DNA BARCODING AND MOLECULAR IDENTIFICATION OF

Amaranthus species USING GENETIC MARKER ITS: FOUND IN IKOT

AKPADEN IN AKWA IBOM STATE

A RESEARCH PROJECT

BY

UDO, VERONICA MICHAEL

AK16/NAS/BIO/102

SUBMITTED TO
DEPARTMENT OF BOTANY
FACULTY OF BIOLOGICAL SCIENCES
AKWA IBOM STATE UNIVERSITY
IKOT AKPADEN, MKPAT ENIN L.G.A

APRIL, 2023
 DNA BARCODING AND MOLECULAR IDENTIFICATION OF
Amaranthus species USING GENETIC MARKER ITS: FOUND IN IKOT
AKPADEN IN AKWA IBOM STATE

A RESEARCH PROJECT

BY

UDO, VERONICA MICHAEL

AK16/NAS/BIO/102

SUBMITTED TO
DEPARTMENT OF BOTANY
FACULTY OF BIOLOGICAL SCIENCES
AKWA IBOM STATE UNIVERSITY
IKOT AKPADEN, MKPAT ENIN L.G.A

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

AWARD OF BACHELOR OF SCIENCE (B.SC) IN BOTANY

APRIL, 2023

i
 DEDICATION

This research work is dedicated with deep affection and gratitude to God

Almighty for this infinite love, grace and protection throughout the period of

this work, and also to my dearest parents Mr./Mrs. Michael, Michael Udo whom

through their advice, love, care and financial assistance has brought me where I

am today, I pray that the good Lord will continue to keep her in good health.

Amen.

ii
 ACKNOWLEDGEMENT

I seize this opportunity and appreciate my Head of Department, Dr. Joseph E.

Okon for his dynamic leadership and fatherly role towards the completion of my

studies, my able supervisor, Dr. Lovina I. Udoh for her effort, advice, and

support in ensuring proper conduct of this research work. I also appreciate the

candid effort of my lecturers in the Department of Botany and Faculty of

Biological Sciences in general.

My deepest appreciation goes to my humble, loving and supporting parents, Mr.

and Mrs. Michael Michael Udo, siblings, Utibe Michael Udo, Michael Michael

Udo and Anthony Michael Udo for their prayers, love, support and

encouragement towards achieving this academic pursuit and who have proven

to be worth more than a million gold.

I cannot fail to recognize the contribution of my colleagues and friends, Udo,

Otobong Godwin, Udoatai, Nkpouto Augustine, Akpainyang, Hephzibah Ekom

and Anan, Moses Etim for their support throughout my studentship and I pray

may the good Lord bless and reward you all in your endeavours.

iii
 TABLE OF CONTENTS

Title i

Dedication ii

Acknowledgement iii

Table of contents iv

List of tables vi

List of figures vii

Lists of plates viii

Nomenclature ix

Declaration x

Certification xi

Abstract xii

CHAPTER ONE

1.0 Introduction 1

1.1 Background of the study 1

1.2 DNA 2

1.3 DNA Barcoding 8

iv
1.4 Statement of the Problem 9

1.5 Aim and objectives 10

1.6 Research objectives 10

1.7 Statement of the problem 11

1.8 Significance of the study 12

CHAPTER TWO

2.0 Literature Review 13

2.1 Introduction 13

2.2 Ecology 14

2.2 Taxonomy 15

2.3 DNA Barcoding 17

2.4 Internal Transcribed Spacer (ITS) 20

2.5 Use of barcode genes for species identification 22

CHAPTER THREE

3.0 Materials and methodology 24

3.1 Study site 24

3.2 Materials 24

3.3 Sample collection 25

v
3.4 DNA Extraction 26

3.5 Qualitative Analysis - Agarose Gel Electrophoresis 27

3.6 DNA Quantification 27

3.7 Gene sequencing 27

3.8 Phylogenetic Relationship among the Genes under Study 29

CHAPTER FOUR

4.0 Results and Discussion 30

4.1 DNA Extraction and Quantification 30

4.2 PCR Analysis 31

4.3 Sequence Editing and BLASTn Analysis 32

4.4 Phylogenetic Analysis 35

CHAPTER FIVE

5.0 Conclusion and recommendations 37

5.1 Conclusion 37

5.2 Recommendation 38

REFERENCES 39

vi
 LIST OF TABLES

Table Description Page No.

4.1 ITS BLAST Hits with Highest Identities in the Gene Bank 47

vii
 LIST FIGURES
Figures Description Page No.

4.4 Distribution of the top 100 BLAST hits in the

genebank showed good quality alignments greater than
200 nucleotides. 46

4.6 Alignment for Amaranthus hybridus 47

4.7 Evolutionary Relationships of Taxa for ITS 49

viii
 LISTS OF PLATES
Plate Description Page No.

