234.
Sudupak MA. 2004. Inter and intra-species Inter Simple
Sequence Repeat (ISSR) variations in the genus Cicer.
Euphytica., 135:229-238.
Terzopoulos PJ and Bebeli PJ. 2008. DNA and
morphological diversity of selected Greek tomato
(Solanum lycopersicum L.) landraces. Sci Hortic.,
116(4):354-361.
Das and Borah, 2012
Journal of Research in Plant Sciences (2012) 1(2): 146-152 152
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Intraspecific variation in Solanum xanthocarpum schard. and wendl.revealed by ISSR marker

  • 1. Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker Keywords: Genetic diversity, Solanum xanthocarpum, ISSR. ABSTRACT: Inter-simple sequence repeat (ISSR) analysis was performed in seven accessions of Solanum xanthocarpum Schard. and Wendl. of Assam to evaluate the applicability of this analysis for assessing the intraspecific variation. The value of similarity indices ranged from 0.375 to 0.125. The similarity result indicates the presence of high level of genetic diversity among the accessions of Solanum xanthocarpum Schard. and Wendl. UPGMA cluster analysis revealed clear grouping among the populations. The primers showed abilities in detecting genetic diversity across wild accessions of Solanum xanthocarpum Schard. and Wendl. Thus, ISSR-PCR technology can be used to study genetic variation and genetic relationships in the genus Solanum xanthocarpum Schard. and Wendl. 146-152 | JRPS | 2012 | Vol 1 | No 2 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.plantsciences.info Journal of Research in Plant Sciences An International Scientific Research Journal Authors: Ajoy Kumar Das1 , Sailendra Prasad Borah2 . Institution: 1. Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, India, 781 039. 2. Department of Botany, Gauhati University, Guwahati, Assam, India, 781 014. Corresponding author: Ajoy Kumar Das. Email: ajoykdas@iitg.ernet.in Phone No: +91 99579 96983. Web Address: http://www.plantsciences.info documents/PS0035.pdf. Dates: Received: 15 Aug 2012 Accepted: 15 Sep 2012 Published: 13 Dec 2012 Article Citation: Ajoy Kumar Das, Sailendra Prasad Borah. Intraspecific variation in Solanum xanthocarpum Schard. and Wendl. revealed by ISSR marker. Journal of Research in Plant Sciences (2012) 1(2): 146-152 Original Research Journal of Research in Plant Sciences JournalofResearchinPlantSciences An International Scientific Research Journal
  • 2. INTRODUCTION The genus Solanum xanthocarpum Schard. and Wendl. is an important medicinal perennial herb belonging to the family Solanaceae. This prickly herb is a good source of important alkaloids such as Solasodine, solasonine, solamargine and diosgenin. Solasodine extracted from this plant has antispermatogenic activity. Solamargine found in this herb has anticancer properties. Diosgenin extracted from Solanum xanthocarpum Schard. and Wendl. is found to be effective in suppressing FAS expression in HER 2 over expressing breast cancer cells (Singh and Singh, 2010). Glucoalkaloid solanocarpine, sterol carpesterol, solanocarpidine and traces of isochlorogenic, neochronogenic, chronogenic and caffeic acids are also found in the fruits of this plant. In India, this plant is used for curing various ailments since ancient times. Powder made from this herb is very useful in asthma, breathlessness and allergies of respiratory tracts. It is also used as carminative, digestive, alternate and astringent. As the herb is a stimulant to the heart and is a blood purifier, it is extremely beneficial in the treatment of cardiac diseases associated with edema. The fruit is useful as an aphrodisiac in males and the seeds, in women for irregular menstruation and dysmenorrheal. It also promotes conception in females. The roots of this plant are largely used in catarrhal and febrile