RESEARCH ARTICLE
Bhartiya Krishi Anusandhan Patrika, Volume 38 Issue 4: 397-402 (December 2023)
Biochemical Analysis and DNA Barcoding of Millet Echinochloa
frumentacea
A.R. Panigrahy1, P.M. More1, S. Prashant1, S.S. Nair1, K.S. Chitnis1
10.18805/BKAP677
ABSTRACT
Background: Millets are small grains that are rich in nutrients. In recent times, millet-based foods have been increasingly
recommended for a healthy diet. Many millets are not annotated or DNA barcoded yet.
Methods: In this study, comparative biochemical analyses especially that of starch and total protein of Echinochloa frumentacea,
called as Indian barnyard white millet (Varai), from geographically different locations like Tamil Nadu and Maharashtra have been
done. Their DNA barcoding has also been done to identify them on the basis of molecular data.
Result: It was observed that starch granules were more abundant in Tamil Nadu variety as compared to Maharashtra variety. Blue
value, indicative of amylose: amylopectin ratio was found to be low in Varai, indicating that Varai has low starch digestibility and its
starch releases glucose slowly, thus making it a low glycaemic index food. Protein content was higher in Tamil Nadu variety, but
overall Varai had a lower protein content as compared to other millets. Ribulose-1,5-bisphosphate carboxylase/oxygenase large
subunit (rbcL) gene from plastid was isolated, amplified by PCR, sequenced and the sequence was submitted to GenBank, NCBI. The
gene was identified to be that of Echinochloa frumentacea and was given the accession numbers by GenBank as OR027010 (Varai,
Maharashtra) and OR027011 (Varai, Tamil Nadu). This study indicated a distinct biochemical difference related to the geographical
location of millets. This study helped barcoding of Echinochloa frumentacea Indian varieties using rbcL gene. This will further help in
studies of phylogeny and evolution and also that of the relatedness of Echinochloa sp within and as compared to other millets.
Key words: Biochemical analysis, DNA barcoding, Echinochloa frumentacea, Millets, rbcL gene, Varai.
INTRODUCTION
Millets are small grains that have a rich nutrient profile.
Nowadays, millet-based foods have been increasingly
recommended for a healthy diet and to solve many health
issues (Anitha et al., 2022). Millet consumption could thus
help achieve SDG 3 (Sustainable Development Goal 3Good health and well-being). Modern food systems may
not offer essential nutrients. It is suggested that change in
the food habits, including the promotion of local foods like
millets, is essential (Pradhan et al., 2021). Echinochloa
frumentacea, commonly called as Varai, or Indian barnyard
millet, belongs to the family Poaceae and subfamily
Panicoideae. It includes 250 annual and perennial species, of
which E. frumentacea (Indian barnyard millet) and E. esculenta
(Japanese barnyard millet) are the most important and
widely cultivated (Farooq and Siddique, 2023). Varai is
grown on fertile, free-draining, sandy loam soils. It is mainly
grown in Kharif season, grown best in tropical conditions.
Panozzo et al. (2021) have DNA barcoded 2 species of
Echinochloa namely E. crus-galli and two in E. oryzicola
but not frumentacaea. Hoste et al. (2022) have given a key
for correct identification of Echinochloa species including
frumentacea and have emphasized the need of efforts in
morphology-based taxonomy, genomics and phylogenetics
to overcome the confusion among the Echinochloa sp which
are major weeds in rice and maize fields. Ceaser and
Maharajan (2022) have stated that genome sequences of
many millets have not been annotated, which may hamper
millet research, which is very important for food security and
Volume 38 Issue 4 (December 2023)
1
Department of Life Science, Ramnarain Ruia Autonomous College,
University of Mumbai, Mumbai-400 019, Maharashtra, India.
Corresponding Author: K.S. Chitnis, Department of Life Science,
Ramnarain Ruia Autonomous College, University of Mumbai,
Mumbai-400 019, Maharashtra, India.
Email: kanchanchitnis@ruiacollege.edu
How to cite this article: Panigrahy, A.R., More, P.M., Prashant,
S., Nair, S.S. and Chitnis, K.S. (2023). Biochemical Analysis and
DNA Barcoding of Millet Echinochloa frumentacea. Bhartiya Krishi
Anusandhan Patrika. 38(4): 397-402. doi: 10.18805/BKAP677.
Submitted: 06-09-2023
Accepted: 07-12-2023
Online: 22-12-2023
attaining UN SDGs. Gao et al. (2022) have done a comparative
analysis of whole chloroplast genomes of Echinochloa sp.
