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Echinochloa Spp. Complex) Using Morphological and Molecular Markers

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Echinochloa Spp. Complex) Using Morphological and Molecular Markers AL SC R IEN TU C A E N F D O N U A N D D A E I T Journal of Applied and Natural Science 8 (3): 1643 - 1648 (2016) L I O P JANS N P A ANSF 2008 Assessment of genetic diversity in Indian Barnyard millet ( Echinochloa spp. complex) using morphological and molecular markers M. P.Moharil 1, Dipti Gawai 1, N. Dikshit 2* , M.S. Dudhare 1 and P. V. Jadhav 1 1Biotechnology Centre, Dr. Panjabrao Deshmukh Agricultural University, Akola-444104 (Maharashtra), INDIA 2 ICAR-National Bureau of Plant Genetic Resources, Dr.PDKV campus, Akola-444104 (Maharashtra), INDIA *Corresponding author. E-mail: [email protected] Received: December 27, 2015; Revised received: June 14, 2016; Accepted: August 24, 2016 Abstract: In the present study, morphological and molecular markers (RAPD primers) were used to analyze the genetic diversity and genetic relationships among 21 accessions of Echinochloa spp. complex comprising the wild and cultivated species collected from Melghat and adjoining regions of Vidarbha, Maharashtra. The availability of diverse genetic resources is a prerequisite for genetic improvement of any crop including barnyard millet. A high degree of molecular diversity among the landraces was detected. Among the 21 genotypes, two major groups (A and B) were formed, at 67.28 % similarity, which clearly encompasses 15 accessions of E. frumentacea and 6 ac- cessions of E. colona . Higher similarity was observed in accessions of E. frumentacea. The accessions IC 597322 and IC 597323 also IC 597302 and IC 597304 showed more than 94% similarity among themselves. The classifica- tion of genetic diversity has enabled clear-cut grouping of barnyard millet accessions into two morphological races (E. frumentacea and E. colona ). Keywords: Barnyard millet, Genetic diversity, Genetic relationship, RAPD marker INTRODUCTION yield potential without compromising on the nutri- tional front. Measurement of genetic diversity also Barnyard millet, Echinochloa sp. (2n= 36, 54) is a self allows the estimation of important population parame- pollinating crop which belongs to family Poaceae and ters e.g., characterization of the geographic structure or is grown as cereal or fodder crop. It is one of the oldest connectivity of populations (Gupta et al ., 2009). Mo- domesticated millets in the semi-arid tropics of Asia lecular traits have virtually no environmental compo- and Africa (Holm et al. 1977). The genus comprises nent and large numbers of variables are produced. As a approximately 50–60 species (Michael 1994) and in- result, qualitative and quantitative traits can be more cludes important native species (Watson and Dallwitz efficiently introduced into plant breeding programmes. 1992), species cultivated as fodder and grain crops, One technique which can provide useful data for the and significant weed species such as E. crus-galli comparison of plant types is random amplified poly- ( barnyard grass) and E. colona ( awnless barnyard morphic DNA (RAPD) analysis. This is a modified grass) (Holm et al . 1977). Echinochloa utilis is a tem- PCR technique involving the amplification of whole- perate annual hexaploid (2n=6x=54) and is cultivated plant DNA extracted from leaves or other plant organs. in Japan, Korea etc. E. frumentacea (2n=36, 54) is In the RAPD technique, multiple 10 base pair (bp) cultivated in India, Pakistan and Nepal (Yabuno, oligonucleotide primers are added each to an individ- 1962). The wild ancestor of Echinochloa frumentacea ual sample of DNA which is then subjected to PCR. is the tropical grass Echinochloa colona (L.) Link. This technique is sensitive, fast, requires the use of no Barnyard millet is the fastest growing crop among all radioactive probes, and is easily performed. In the pre- millets and can be harvested in a short period of nine sent paper, an attempt has been made to assess the ge- weeks. Its grains contain 11.2(g) protein, 10.1(g) crude netic diversity in Indian Barnyard millet ( Echinochloa fiber, 4.4(g) minerals, 15.2(mg) Iron, 11(mg) calcium frumentacea (Roxb.) Link) and its progenitor wild per 100gm grain (MINI, India). Also it is a nutritive species ( E. colona (L.) Link) from Melghat Biosphere fodder for animals. Studies on nutrient composition, Reserve using morphological and molecular markers. glycaemic index and health benefits of barnyard mil- lets indicated that it is beneficial for type-II diabetes MATERIALS AND METHODS (Ugare et al., 2011). The crop is valued for its drought Plant material: During a survey and exploration pro- tolerance, good yield and superior nutritional value. gramme during 29 th October - 5th November 2012, a Study of biodiversity and available natural resources total of 21 accessions of barnyard millet and their wild are essential for identifying genotypes having higher relatives were collected (Table 1). The passport data ISSN : 0974-9411 (Print), 2231-5209 (Online) All Rights Reserved © Applied and Natural Science Foundation www.jans.ansfoundation.org M.P.Moharil et al. / J. Appl. & Nat. Sci. 8 (3): 1643 - 1648 (2016) (place of collection i.e. village , block, latitude, longi- by 40 cycles of 92 oC for 1 min., 26.8 to 42.4 oC de- tude, altitude) and vernacular name of the species col- pending on primer for 1 min., 72 oC for 2 min. and syn- lected are mentioned (Table 2).Two sets of different ac- thesis 72 oC for 7 min. cessions of both the species ( E. frumentacea and E. co- PCR amplification products were analyzed by electro- lona ) were examined. 