affection, cough, chest pain, flatulence, sore throat, toothache and constipation and in bronchial diseases. Leaves of this plant are also used in rheumatic pain. Solanum xanthocarpum Schard. and Wendl. is non toxic and safe for human use and is regarded as a valuable plant in both ayurvedic and modern drug development areas for its versatile medicinal uses. The taxonomic description of the genus Solanum, the largest and hyper diverse genus among the members of the family Solanaceae accumulated so far is mainly base on morphometrical characters and has left many issues unresolved. The nomenclature of S. xanthocarpum had remained controversial in the past. Phenotypically this species is highly polymorphic. Morphological character such as presence of hairs on the stem and petiole, point of origin of spines, arrangement of spines, length of filaments, flower colour etc. of this plant shows polymorphism. Even, many taxonomists confused this species with other Solanum species. The status of this critical taxa can be justified by using molecular tools. Again, the existence of genetic variability among the species is very important for any plant improvement program. The analysis of genotypes derived from different geographical areas is important to study genetic diversity. Though morphological characters are used for distinguishing the plants but it is often restricted as Das and Borah, 2012 147 Journal of Research in Plant Sciences (2012) 1(2): 146-152 Table 1 Plant materials used in the study Name of the species Acc. No. Locality Geographical location(Altitude/latitude) Solanum xanthocarpum Schard. And Wendl. 1 = Sx01 Jalukbari, Assam 26°8'38.8914''N and 90°39'41.3809''E 2 = Sx02 Goalpara, Assam 26°10'0''N and 90°37'0''E 3 = Sx03 Golaghat, Assam 26°30'36''N and 93°85'0''E 4 = Sx04 Rani, Assam 26°3'43.84872''N and 91°36'3.90852''E 5 = Sx05 Khetri, Assam 26°10'00''N and 92°04'00''E 6 = Sx06 Shillong, Meghalaya 25°34'0''N and 91°53'0''E 7 = Sx07 Karbi Anglong, Assam 25°33' N and 92°10' E Figure 1 Ethidium bromide stained agarose gel showing genomic DNA of seven accessions of Solanum xanthocarpum Schard. and Wendl. M = Marker.
  • 3. theses character may not be evident at all stages of development. Now days, variety of genetic markers are used to assess the genetic variation and identification of species. Molecular tools provide valuable data in exploiting the genetic variability that exist among different genotypes through their ability to detect variation at DNA level. The use of molecular markers has been developed as powerful tools, for diversity analysis in establishing relationship between cultivars, by many workers all over the world (Awasthi et al., 2004, Rangan et al., 2008). Among other molecular markers, random amplified polymorphic DNA (RAPD) markers and inter - simple sequence repeats (ISSR) markers are widely used to exploit the variation existed among the plants species. But though, RAPD is technically simple and less expensive, ISSR is highly reproducible, more reliable and very useful for population genetics, variety identification, gene tagging, germplasm evaluation and phylogenetic studies, conservation genetics. Due to these advantages, it might provide valuable data in exploring Das and Borah, 2012 Journal of Research in Plant Sciences (2012) 1(2): 146-152 148 Table 2 Characters of Primers used in the study Marker Primer code Sequence (5´- 3´) Annealing Temperature(ºC) ISSR UBC 812 GAGAGAGAGAGAGAGAA 50.9 UBC 814 CACACACACACACACACAA 55.0 UBC 840 GAGAGAGAGAGAGAGAAT 58.0 UBC 848 CACACACACACACACAAG 50.9 Figure 2 Polymorphism on the band profiles of genotypes of Solanum xanthocarpum Schard. and Wendl. revealed by the PCR Amplification generated by UBC 812 (A), UBC 814(B), UBC 840 (C) and UBC 848 (D) resolved on 2.0% gel. M = Marker.
