2. GENETIC DIVERSITY IN SOYBEAN (GLYCINE MAX (L.))
BY USING RAPD MARKER
SUBMITTED BY
MR.GITE ABHIJEET SUNIL
(BTL-27/2018)
RESEARCH GUIDE CO-GUIDE
DR.DAS S. PROF.SHELKE M.R.
Centre for Biotechnology, College of Agricultural
PIMS, Loni . Biotechnology, Loni .
SUBMITTED TO
DEPARTMENT OF PLANT BIOTECHNOLOGY
COLLEGE OF AGRICULTURAL BIOTECHNOLOGY, LONI 413736.
TAL-RAHATA, DIST-AHMEDNAGAR
3. 1) Introduction
2) Importance of ResearchWork
3) Objective
4) Place of Research Work
5) Materials
6) Methodology
7) Result and Discussion
8) Summary and Conclusion
9) Outcome of the Programme
10) References
CONTENTS
4. Botanical Classification :-
Kingdom - Plantae
Division - Magnoliophyta
Class - Magnoliopsida
Order - Fabales
Family - Fabaceae
Genus - Glycine
Species - Glycine max
Botanical Name - Glycine max L.
1.INTRODUCTION :-
5. Soybean (Glycine max) is an annual crop and belongs to the family of
Fabaceae.
It is an important legume crop, known for its high quality of protein and
oil content, and beneficiary secondary metabolites such as saponin,
phenolic compound (Tidke et al., 2017).
It contains 40-42% protein, 18-22% oil comprising of 85% unsaturated
fatty acids and 15% saturated fatty acids, 28% Carbohydrate and other
nutrients.
It also consist many therapeutic components and has increased its
importance in industrial, agricultural and medicinal sectors.
It is an important crop in the world in terms of production and usage.
Production of Soybean in India is dominated by Maharashtra and Madhya
Pradesh which contribute to 89 % of total Soybean production in country.
Soybean :-
6. Genetic diversity is defined as the biodiversity within species and it is the
foundation of plant breeding. Genetic diversity is an essential requirement
of plant breeding and the basis of genetic development.
RAPD is a technique based on the PCR method to identify genetic
variation and it is dominant marker.
RAPD has been used as an assessment of intraspecific variation since 1990.
Moreover, in recent years, RAPD was also used for genetic mapping,
taxonomic study and even used to detect genetic mutation in the treated
plant.
RAPD is well established genetic tool which provides a simple and fast
approach to detect DNA Polymorphism for cultivars identification and
diversity analysis.
7. 2.IMPORTANCE OF RESEARCH WORK :
This study will be useful as basic information for further soybean
breeding programs, since it will make it easy to select where the
soybean which has high genetic variation can be used for
breeding.
It is important tool for evaluating genetic diversity.
Understanding the genetic diversity of soybean germplasms is
essential to broaden the genetic base and to further utilize it in
breeding program .
Generations of new and improved cultivars can be enhanced by
new sources of genetic variation; therefore criteria for parental
stock selection need to be considered not only by agronomic
value, but also for genetic dissimilarity.
12. I. Plant Material :-
Seeds of Soybean genotypes were used .
II. Methodology :-
a) Seed Germination
b) DNA Extraction
c) Agarose Gel Electrophoresis
d) Quantification of DNA
e) PCRAmplification
f) Resolution of amplified product
g) Scoring of amplified fragments
13. I. Plant Material
Soybean Genotypes :-
The genotypes of soybean (Glycine max (L.)) was collected from MPKV,
Agricultural Research Station (ARS), Rahuri. Maharashtra and used as an
experimental material.
Sr. No. Names of Genotypes
1. IVT Code-24
2. NRC-147
3. JS-335
4. BAVS-102
5. DSB-34
6. MACS-450
Table 1: List of Selected Soybean Genotypes
14. II. Methodology
a) Germination of Soybean Seeds :-
The seeds of different varieties of Soybean were grown in germination
tray.They were allowed to grow with regular irrigation until appropriate growth
essential for DNA isolation wasobtained.
Plate 1: Soybean plantlets grown in germination tray
15. b) DNA Extraction :-
The plant genomic DNA was isolate by using the modified CTAB
method described by Saghai-Maroof et al. (1984).
Table 1. Reagents of 2% DNA Extraction buffer
Extraction buffer were modify with β-mercaptoethanol (0.2%) and
PVP (0.3%).