1.1 A. Blitum 4

1.2 A. deflexus 4

1.3 A. Hypochondriacus 4

1.4 A. Hybridus 4

1.5 A. Cruentus 4

1.6 A. Viridis 4

3.1 Sample of Amaranthus hybridus 38

4.1 Extracted DNA from Amaranthus hybridus 43

4.2 Agarose gel electrophoresis amplification of PCR product

and DNA of the Amaranthus hybridus. 44

4.3 Amplified PCR product of about 700bp for ITS gene

compared against a 100bp DNA ladder. 45

ix
 DECLARATION

I declare that this project on the DNA BARCODING AND MOLECULAR

IDENTIFICATION OF Amaranthus species USING GENETIC MARKER

ITS: IN IKOT AKPADEN IN AKWA IBOM STATE, is an original work

written by me under the supervision of Dr. Lovina I. Udoh, Department of

Botany, Faculty of Biological Sciences, Akwa Ibom State University, Ikot

Akpaden. The information derived from the literature has been duly sited in the

text and references are provided.

_____________________ __________________
Udo, Veronica Michael Signature/Date
(student)

x
 CERTIFICATION

This is to certify that this research project titled DNA BARCODING AND

MOLECULAR IDENTIFICATION OF Amaranthus species USING

GENETIC MARKER ITS: IN IKOT AKPADEN IN AKWA IBOM STATE,

is an authentic record of the worked carried out by UDO, VERONICA

MICHAEL, AK16/NAS/BIO/102 under the supervision and guidance of Dr.

Lovina I. Udo, in partial fulfilment of Bachelor of Science Degree in the

Department of Botany Akwa Ibom State University Nigeria.

_____________________ __________________
Dr. Joseph E. Okon Signature/Date
(HOD)

_____________________ __________________
Dr. Lovina I. Udoh Signature/Date
Supervisor

_____________________ __________________
External supervisor Signature/Date

xi
 NOMENCLATURE

DNA Deoxyribonucleic acid

ITS Internal transcribed space

matK Maturase K

rbcL Ribulose 1,5 - biphosphate carboxylase

trnL noncoding introns

CBOL Consortium for the Barcoding of Life

PCR Polymerase Chain Reaction

NCBI National Centre for Biotechnology Information

UV Ultra-Violet

TAE Tris-Base Acetate

PNA Peptide Nucleic Acid

LNA Locked Nucleic Acid

GNA Glycol Nucleic Acid

TNA Threose Nucleic Acid

xii
 ABSTRACT

The Amaranthus genus consists of as many as 70 species, many with similar

morphology. These Amaranthus species are an emerging and promising
nutritious traditional vegetable food source. The development and validation of
a DNA barcode specific to key Amaranths would aid in plant identification. The
use of molecular marker for plant identification has been considered to be a
most authentic method. This research study was focused on the use barcode
marker plant ITS2 for DNA Barcoding of the Amaranthus species. Total
genomic DNA was isolated from young leaves of the Amaranthus hybridus and
PCR approach was used to amplify ITS genes resulting in an amplicon size of
about 500bp. The amplicon was sequenced and authenticated using BLASTn
algorithm in NCBI, this retrieved similar gene regions of different species of
Amaranthus. According to the best match in BLAST, ITS identified the plant
under study as hybridus. The ITS2 had query coverage of 100% for the
Amaranthus hybridus under studied. Thus, ITS2 gene had the highest
amplification and sequencing success. Overall, the research objective is to
provide a molecular approach to identify and differentiate Amaranthus species
found in Ikot Akpaden in Akwa Ibom State. The study will contribute to the
understanding of the genetic diversity and conservation of Amaranthus species
in the region.

xiii
 CHAPTER ONE
INTRODUCTION

1.1 Background of the study

Amaranth is the name given to a group of approximately 70 species of annual or

short-lived perennial plants in the genus Amaranthus including several species

of aggressive edible weeds. Amaranths are branching broad-leaved plants with

egg-shaped or rhombic leaves which may be smooth or covered in tiny hairs.

The leaves have prominent veins, can be green or red in color and have long

petioles. The plants produce single flowers on terminal spikes which typically

red to purple in color. Amaranths can reach up to 2.5 m (6.6 ft) in height and are

usually grown as annuals, harvested after one growing season. Amaranth may

also be referred to as Chinese spinach and their origin is unclear due to their

worldwide distribution (Plant Village, 2019). The Amaranthus species belong to

the Caryophyllales order, Amaranthaceae family, Amaranthoideae subfamily

and Amaranthus genus. The Caryophyllales contains 33 families, 692 genera

and 11,155 species.