Omonhinmin and Onuselogu (2022), in their article have
emphasized the importance of Ribulose-1,5-bisphosphate
carboxylase/oxygenase large subunit rbcL gene as global
molecular data repositor, which can help in finding
relatedness of plants and study evolutionary relationships.
The aim of this study was to select and collect millets
from various regions of India and perform biochemical
analyses on them, including microscopic analysis of starch
granules in millets, extraction and estimation of total proteins
and estimation of the amylose: amylopectin ratio via Blue
Value determination. The other aim of this study was to
sequence plastid rbcLgene and to barcode Echinochloa
frumentacea Indian varieties from Maharashtra and Tamil Nadu.
397
Biochemical Analysis and DNA Barcoding of Millet Echinochloa frumentacea
MATERIALS AND METHODS
DNA barcoding using rbCLa gene
This project was conducted during the period from June
2022 to Augsut 2023, at the Department of Life Science,
Ramnarain Ruia Autonomos College, Mumbai. The
genomic studies were conducted in collaboration with
GeneOmbio Technologies Pvt Ltd, Pune.
Genomic DNA isolation
Selection and collection of millets from different regions
in India
Plant rbcL region gene was amplified using standard PCR
reaction. The primer pair rbcL-F and rbcL-R (Table 1) was
used in PCR reaction with an annealing temperature of
57 C. After amplification, products were purified by using
exosap kit (Invitrogen) and were directly sequenced using
an ABI PRISM BigDye Terminator V3.1 kit (Applied
Biosystems, USA). The sequences were analyzed using
Sequencing Analysis 5.2 software. BLAST analysis was
performed at BlastN site at NCBI server (http://
www.ncbi.nlm.nih.gov/BLAST). DNA sequencing was
performed using one of the PCR primers. The PCR reaction
was performed in Applied Biosystems 2720 thermal cycler.
Thermal cycling program for PCR used was; initial
denaturation at 95C for 5:00 min, denaturation at 94C for
0:30 sec, annealing at 57C for 0:30 sec, extension at 72C
for 0:30 sec, followed by final extension at 72C for 10 min.
This was repeated for 35 cycles and final hold at 4C until use.
Following PCR mix was prepared for all DNA samples
along with a negative PCR control and a positive control
(Certified Reference Material). Final volume of each reaction
was 25.0 μl. The reaction mix was prepared for all samples
and added into 200 μl PCR tubes. Genomic DNA was
added later to each tube. PCR reaction mixture used was
Genomic DNA 5 μl, 10X PCR buffer 2.5 μl, 50 mM MgCl2
0.75 μl, 0.5 mM dNTP Mix 0.5 μl, 10 pmole primer solution
1.00 μl, DMSO 1.25 μl, Taq DNA polymerase (5.0 units/ μl)
0.2 μl and Nuclease free water 13.8 μl.
Agarose gel (2%w/v) spiked with nuclear stain dye
Labsafe (1:65000 diluted) was prepared Agarose (LE,
Analytical Grade, Promega Corp., Madison, WI 53711 USA)
in 0.5 TBE buffer. 5.0 ml of PCR product was mixed with
1 ml of 6 Gel tracking dye. 5 ml of gScale 100 bp+3k
DNA Ladder (ExcelBand, SMOBIO) was loaded in one
lane for confirmation of size of the amplicon using
reference ladder. The DNA molecules were resolved at
5V/cm until the tracking dye was 2/3 distance away from
the lane within the gel. Bands were detected under a UV
Trans illuminator. Gel images were recorded using BIORAD GelDoc-XR gel documentation system. The PCR
product of size 599 bp was expected to be generated
through this reaction.
Millet Echinochloa frumentacea (Varai) was obtained from
local markets in Mumbai (Maharashtra) and Chennai (Tamil
Nadu). Varai is generally grown on fertile, free-draining,
sandy loam soils. It is mainly grown in Kharif season. It is
a short duration crop, grown best in tropical conditions.
Biochemical analysis
Microscopic examination of millet starch granules
The samples were soaked overnight in water and then
sectioned, stained by dilute iodine and observed under the
microscope (45) for analysis of starch granule shape
and abundance.
Extraction and estimation of total proteins
The millets were powdered. 1 g of each sample was
weighed, 4 ml of hexane was added and the mixture was
kept for 4 hours. This was the defatting step. Then they
were centrifuged at 4000 rpm for 40 min, the supernatant
was removed and in the pellet, 8 ml distilled water was
added and kept for 4 hours. Again, the samples were
centrifuged and the supernatants were estimated for their
protein content by Lowry‘s method (Plummer, 2013).