  • 4. the genetic variation and distinction among the genotypes of S. xanthocarpum. In this study, four ISSR molecular markers were used in the evaluation of the genetic diversity of seven accessions of S. xanthocarpum Schard. and Wendl. of Assam and the efficiency of ISSR markers in the discrimination of accessions was evaluated. MATERIALS AND METHODS Plant material Seven accessions of Solanum xanthocarpum Schard. and Wendl. were collected from the different parts of Assam. Information about the collected plants is included in Table-1. Fresh, healthy green leaves of these samples were used for DNA isolation and molecular fingerprinting analysis. DNA isolation Genomic DNA was extracted from 0.5 g of youn g fr esh leaves usin g CTAB (Hexadecyltrimethylammoniumbromide) method (Lassner et al.,1989). The purity of genomic DNA was evaluated by measuring absorbance (A260 nm/A280 nm ratio) with a Double Beam UV spectrophotometer. The Purity and integrity of the DNA isolated were determined by agarose gel (0.8%) electrophoresis stained with ethidium bromide and using ladder DNA cleaved as a size standard. Primer used in PCRs For ISSRs, ten primers obtained from UBC primer set 100/9 (University of British Columbia) were initially screened for their repeatable amplification with seven accessions of Solanum xanthocarpum Schard. and Wendl. After screening, four primers were selected for further analysis based on their ability to detect distinct, clearly resolved polymorphic amplified products. To ensure reproducibility, the primers generating weak products were discarded. Characters of the selected primers are given in Table-2. ISSR amplification ISSR amplification reactions were carried out in 20 μl volume containing 50 ng template DNA, 0.5 U Taq DNA polymerase, 10 mM dNTP, 10 μM primer in 1× reaction buffer that contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, and 0.01% gelatin. Amplification was performed PCR amplification was carried out in a Mini Thermal Cycler (Applied Biosystems 9700). Amplification conditions were one cycle at 94°C for 5 min., and 36 cycles each with 94°C Das and Borah, 2012 149 Journal of Research in Plant Sciences (2012) 1(2): 146-152 Table 3 Degree of polymorphism, percentage of polymorphism (Pol %), polymorphic information content (PIC) and marker index (MI) for ISSR primers in the seven genotypes of Solanum xanthocarpum Schard. and Wendl. Marker Primer code Total No. of bands Total No. of polymorphic bands POL% PIC MI(Pol% x PIC) ISSR UBC 812 9 9 100 0.42 42.0 UBC 814 10 10 100 0.42 42.0 UBC 840 12 11 91.6 0.49 43.9 UBC 848 8 8 100 0.48 49.0 Sx01 Sx02 Sx03 Sx04 Sx05 Sx06 Sx07 Sx01 1.000 Sx02 0.375 1.000 Sx03 0.333 0.227 1.000 Sx04 0.360 0.370 0.308 1.000 Sx05 0.227 0.200 0.125 0.233 1.000 Sx06 0.227 0.304 0.286 0.276 0.364 1.000 Sx07 0.316 0.333 0.250 0.296 0.273 0.333 1.000 Table 4 Similarity matrices of seven genotypes of Solanum xanthocarpum based on ISSR profiling.
  • 5. for 30 sec., 42°C for 30 sec., and 72°C for 1 min. The amplified products were loaded on 2% agarose gel and separated in 0.5× TBE buffer at 75 V and documented using a gel documentation and image analysis system (Make: Gel Doc. 2000, Bio Red). Data scoring and analysis Duplicate samples from each individual were tested and, only well resolved and reproducible bands amplified in both cases, were considered for the scoring and data analysis. The numbers of polymorphic and monomorphic amplification products were determined for each primer for seven accessions of Solanum xanthocarpum Schard. and Wendl. Scoring was done as 1 for the presence and 0 for the absence across the genotypes. The similarity matrix was computed to estimate all pair wise differences in similarity matrices of the amplification product, using sequential, hierarchical clustering option of the SPSS version 11.0 software package (Information Technology Science Centre, Lingnan University, 2002). Level of similarity among species was established as percentage of polymorphic bands and a matrix of genetic similarity was compiled using the Dice’s coefficient (Dice, 1945). The program also generated a dendrogram which grouped the species on the basis of Nei Genetic distance (Nei, 1978) using unweighted pair group method with arithmetic average (UPGMA) cluster analysis (Sneath and Sokal, 1973). RESULTS Genomic DNA isolation The genomic DNA of nine genotypes of Solanum xanthocarpum Schard. and Wendl. was extracted. The genomic DNA extracted were pure and intact bands of each sample was obtained when run in 0.8% agarose gel (Figure 1). ISSR amplification The total number of DNA bands amplified with the genotypes as well as the number of polymorphic bands along with percentage of polymorphic bands (POL%), polymorphic information content (PIC) and marker Index (MI) is presented in Table 3. A total of 39 mappable ISSR markers were generated by four primers. Out of these, 38 were polymorphic (97.43%).The number of amplification products obtained per primer is in the range of 8 to 12, with the primer UBC 848 producing the minimum number (8) and UBC 840 