This diversity is greater than most highly cultivated crops despite the

underdeveloped and relatively lack of support (National Research Council

(NRC), 1989). Southern Asia (Indo-Burma region) and Central and South

America are considered the centre of origin for most of the varieties that have

been held under cultivation since time immemorial (De Candolle A, 1984;

1
Vavilov, 1927). Spread to other parts of the world likely began with colonisation

and exploration. Wild species still occur in parts of Asia, Africa and America.

Most Amaranths are classified either as a leafy or grain (pseudocereal)

Amaranth based on the part that is most often used, i.e., edible seeds or leaves

(Sauer, 1967). Examples of leafy and grain Amaranths are A. tricolor L. and A.

cruentus L. respectively. In addition, some Amaranth like A. tricolor is valued

as ornamental, while A. palmeri S. Wats. is considered a weed of economic

importance. Because of their huge genetic and morphological variability, they

are considered a taxonomically difficult group (Chidozie, 2020).

Amaranth leaves and stems are commonly eaten after cooking in a manner

similar to spinach. There are four main species which are cultivated as

vegetables; A. cruentus, A. blitum, A. dubius, and A. tricolor. Several species,

such as A. caudentis, A. cruentis and A. hypochondriacus are grown as a grain

crop in places such as Mexico, Nepal and India and are used to produce cereals

and snacks.

Amaranth grows best in a well-draining soil in full sunlight. Most species of the

plant will thrive in soils with a neutral pH, whereas some are adapted to grow in

acidic soil. The plants are drought and heat resistant but will not tolerate frost.

Propagation Amaranths are propagated from seed and can be started indoors for

transplanting or direct seeded. Transplants can be started approximately 6 to 8

weeks before the forecasted last frost but seeds should not be sown outdoors

2
until all danger of frost has passed. Seeds should be sown to a depth of 1–2 cm

(0.4–0.8 in) in rows spaced 50 cm (20 in) apart. The plants should be thinned to

a final within row spacing of 20 cm (8 in). General care and maintenance

Amaranth is easy to care for and requires little maintenance. While the seedlings

are young, it is important to remove any weeds from around the plants to

prevent competition. Applying a layer of mulch will help to prevent weeds and

conserve soil moisture.

The plants will benefit from supplemental irrigation during dry periods and the

addition of fertilizer once or twice throughout the growing season. Harvest

Grain Amaranth varieties are usually ready to harvest after about three months.

The flowers can simply be cut from the plant using a pair of scissors and set in a

warm, dry place to finish drying out. When the flowers are dry, seeds can be

removed by brushing or by beating the flowers in a bag. Passing the beaten

flowers through a fine screen mesh can help to remove the seeds from the chaff.

The species commonly seen Ikot Akpaden, Mkpat Enin Local Government

Area, Akwa Ibom State are; A. blitum, A. caudatus, A. cruentus, A. deflexus, A.

dubius, A. graecizans, A. hybridus, A. hypochondriacus, A. retroflexus, A.

spinosus, A. thunbergii, A. tricolor and A. viridis.

3
Plate 1.1: A. Blitum (Wikipedia, 2020) Plate 1.2: A. deflexus
(Wikipedia, 2022)

Plate 1.3: A. Hypochondriacus Plate 1.4: A. Hybridus (Vidal Force,

(Vidal Force, 2019) 2019)

Plate 1.5: A. Cruentus 4 Plate 1.6: A. Viridis (Gardenia, 2020)

(Scamperdale, 2022)
1.2 DNA

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic

instructions used in the development and functioning of all known living

organisms (with the exception of RNA viruses). The main role of DNA

molecules is the long-term storage of information. DNA is often compared to a

set of blueprints, like a recipe or a code, since it contains the instructions needed

to construct other components of cells, such as proteins and RNA molecules.

The DNA segments that carry this genetic information are called genes, but

other DNA sequences have structural purposes, or are involved in regulating the

use of this genetic information.

DNA consists of two long polymers of simple units called nucleotides, with

backbones made of sugars and phosphate groups joined by ester bonds. These

two strands run in opposite directions to each other and are therefore anti-

parallel. Attached to each sugar is one of four types of molecules called bases. It

is the sequence of these four bases along the backbone that encodes

information. This information is read using the genetic code, which specifies the

sequence of the amino acids within proteins. The code is read by copying

stretches of DNA into the related nucleic acid RNA, in a process called

transcription.

Within cells, DNA is organized into long structures called chromosomes. These

chromosomes are duplicated before cells divide in a process called DNA

5
replication. Eukaryotic organisms (animals, plants, fungi, and protists) store

most of their DNA inside the cell nucleus and some of their DNA in organelles,

such as mitochondria or chloroplasts. In contrast, prokaryotes (bacteria and

archaea) store their DNA only in the cytoplasm. Within the chromosomes,

chromatin proteins such as histones compact and organize DNA. These compact

structures guide the interactions between DNA and other proteins; helping

control which parts of the DNA gets transcribed.