Estimation of the amylose: Amylopectin ratio by
determination of the Blue Value (Nwokocha, 2014)
Millets were soaked overnight and then ground in mortar
and pestle using distilled water, later filtered through
muslin cloth. After the starch settled, the filtrate was
decanted and then the starch was dried. 0.1 g dry starch
sample was weighed in a tube, 1 ml ethanol (95%) was
added followed by 9 ml of 1 M NaOH solution and heated in
a boiling water bath for 40 min to solubilize the starch. The
starch solution was cooled and transferred into a 100 ml
standard volumetric flask and the volume was made up to
the 100 ml mark with distilled water. 2.5 ml of starch solution
was taken into a 50 ml standard flask; 0.5 ml of 1 M acetic
acid was added, followed by 1 ml of stock iodine (0.2 g I2
and 2.0 g KI per 100 ml) and the solution was made up to
the 50 ml mark with distilled water. The color was allowed to
develop for 20 minutes and then the absorbance reading was
measured at 620 nm using a UV/visible spectrophotometer.
In the reference cell, an iodine solution of the same
concentration as above but without starch was used. The
blue value was calculated according to the method of Gilbert
and Spragg (1964) using the formula:
Blue value =
398
Absorbance at 620 nm 4
Concentration (mg/dl)
DNA was isolated using Macherey Nagel Nucleospin kit,
as per manufacturer’s instructions.
PCR of rbcL gene
Purification of PCR products
To remove unused dNTPs and primers from the reaction
mixture, 10 ml PCR product was used for ExoSAP purification.
ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo
Fisher) was used for enzymatic cleanup of amplified PCR
product. Excess primers and nucleotides were hydrolysed
in a single step. Purified PCR samples were further used
for DNA sequencing.
Bhartiya Krishi Anusandhan Patrika
Biochemical Analysis and DNA Barcoding of Millet Echinochloa frumentacea
DNA sequencing
ExoSAP purified PCR products (50 ng) were used for DNA
sequencing. ABI BigDye ® Terminator v3.1 Cycle
Sequencing reaction kit (Applied Biosystems, USA), was used.
Sequencing reaction composition for 10 μl sequencing used
was; PCR product DNA (3.00 μl), Sequencing buffer (1.90 μl),
RR-100 (Ready Reaction Mix) (0.25 μl), 10pmole sequencing
primer (1.00 μl) and Nuclease free water (3.85 μl).
Sequencing reaction was run in 2720 Thermal Cycler
(Thermo Fisher) in standard sequencing program: 25
cycles of (96C for 10 sec, 50C for 10 sec, 60C for 4 min),
then ramp to 4C.
Cycle sequencing PCR products were then purified by
EDTA-Ethanol precipitation protocol. The cleaned-up
sequencing products were dried at 37 C for 30 minutes
and then dissolved in HiDIFormamide solution (10 μL).
The reaction tubes were then subjected to denaturation
at 95C for 3 min and snap chilling at 4C. These products
were loaded on Applied Biosystems DNA sequencing
machine for capillary electrophoresis. Machine: 3130 Genetic
analyzer Automated DNA sequencing machine Softwares
used: Sequencing Analysis 5.1; ChromasPro v3.1.
DNA sequence analysis
DNA sequences were generated in FASTA format in
sequencing machine and further analyzed by Sequencing
Analysis 5.1 software. Using forward and reverse strand
sequences a contig of trimmed sequence was generated.
For each sample one FASTA sequence was thus generated
and further analyzed. BLAST analysis-Sequencing similarity
of the samples sequence with Genbank Database
sequences was analysed by nucleotide BLAST. Clustal WClustal W alignment was used for comparing different
sequences and finding out similarity between them. MEGA
6-software was used for construction of phylogenetic tree
for the sequences by including the nearest matching
reference sequences from NCBI Genbank nucleotide
sequence database (Saitou, 1987; Kumar, 2018).
low Blue Values (Table 3), which indicated presence of
higher amounts of amylopectin in its starch, less
digestibility of starch, a slower glucose release and thus a
low glycaemic index. Regional variation in Blue Values is
observed. Villas et al. (2019), have concluded in their study
that high amylose content makes digestion easier while
high amylopectin interferes in the digestion, thus molecular
structure has a strong influence on starch digestibility.
Agarose gel electrophoresis of plant genomic DNA
Genomic DNA was extracted and detected by Agarose Gel
Electrophoresis (Fig 3).
