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Studies on the Genetic Diversity of Lentil (lens Culinaris Ssp. Culinaris) Using Rapd Markers

A K Ghose
Biotech. Div., BSRI, Ishurdi, Pabna

M S Haque
Dept. of Biotech., BAU, Mymensingh

S N Begum
Biotech. Div., BINA, Mymensingh

R Barua
ARD, BRRI, Gazipur

M K Hasan
CSISA BD, CIMMYT- Bangladesh

M S I Sagar
Department of Biotech., BAU, Mymensingh

Abstract/ Executive Summary:

Random Amplified Polymorphic DNA (RAPD) technique was used to assess genetic diversity and relationship among ten lentil varieties, namely Binamasur-1, Binamasur-2, Binamasur-3, Binamasur-4, BARImasur-1, BARImasur-2, BARImasur-3, BARImasur- 4, BARImasur-5 and BARImasur-6. Genomic DNA was extracted from young leaves using mini CTAB method. Three individuals from each of the 10 varieties were analyzed using 3 random primers. Amplification with 3 (out of 10) random primers generated 20 RAPD markers of which 13 (65%) were considered as polymorphic. The highest proportion of polymorphic loci was 15% for Binamasur-2, Binamasur-3 and BARImasur-2. Nei’s gene diversity value (0.9878) was the highest in case of BARImasur-6 and BARImasur-4 varietal pair whereas BARImasur-6 Vs Binamasur-1 was monomorphic in nature. Results of this investigation indicated relatively high level of genetic variation in Binamasur-1 and BARImasur-4 and BARImasur-6 compared to other varieties. High level of varietal differentiation (Gst=0.8055) and low level of gene flow (Nm=0.0122) estimates across all loci reflected that the level of genetic variation existed among all the 10 lentil varieties. The similarity indices for individual cultivars (Si) were higher (average 95.20%) than inter-cultivar similarity (Sij) (average 83.74%). The inter-cultivar similarity (Sij) for BARImasur-4 Vs BARImasur-6 was higher (94.07) whereas Sij value was lowest for Binamasur-1Vs BARImasur-6 (63.07). The unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei's genetic distance, grouped the 10 cultivars into two main clusters. Binamasur-1 grouped into cluster I and the remaining 9 cultivars grouped into cluster II. The cultivar BARImasur- 4 was close to the cultivar BARImasur-6 with the lowest genetic distance (0.0122) and the highest genetic distance (0.4907) was found between Binamasur-1 and BARImasur-6. The RAPD markers were found to be useful tool in molecular characterization of lentil varieties which could be utilized by the breeders for the improvement of lentil cultivars.

Keywords: Lentil, Genetic Diversity, RAPD Marker

Location: Biotech. Div., BSRI, Ishurdi, Pabna

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Lentil

Objectives:

To achieve the objectives like, characterization of lentil germplasms at molecular level through RAPD markers; determining the variation between the individuals among different varieties and discriminate the different varieties based on different levels of nuclear variation.

Methodology:

Study materials and sample collection - Ten varieties of lentil were used in the study. Out of the 10 varieties, 4 were BINA varieties (Binamasur-l, Binamasur-2, Binamasur-3, Binamasur-4) and 6 were BARI varieties (BARImasur-1, BARImasur-2, BARImasur-3, BARImasur-4, BARImasur-5 and BARImasur-6). Young, vigorously growing fresh leaf samples were collected from 28 day old seedlings to extract genomic DNA. Initially healthy portion of the youngest leaves of the seedlings were cut apart with sterilized scissors and washed in distilled water and ethanol and dried on fresh tissue paper to remove spore of microorganisms and any other source of foreign DNA. The collected leaf samples were then put into polythene bags and kept on ice in an ice box. After that the polythene bags were wrapped by aluminium foil and stored at -800C freezer. Genomic DNA isolation The mini CTAB method was used to collect total DNA. The leaf samples were cut into 2-3 cm pieces and the sample was grounded. 670 µl extraction buffer and 50 µl 20% SDS were added. Vortexed for 20 second and incubated for 10 minutes at 65^oC in the hot water bath. 100 µl 5M NaCl was added and inverted gently to suspend the samples evenly then added 100 µl CTAB and mixed well. Again vortexed for 20 second and incubated for 10 minutes at 65oC in the hot water bath. 900 µl chloroform (chloroform: isoamylalcohol = 24:1, v/v) was added and mixed well. The samples were spinned down at 12000 rpm for 15 minutes. Then transferred the supernatant into a new eppendorf tube and 600 µl ice-cold isopropanol was added to the supernatant and shakened. Spinned down at 12000 rpm for 15 minutes by centrifuge. Supernatant was discarded and washed the pellet with 200 µl 70% ethanol. Then spinned down at 12000 rpm for 5 minutes, removed the ethanol and then the pellets were allowed for air-drying for1 hour. The pellet was then suspended in 30 µl 1X TE buffer. Finally, the DNA samples were stored at - 20°C.

Source of Information: Eco-friendly Agril. J. 7(09): 85-92, 2014 (September); ISSN 1999-7957

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present work was the preliminary study to assess genetic variation of lentil varieties and it had some limitations in terms of limited number of individuals and varieties as well as number of primers used. The results indicated that Binamasur-1 and BARImasur-4 are maintaining higher genetic variation than other varieties. The present study might be used as a guideline for further for sustaining the genetic qualities of lentil in Bangladesh.

Knowledge Management: Journal