15 accessions of E. frumentacea phoresis on 1. 5% agarose gel stained with ethidium and 6 accessions of E. colona were used for the study. bromide solution (0.5 µg/ml) and run in TBE buffer at Morphological characters: All the 21 genotypes were 70-80 Volts. A 1kb ladder plus maker (Fermentas) was sown in the fields in a Randomized Block Design used as a molecular weight standard. The documenta- (RBD) at ICAR-National Bureau of Plant Genetic Re- tion was done using Gel Doc system (Alpha innotech). sources Regional Station, Dr.PDKV campus, Akola Data analysis : Field data for morphological charac- (Maharashtra) for two consecutive years 2012 and ters was evaluated and represented graphically by plot- 2013. Crop management was done according to the ting a graph of the various characters used for the recommended agronomic practices (N:P:K 40:20:0). study. Sowing was done in the plots (3m × 1m), plant to plant Molecular data was analyzed from the banding pattern distance was maintained at 10 cm, while row to row of RAPD markers. The presence of band was scored distance was kept at 30.0 cm. Various morphological from the photograph. Only clear and reproducible characters including, plant height, leaf length, leaf bands were given consideration. These bands were width, flag leaf length, flag leaf width, inflorescence considered as polymorphic, when they were absent in length, inflorescence width, number of racemes / some sample in frequency greater than 1% and change inflorescence, raceme length, color of inflorescence, in band intensity were not considered as polymor- days to maturity, inflorescence shape, inflorescence phism. Clear bands were scored as present (1) or ab- compactness, shape of lower raceme, length of pedun- sent (0) at particular position or distance migrated on cle, arrangement of spikelet, degree of branching, de- the gel. The data matrix of 1’s and 0’s been prepared gree of lodging and 100 seed weight were taken to from the scorable bands and was entered into the data assess genetic variability in accessions. analysis package. DNA isolation and PCR amplification: Seeds were The similarity index was used to calculate the genetic grown in the green house and leaf material was har- distance values and to construct the dendrogram. The vested from 3-4 week old seedlings. Total cellular dendrogram provides a visual representation of the DNA was isolated from leaf material of individual differences in the population of various accessions. plants following the procedure of Doyle and Doyle The NTSYS-pc software ver. 2.02 was used to esti- (1987, 1990) using CTAB with few modifications. mate genetic similarities with the Jaccard’s coefficient. Measurement of DNA purity and quality: The quan- The matrix of generated similarities was analyzed by tity of DNA was measured using a UV Spectropho- the unweighted pair group method with arithmetic av- tometer at 260 nm. The purity of DNA was determined erage (UPGMA), employing Sequential, Agglomera- by calculating the ratio of absorbance at 260 nm to that tive, Hierarchical and Nested Clustering (SAHN) clus- of 280 nm (Sambrook et al.,1989).The DNA concen- tering module. tration was determined by the formula: DNA concen- Size of fragments in base pairs was detected with the tration = OD260 × 50 µg/ml × dilution factor help of 1 Kb plus ladder (Table 5). Percent polymor- (Linacero et al.,1998). phic loci (a locus is defined polymorphic when the Screening of RAPD primers: Decamer oligonucleotides frequency of the marker (allele) is <1.) was calculated were used in this study. 20 RAPD primers (Table 3) were using the formula: screened for polymorphism. Primers that gave clear and polymorphic profiles were chosen for further study. DNA amplification of selected primers : DNA sam- ples for PCR amplification were diluted to 30 ng/ µl with T.E buffer. Twenty random decamer primers RESULTS AND DISCUSSION were used to study polymorphism. Out of these, 11 Estimation of genetic diversity based on morpho- primers gave polymorphic bands and were used for logical characters: During the present study, a wide further study. range of diversity was observed between E. frumentacea The PCR amplifications were carried out in a 25 µl and E.colona accessions. The data was analyzed by plot- reaction mixture containing 30 ng of genomic DNA, ting a column chart of the two species against different 0.5uM primer, 1X PCR reaction buffer morphological characters viz.
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