Sr. No. Components Quantity
1. 10 % CTAB 20 ml
2. 4 M NaCl 35 ml
3. 1M Tris HCl (pH 8.0) 10 ml
4. 1 M EDTA(pH 8.0) 4 ml
5. 0.2% β-mercaptoethanol 200 µl
6. 0.3 % of PVP 300 mg
5. Double distilled sterile water 31 ml
16. These tubes were kept in centrifuged machine
at 13,000 rpm for 11 min at 4OC. Then
supernatant was collected in a fresh tube and
equal volume of chloroform: isoamylalcohol
was added.
2% DNA extration buffer was prepared and
kept in water bath at 65℃ for 30 min.
DNA Isolation Protocol :-
Leaf samples were crushed in liquid nitrogen
using mortar and pestle .
Leaf powder was transferred to centrifuge
tube containing 1 ml extraction buffer and
kept in water bath for 65℃ for 45 min.
Plate -2
Sample Crushing
17. The tubes were followed by inversions for 5 min and
then again centrifuged at 13,000 rpm for 11min at 4OC.
The above steps was repeated for 2-3 times.
Plate-3
Addition of Chilled
Isopropanol
Then ⅓rd volume of chilled isopropanol was added in
the collected supernatant and the tubes were kept for
incubation at -20℃ for overnight.
On the next day, the tubes were centrifuged at 13,000
rpm for 11min.Then equal volume of ethanol was
added in the collected pellet and tubes were
centrifuged at 7000 rpm for 5min.
The above step were repeated 2-3 times, then the pellet
was air dried and dissolved in 250 μl of 1X TE buffer
and the isolated DNA was stored at-20°C.
18. 1μl of RNase were add into the
100μl of isolated DNA to remove
RNA contamination.
Then the tubes were incubate at
37OC for 1 hour in hot water bath.
RNase treatment
19. c) Agarose Gel Electrophoresis :-
After RNase treatment DNA band was
separated by agarose gel electrophoresis.
Requirement-
i. ElectrophoresisAssembly.
ii. 0.8% Agarose.
iii. 1X TAE buffer.
iv. Ethidium bromide.
v. 6X Loading dye. Plate -4
Gel electrophoresis
20. Then the mixture was poured into a casting tray and
allow to solidify.
The casted gel was placed in 1X TAE buffer tank
with wells towards cathode.
5μl of DNA of different genotypes was mixed with
1μl of 6X gel loading dye and loaded in gel.
Then the electrophoresis was carried out at 50V for
an hour and after that the gel was view under gel
documentation unit.
0.8% agarose gel was prepared and then the
ethidium bromide dye was added as a staining agent.
Agarose gel electrophoresis Protocol :-
21. d. Quantification of DNA
The concentration and purity of
DNA was checked using 1μl of
DNA of each soybean varieties
by using Bio-Spectrometer.
Quantification of DNA
Plate -5
22. RAPD Primers were used for PCR Amplification.
e.Polymerase Chain Reaction
Components
Final
Concentration
Volume for one tube
(μl)
10X PCR buffer with MgCl2 2 X 2.0
10 mM dNTPs 0.8 mM 1.6
Primer (100 pmol) 5 pmol 1.0
Taq DNA polymerase (5U) 1 U 0.2
Sample DNA 30-60 ng/μl 2.0
Sterile double distilled water - 13.2
Total - 20 μl
Components of PCR -
Table 2:The components, stock and volume of PCR reaction mixture
23. Sr.
No.
Steps
Temperature
(0 C)
Duration
(min)
Number of
cycles
1. Initial Denaturation 94 10
2. Denaturation 94 1
35
3. Annealing
59
1
4. Extension 72 1
5. Final extension 72 10
6. Hold 4
Temperature Profile :
Table 3: Temperature Profile for PCRAmplification.
Optimization of PCR conditions
24. f. Resolution of Amplified Products
The amplified products was load on 3.5% agarose gel at 100 V for
2 hour and observed under gel documentation.
The amplified products for all the primers was compare with each
other and bands of DNA fragments was score as ‘1’ for presence
and ‘0’for absence and ‘9’for missing.
Percent polymorphism was calculate by using the formula :-
Number of polymorphic bands
Percent polymorphism = × 100
Total number of bands
25. 7. Result & Discussion
Plate.6. Genomic DNA on 0.8% agarose gel
1) DNA Isolation :
Genomic DNA was extracted using CTAB extraction method.
The results of 0.8% agarose gel electrophoresis showed good quality of
DNA.
Sr. No. Varieties Name
1. IVT Code-24
2. NRC-147
3. JS-335
4. BAVS-102
5. DSB-34
6. MACS-450
Table 4:Name of varieties
1 2 3 4 5 6
26. Sr. No Name of Variety
Concentration
of DNA
(ng/μl)
O.D
(260/280)
1. IVT Code-24 471.1 1.7
2. NRC-147 183.4 1.8
3. JS-335 236.5 1.7
4. BAVS-102 158.0 1.8
5. DSB-34 292.3 1.8
6. MACS-450 502.3 1.7
2) DNAQuantification:
Table 5. Quantification of DNA
The quantity of DNA was checked by using Bio-Spectrometer.