Polymerase Chain reaction
Samples showed PCR amplicon of desired size of 599
bp on agarose gel (Fig 4).
DNA sequencing
Source: Chloroplast Echinochloa frumentacea.
Organism: Echinochloa frumentacea.
Eukaryota; Viridiplantae; Streptophyta; Embryophyta;
Tracheophyta; Spermatophyta; Magnoliopsida; Liliopsida;
Poales; Poaceae; PACMAD clade; Panicoideae;
Panicodae; Paniceae; Boivinellinae. Echinochloa.
Varai Maharashtra
Varai Tamil Nadu
Fig 1: Comparative microscopic analysis of starch granules of
Varai from Maharashtra and Tamil Nadu (45).
RESULTS AND DISCUSSION
Biochemical analysis
Microscopic analysis of starch granules
There was a distinct geographical difference in the shape,
size and abundance of starch granules in the samples
collected from Maharashtra and Tamil Nadu (Fig 1).
Protein estimation
Total protein was calculated using the equation from the
standard protein graph (Fig 2). Varai from Maharashtra
showed lesser total protein content as compared to that
from Tamil Nadu (Table 2). Overall Varai has a lower protein
content as compared to other millets (Gopalan, 2011).
Estimation of amylose: amylopectin ratio by determination
of Blue Value
Blue Value indicates the amylose to amylopectin ratio, which
in turn indicates starch digestibility. Varai illustrated very
Volume 38 Issue 4 (December 2023)
Fig 2: Std protein graph by Lowry‘s method.
Table 1: Forward and Reverse primers for rbcL PCR.
Amplicon
Name
Sequence 5-3
Bases
rbcL-F
ATGATAACTCGACGGATCGC
20 bases
599 bp
rbcL-R
CTTGGATGTGGTAGCCGTTT
20 bases
599 bp
size
399
Biochemical Analysis and DNA Barcoding of Millet Echinochloa frumentacea
Table 2: Protein content of Varai (mg/ml per gram of millet).
Protein
Distilled
OD at
Calculated protein content based
sample (ml)
water (ml)
670 nm
on std equation y= 4.7511
Varai T
0.1
0.9
0.23
0.048
0.48
Varai M
0.1
0.9
0.07
0.015
0.15
Sample
10 mg/ml
Origin
Table 3: Blue values of Varai.
Absorbance at
Calculated Blue Value (Abs 4)
620 nm
/100=Abs 0.04 mg/dl
Varai M
0.07
0.0028
Varai T
0.09
0.0036
Sample
Dilution factor
Fig 3: Agarose gel electrophoresis of genomic DNA performed
on 1% (w/v) gel.
1 actaaagcaa gtgttggatt taaagctggt gttaaggatt ataaattgac ttactacact
61 ccggagtacg aaaccaagga tactgatatc ttggcagcat tccgagtaac
tcctcagccc
121 ggggttccgc ctgaagaagc aggggctgca gtagctgcgg aatcttctac
tggtacatgg
181 acaactgttt ggactgatgg acttaccagt cttgatcgtt acaaaggacg
atgctatcac
241 atcgagcccg ttcctgggga ggcagatcaa tatatctgtt atgtagctta
tccattagac
301 ctatttgaag agggttctgt tactaacatg tttacttcca ttgtgggtaa
cgtatttggt
361 ttcaaagccc tacgcgctct acgtttggag gatctacgaa ttcccattgc
ttatgcaaaa
421 actttccaag gtccgcctca cggtatccaa gttgaaaggg ataagttgaa
caagtatggt
481 cgtcctttat tgggatgtac tattaaacca aaattgggat tatccgcaaa
aaattacggt
541 agagcgtgtt atgagtgtct acg
/translation= TKASVGF KAGVKDYKLTYYTPEY
ETKDTDILAAFRVTPQ PGVPPEEAGAAVA AESSTGT WT
TVWTDGLTSLDRY KGRCYHIEP VPGEADQYI CY VAY PL
DLFEEGSVTNMFTSIVGNVFGFKALRALRLEDLRIPIAYAK
TFQGPPHG IQVER DKL NKYGRPLLGCTI KPKLGLSAK
NYGRACYECLR
>13722 Varai T (Assembled Contig). Sequence was
submitted to GenBank. Accession number given by
GenBank OR027011.
Base count 148 a 111 c 130 g 159 t
Origin
Lane1: 100-1000+3k DNA marker; Lane 2: NTC (Negative
test control). Lanes 3-6: 13721-13724 sample PCR Product.