producing the maximum number (12) of bands. Except primer UBC 840 all the primers showed 100% POL percentage. The PIC value ranged from 0.42 to 0.49. Marker index (MI) value was found to be ranging from 42.0 to 49.0. Primer UBC 848 showed maximum value of MI i.e. 49.0 (Table 3). The amplification products obtained by all the primer used in the study are exhibited in Figure 2, which exemplified the typical ISSR banding patterns observed. Gene diversity analysis with ISSR marker The ISSR data revealed that the genetic similarity indices ranged from 0.125 to 0.375 (Table No. 4). The minimum similarity (0.125) was found between Solanum xanthocarpum (Sx03) collected from Golaghat and Sx05 collected from Khetri. Maximum level of similarity indices (0.375) was found between Sx01 and Sx02 collected from Jalukbari and Goalpara respectively (Table 4). The genetic similarity value ranged from 0.125 to 0.375 suggesting a wide genetic base within the genotypes used in the present investigation. Dendrogram based on the similarity Das and Borah, 2012 Journal of Research in Plant Sciences (2012) 1(2): 146-152 150 Figure 3 UPGMA dendrogram based on ISSR data for the studied population
  • 6. matrices of ISSR-PCR banding patterns clearly distinguished the samples of Solanum xanthocarpum Schard. and Wendl. collected from different parts of Assam into two clusters (Figure 3). Cluster-I has four accessions comprising Sx01, Sx02, Sx04, and Sx03 collected from Jalukbari, Goalpara, Rani and Golaghat. While three accessions (Sx05, Sx06 and Sx07) that were collected from Khetri, Shillong and Karbi Anglong formed cluster-II. A close association was observed between Sx02 and Sx04 and in between Sx05 and Sx06. DISCUSSION Assessment of genetic variability within a germplasm is of interest, for practical applications such as, for conservation of genetic resources management and for breeding purposes, to predict the ability to combine or to rapidly verify the breeding material. Again, identification of the species is of fundamental importance in diversity studies in a variety of ways. For evaluation of species diversity, it is essential that individuals can be classified accurately. The identification of taxonomic units, whose genetic constitution is distinct from their more abundant relatives, is important in the development of appropriate conservation strategies. Most of the important members of Solanum show high polymorphism in morphological character possessing taxonomic confusion. ISSR molecular markers have been used successfully in germplasm bank characterization (Sudupak, 2004, Carvalho et al., 2005, Essadki et al., 2006 and Terzopoulos and Bebeli, 2008) especially in the assessment of the differences among species or varieties belonging to the same genus. The present study revealed the genetic diversity within a collection of Solanum xanthocarpum Schard. and Wendl. germ-plasm representing different geographical regions of Assam using ISSR marker. The ISSR data generated in these population insights into the existing diversity of the species. In this study, ISSR markers revealed moderate levels of polymorphism with an average of nine polymorphic bands per primer. Similarity indices ranged from 0.125 to 0.375, also emphasizing the effectiveness of ISSR markers. Cluster analysis based dendrogram prediction using ISSR markers clearly distinguished the experimental species. This indicates that the markers have high discrimination ability of the groups of Solanum xanthocarpum species. Evaluation of diversity would also have immense significance for in situ conservation of this medicinally important species especially for further genetic improvement programmes. Moreover, the results suggest that the application of molecular fingerprinting enables a rapid and sensitive method in detecting genetic variations among the different populations of S.m xanthocarpum. Our results confirm that DNA analysis by ISSR is an efficient method for the exploration of genetic diversity in S. xanthocarpum populations. To our knowledge, this is the first report by ISSR markers to assess the great variability that exist among germplasm of members of Solanum xanthocarpum from Assam. CONCLUSION Genetic diversity that refers to the total number of genetic characteristics in the genetic makeup of a species is the most fundamental component of biological diversity. Its analysis is a key element for any kind of genetic improvement and conservation programme. Again, maintaining diversity gives the population a buffer against change, providing the flexibility to adapt. Moreover, the results suggest that DNA analysis by ISSR marker can be considered as a valuable method for discrimination of S. xanthocarpum. And this study might help in formulating new drugs in the field of pharmacology. To our knowledge, this is the first report to assess the variability among germplasm of Solanum xanthocarpum by using ISSR markers from Assam. Das and Borah, 2012 151 Journal of Research in Plant Sciences (2012) 1(2): 146-152
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