It was found to be in the range of 158.0 ng/µl -502.3 ng/µl.
27. Present investigation was undertaken to analyze the genetic diversity in
soybean genotypes by using RAPD marker.In this study, DNA was isolated
from the young leaves of six soybean genotypes by using modified CTAB
DNA extraction method.The quality of DNA was tested using qualitative
and quantitative analysis.Good intact bands on agarose gel certified its
quality.The RNase treatment was given to the sample to avoid any RNA
contamination. During the quantification of DNA , the ratio of the 260/280
spectrophotometric measurements from 1.7-1.8 indicates the presence of
the pure DNA and determining its quality. This study can useful for
selection of parents to develop new hybrids.
8.Summary and Conclusion
28. While working on “Genetic diversity in Soybean (Glycine max(L))
using RAPD marker” I have learnt the following techniques :-
Isolation of DNA
Purification and Quantificationmethods
Electrophoresis
Preparation of chemical solutions
Handling Instruments
General laboratory practices
9. Outcome of the Programme
29. Agam M.,Kusmiyati F., Anwar S. and Herwibawa B.(2020).Diversity analysis in
soybean (Glycine max [L.]) mutant lines grown in saline soil using agronomic traits
and RAPD markers. The 1st International Conference on Genetic Resources and
Biotechnology IOP Conf. Series: Earth and Environmental Science; 482:1-7.
Aziza S., E.Kholy. (2013). Evaluation of Genetic Variation among Soybean (Glycine
max L.) Cultivars using SDS-PAGE and RAPD Markers. Egypt. J. Botany;14(3):33-
48.
Bimal S., Aggarwal A., and Sain N., (2013). Molecular Characterization and Diversity
Analysis of Soybean Varieties Against Soybean Rhizoctonia Aerial Blight Resistance.
International Journal of Bioinformatics and Biological Science ;1(.3&4):339-351.
Brown G.,Thompson J.,Nelson R.,and Warburton M.(2000).Evaluation of Genetic
Diversity of Soybean Introductions and North American Ancestors Using RAPD and
SSR Markers.Crop Science;40:815–823.
10.REFERENCES
30. Jain R., Joshi A. and Jain D .(2017). Molecular Marker based Genetic Diversity
Analysis in Soybean [Glycine max (L.) Merrill] Genotypes. International Journal of
Current Microbiology and Applied Sciences ; 6 (6) : 1034-1044.
Mundewadikar D., Deshmukh P.(2014). Genetic Variability and Diversity Studies in
Soybean [Glycine max (L.) Merrill] using RAPD Marker. International Journal of
Scientific and Research Publications ; 4(9):2250-3153.
Nkongolo K., Alamri S. and Michael P. (2020). Assessment of Genetic Variation in
Soybean (Glycine max) Accessions from International Gene Pools Using RAPD
Markers: Comparison with the ISSR System. American Journal of Plant Sciences;
11:1414-1428.
Omar A., Mohamed A., Nasr M.,Hameed K., Alabsawy E., Hamdi A., and Zaye E.,
(2018). Molecular Characterization of Soybean (Glycine max (L).Merr) Genotypes
Tolerant and /or Susceptible to Cotton Leaf Worm (Spodoptera littoralis). American
Journal of Biochemisry and Biotechnology ; 8(1): 34-47.
31. Panjoo M., Nazarian F,Ismaili A. and Ahmadi H.(2014). Evaluation of Genetic
Diversity among Soybean (Glycine max) Genotypes, Using ISJ and RAPD
Molecular Markers. Journal of Plant Physiology and Breeding ;4(2): 55-65.
Sammour R., Mustafa A.(2013). Genetic variation in soybean (Glycine max (L.)
Merrill) germplasm. Research and Reviews in Biosciences,7(1):19-26.
Tidke S.D., and Jadhav A.J., (2018). Molecular Characterization of Different
Varieties of Glycine max L. Using RAPD Marker. Trends in Biosciences ;11(25):
3342-3346.
Wahyudi D., Hapsari L., Sundari.(2020). RAPD Analysis for Genetic Variability
Detection of Mutant Soybean (Glycine max (L.) Merr. Journal of Tropical
Biodiversity and Biotechnology ; 5(1) : 68 -77.
32.
33.