Lane 7: CRM (Certified Reference Material) or Standard
positive control for plant.
Fig 4: PCR Image: 2% (W/V) Agarose gel electrophoresis:
Following sequences were generated for the sample
>13721 (rbcL) Varai M (Assembled Contig). Sequence was
submitted to GenBank. Accession number given by
GenBank OR027010;
Base count- 154 a 113 c 133 g 163 t
400
1 ggatttaaag ctggtgttaa ggattataaa ttgacttact acactccgga
gtacgaaacc
61 aaggatactg atatcttggc agcattccga gtaactcctc agcccggggt
tccgcctgaa
121 gaagcagggg ctgcagtagc tgcggaatct tctactggta catggacaac
tgtttggact
181 gatggactta ccagtcttga tcgttacaaa ggacgatgct atcacatcga
gcccgttcct
241 ggggaggcag atcaatatat ctgttatgta gcttatccat tagacctatt
tgaagagggt
301 tctgttacta acatgtttac ttccattgtg ggtaacgtat ttggtttcaa
agccctacgc
361 gctctacgtt tggaggatct acgaattccc attgcttatg caaaaacttt
ccaaggtccg
421 cctcacggta tccaagttga aagggataag ttgaacaagt atggtcgtcc
tttattggga
481 tgtactatta aaccaaaatt gggattatcc gcaaaaaatt acggtagagc
gtgttatgag
541 tgtctacg
Bhartiya Krishi Anusandhan Patrika
Biochemical Analysis and DNA Barcoding of Millet Echinochloa frumentacea
Fig 5: Relatedness of Varai with other millets.
Fig 6: Phylogenetic tree of E. frumentacea (OR027010 and OR027011), E. colona (NC_ 032383) and E. ugandensis (NC_036127).
translation=GFKAGVKDYKLTYYTPEYETKDTDILAAF
RVTPQPGVPPEEAGAAVAAESSTGTWTTVWTDGLTSLDRY
KGRCY HIEPVPG EADQYICY VAYPLDLFE EG SVTNMF
TSIVGNVFGFKALRALRLEDLRIPIAYAKTFQGPPHGIQVERDKLN
KYGRPLLGCTIKPKLGLSAKNYGRACYECLR
In this study, the obtained sequences of Varai varieties
were compared with sequences of other millets from NCBI
database and phylogenetic tree was obtained (Fig 5). It
illustrated that all millets have evolved together, whereas
Amaranthus (Rajgira) has diverged from the millets,
Amaranthus being correctly called as pseudo millet. Both
the varieties of Varai were 100% identical when compared
with each other. Varai showed distinct evolutionary deviation
from Eleusine coracana (Ragi) (Fig 5).
Gao et al. (2022), based on the results of genetic
relationships, divided 10 species of barnyard grass into four
groups. The first group comprised E. oryzicola, E. crus-galli
var. zelayensis, E. glabrescens and E. stagnina; the second
group included E. crus-galli var. crus-galli and E. esculenta;
the third group contained E. haploclada alone and the fourth
Volume 38 Issue 4 (December 2023)
group consisted of E. ugandensis, E. colona and E. frumentacea.
Fig 6 confirms the relatedness of these varieties.
DNA sequencing can help in improving agronomic
traits, value addition in food, feed and nutritional security
through recombinant technology. It can help in gene
manipulation to create drought resistant crops. Thus it can
lead the way to sustainable agriculture pertinent to the
United Nations Accord of Sustainable Development Goals.
CONCLUSION
This study indicated a distinct biochemical difference related
to the geographical location of millets. It has contributed to
DNA barcode Echinochloa frumentacea Indian varieties
using rbcL gene. The findings in this study will further help
in studies of phylogeny and evolution and also that of the
relatedness of Echinochloa sp within and as compared to
other millets. It can be helpful in generating biotechnological
interventions in food security and generating drought resistant
crops. Besides, this study emphasized the importance of
millets, 2023, being declared as International Year of Millets.
401
Biochemical Analysis and DNA Barcoding of Millet Echinochloa frumentacea
ACKNOWLEDGEMENT
DNA barcoding was done in GeneOmbio Technologies Pvt
Ltd, Pune, their help is highly acknowledged.
Financial support
This project was completed using the funds obtained under
Seed Grant Funding (2022-2023) from Ramnarain Ruia
Autonomous College and Ruia College Alumni Association.
Ethics statement
NA.
Conflict of interest
The authors declare that they do not have any conflicts
of interest.
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