34. Semester : VII (New)
Academic
Year
: 2021-22
Course No. : ET-472 Course Title : Educational Tour
Credits : 1(0+1) Total Marks : 50
Name of the Student : Mr. Gite Abhijeet Sunil
Registration No. : BTL- 27/2018
Name of the College :
College of Agricultural Biotechnology, Loni,
Tal. Rahata, Dist. Ahmednagar.
A Report on
EDUCATIONAL TOUR
36. The ICAR-National Research Centre on Pomegranate, Solapur (Maharashtra),
was established on June 16, 2005 by the Indian Council of Agricultural Research,
New Delhi, as a step to strengthen research and development infrastructure for
pomegranate crop.
Though Universities and institutes all over the world are having research
programmes on pomegranate, this is the only institution working solely on
pomegranate.
The centre was established to fulfill the growing demand for pomegranate both in
domestic and export market through tapping the immense production potential
prevailing in the country.
The perspective plan emphasizes the methods of achieving the targets through
scientific, profitable, ecofriendly and innovative technologies for cultivation, both
in traditional as well as non traditional areas thereby, expanding pomegranate area
and production.
37. Institute Profile
ICAR-National Research Centre on
Pomegranate, Solapur.
Director Dr. R. A. Marathe.
Established on June 16, 2005.
Location NH-65, Solapur-Pune Highway,
Kegaon,Solapur (Maharashtra).
413 255, India.
Website https://nrcpomegranate.icar.gov.in
39. Vision and Mission :-
Vision:-
To generate research publications with high impact factor. The
perspective plan emphasizes the methods of achieving the targets
through scientific, profitable, ecofriendly and innovative technologies
for cultivation, both in traditional as well as non traditional areas
thereby, expanding pomegranate area and production.
Mission :-
To establish an international repository of genetic resources,
develop suitable technologies for pomegranate production and to
improve economic status of farmers in different regions.
40. 1.SAIF (Sophisticated Analytical Instrument Facility):-
ICAR-NRCP houses a variety of major analytical instruments and
other research facilities which are operated and maintained by a
dedicated and qualified group of Scientists.
It is an integral part of institute and operates with an “open access
policy” so that, all can benefit from the services of SAIF.
SAIF is primarily meant to serve educational institute who are unable
to procure and maintain such facilities, but in case of clash of timings
the research work of the ICAR-NRCP will always get priority.
SAIF does not operate to compete with commercial laboratories.
41. 2.Special Equipments :-
The laboratories were equipped with various instruments which
are essential for running the laboratory viz,
Spectrophotometer Water Purification System
Microscope with digital camera Horizontal Shaker
BOD Incubators Microwave Digestion System
Deep Freezer Precision Water Bath
Photosynthesis unit & Leaf area meter Digital Refractometer
Laminar air flow chamber Fermenter
Atomic absorption Spectrophotometer Muffle Furnace
44. 4..Research Farm :-
The research farm is situated in 2 different locations viz., Kegaon & Hiraj
which is about 1.5 km away from each other. The research farm is accessible with the
well connecting roads laid across the length and breadth of the farm. Besides, the
research farm features the following facilities:
Drip irrigation system,
Water harvesting ponds,
Open wells & borewells,
Tractors & accessories,
Sprayers,
Various farm implements,
Solar lighting system,
Barbed wire fencing, etc..
Farm Pond, Hiraj. Fruiting Orchard
45. 5. Agriculture Knowledge Management Unit:-
It is provided with internet connections in order to have
connectivity with ICAR headquarters and other institutes. Several
online journals are accessible through Consortium on e-Resources in
Agriculture (CeRA).
6.Library :
The Centre has developed its library which has enormous
books (2001) and subscribed to 06 Journals of related disciplines.
No. of Books available : 2001
No. of Journals being subscribed : 06
No. of News Papers subscribed : 06
46. Services :-
Consultancy : For pomegranate production , protection, export and
post- harvest management and value addition.
Contract Research : On the specific aspects of Pomegranate can be
taken up.
Training :- Regarding Pomegranate production ,protection and post
harvest technology of pomegranate are organized as per
requirement.
Soil testing :- Testing of potential chemicals and bioformulation
against pests or disease supply of pure cultures.
47. Research Projects :-
Conservation, characterization and sustainable use of diversity in
pomegranate.
Principal Investigators : Dr.(Mrs.).Shilpa Parashuram
Genetic improvement of pomegranate.
Principal Investigators : Dr. K. Dinesh Babu
Development and refinement of integrated production technologies for
improved productivity.
Principal Investigators : Dr. D. T. Meshram
Propagation, bio-hardening and mass multiplication of elite planting
material in pomegranate (Punica granatum L.).
Principal Investigators : Dr. N.V. Singh
48. Development and refinement of integrated crop protection technologies for
improved productivity of pomegranate.
Principal Investigators : Mr. Mallikarjun
Post harvest management value addition and improving knowledge of
stakeholders for increasing production and marketing of pomegranate.
Principal Investigators : Dr. Nilesh N. Gaikwad
Development and refinement of integrated crop protection technologies for
improved productivity of pomegranate.
Principal Investigators : Mr. Mallikarjun
Biological control of wilt complex problem in pomegranate.
Principal Investigators : Dr. U. R. Sangle
50. Kalash Seeds Pvt. Ltd. is a leading seed company mainly in the vegetable sector
in India. Our talented team of scientists, breeders, and researchers bring to you
the best quality seeds. We have been catering to the needs of thousands of Indian
and international professional growers and farmers since 2011 with an experience
of over 50 years.
Kalash Seeds Pvt. Ltd is a leading seed company in India doing Research &
Marketing in tropical vegetable.
Presently among top 5 Companies in India in vegetable seeds industry. Recently
started focusing on SAARC countries.
There is steady growth of turnover as the research products are giving very good
results in the market.
In Indian Vegetable Seed market, Kalash has already achieved market leadership
in chili, pumpkin, beetroot, onion, broccoli, muskmelon.
51. Vision :-
To add to the prosperity of farmers and stakeholders by delivering excellent
research products.
To provide healthy environment for mutual growth of company as well as
employees.
To adopt innovative technologies for developing outstanding products & process
thus making a contribution to countries agricultural growth.
Mission :-
Kalash seeds mission is to “reach every corner of the world to connect each
vegetable growing farmer and provide service to dealers, stakeholders and
farmers with elation and prosperity by providing Seeds for Better Future”.
52. Kalash Seeds Private Limited,Jalana.
Chairman Mr. Suresh Agrawal.
Established on 31 january,2011.
Location Kalash Seeds Pvt. Ltd,
P.O.Box-77, Mantha
Road,Jalna - 431203
(Maharashtra - India).
Website www.kalashseeds.co.in
53. Infrastructure :-
1.Farms and green houses :-
Kalash Seeds has its own farms and green houses, their farms
are located in the state of Maharashtra, Andhra Pradesh, Telangana,
Tamilnadu, Punjab, Himachal Pradesh, and Karnataka. It has strong
network and trusted relationship with farmers help them in quality
seed production.
2.Company resources and facility :-
Involved in Agri-Research, seed production, processing &
marketing
• Over 160 ha. Research farm land, 5 ha. polyhouse & 1 ha.
• Temperature controlled greenhouse.
• Sophisticated laboratories for biotech & phytopathology research.
• Fourteen field research stations in different regions of India.
• A strong team of more than 700 technical & highly qualified
personnel's.
54. 3.High tech processing :-
Kalash Seeds has a well established seed processing centre with well
equipped seed treatments, and packing line. We have the good seed cleaning,
coloring, advanced automated filling, packing machines etc. in the processing
plant.
55. Research and development :-
1.Breeding :-
India is having diverse agro climatic zones and the agriculture practices
are different in different regions. Hence, the requirement of seeds for
commercial production of various crops varies.
With this information better products in different vegetables, cotton,
paddy, etc. are developed and evaluated for their performance in
different agro climatic conditions.
They initiated breeding programs for Non Bt desi cotton, germplasm
collection evaluation, trait identification, and hybrid development is
undertaken for sucking pest and draught tolerant like traits. Similarly,
Open pollinated and F1 hybrid development initiated in rice crop.
56. 2.Biotechnology :-
Kalash Seeds is also focused on delivering best quality product to
farmers and the seeds produced are tested for genetic purity
(quality).
Marker based purity test of seeds for quality control provides a
quicker and authentic alternative to time consuming grow out test
(GOT).
This shortens the time needed to make the seeds available for
farmers with more precision.
57. Marketing and sales :-
Company has its own excellent marketing network throughout India and SAARC
countries.
In India, there are regional offices in all major states.
A strong dealer and distributor network is associated with the company since its
inception.
Currently, Kalash Seeds focuses on SAARC countries, i.e. India, Nepal, Sri Lanka,
Bangladesh, Pakistan, Bhutan and Afghanistan for its vegetable seeds and open
pollinated seed business.
Research partnership :-
Beej Sheetal Research Pvt. Ltd is research partner for breeding and new variety
development in Vegetables.
Also, Initiatives taken for licensing the newly developed varieties orF1 hybrids of
different crops from various public institutions. Hybrid Chili F1 product licensed for
production and sale from Punjab Agriculture University, Ludhiana.