In living organisms, DNA does not usually exist as a single molecule, but

instead as a pair of molecules that are held tightly together (Watson and Crick,

1953). These two long strands entwine like vines, in the shape of a double helix.

The nucleotide repeats contain both the segment of the backbone of the

molecule, which holds the chain together, and a base, which interacts with the

other DNA strand in the helix. A base linked to a sugar is called a nucleoside

and a base linked to a sugar and one or more phosphate groups is called a

nucleotide. If multiple nucleotides are linked together, as in DNA, this polymer

is called a polynucleotide. The backbone of the DNA strand is made from

alternating phosphate and sugar residues (Ghosh and Bansal, 2003). The sugar

in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are

joined together by phosphate groups that form phosphodiester bond between the

third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds

mean a strand of DNA has a direction. In a double helix the direction of the

6
nucleotides in one strand is opposite to their direction in the other strand: the

strands are anti-parallel. The asymmetric ends of DNA strands are called the 5'

(five prime) and 3' (three prime) ends, with the 5' end having a terminal

phosphate group and the 3' end a terminal hydroxyl group. One major difference

between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being

replaced by the alternative pentose sugar ribose in RNA. The DNA double helix

is stabilized primarily by two forces: hydrogen bonds between nucleotides and

base-stacking interactions among the aromatic bases (Yakovchuk et al.,2006).

The four bases found in DNA are adenine (abbreviated A), cytosine (C),

guanine (G) and thymine (T). These four bases are attached to the

sugar/phosphate to form the complete nucleotide, as shown for adenosine

monophosphate. These bases are classified into two types; adenine and guanine

are fused five- and six-membered heterocyclic compounds called purines, while

cytosine and thymine are six-membered rings called pyrimidines. A fifth

pyrimidine base, called uracil (U), usually takes the place of thymine in RNA

and differs from thymine by lacking a methyl group on its ring. Uracil is not

usually found in DNA, occurring only as a breakdown product of cytosine. In

addition to RNA and DNA, a large number of artificial nucleic acid analogues

such as peptide nucleic acid (PNA), Morpholino and locked nucleic acid

(LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA)

7
have also been created to study the proprieties of nucleic acids, or for use in

biotechnology (Verma and Eckstein, 1998).

1.3 DNA Barcoding

Generally, biodiversity is being threatened globally by climate change as well as

human activities and this has aroused concerns about the conservation status.

Although biodiversity is disappearing at a very fast rate, technological

development that can help reverse biodiversity loss is also on the increase but

limited by the fact that it is not easily accessible by countries rich in biodiversity

but constrained in resources. In order to resolve this problem, the use of DNA

barcoding technique is being employed. 'DNA barcoding' is a revolutionary

diagnostic technique in which short DNA sequence(s) can be used for species

identification. The DNA barcode sequence includes about 650 DNA “base-

pairs” (represented by the letters A, C, G and T) which is a tiny portion of the

billions of base pairs that make up the entire genome of many organisms. The

goal of barcoding is that anyone, anywhere, anytime be able to identify quickly

and accurately any species whatever its condition.

Barcoding is done with a well-known gene, not a newly discovered gene and it

is used for identifying specimens, not for biomedical purposes such as

developing pharmaceuticals (Stoeckle et al., 2005). This technology is being

promoted by the International Consortium for Barcoding of Life (CBOL),

8
which comprises major natural history museums, herbaria and other scientific

organizations, to enable the rapid and inexpensive identification of the estimated

10 million species on earth (Stoeckle et al., 2005). The stages involved in flora

barcoding are shown in plate 1.2. The scientific benefits of DNA barcoding are

enormous, including: enabling rapid species identification, including any life

stage or fragment, facilitating species discoveries based on cluster analyses of

gene sequences (e.g. cox1 in animals; rbcL and matK in plants), promoting the

development of handheld DNA sequencing technology that can be applied in

the field for biodiversity inventories and providing insight into the diversity of

life. The advantages that countries rich in biodiversity can gain are huge,

namely: the ability to identify specimens quickly and cheaply, better ability to

control the movement of species across national borders, opportunity for

training of students/researchers, involvement of local researchers in global

networks and biodiversity initiatives, and opportunity to improve the national

research infrastructure of specimen collections, molecular labs, and biodiversity

databases (Stoeckle et al., 2005). Due to the high rate of speciation and

hybridization of plant species leading to the formation of new form and change

in the genetic composition of plant species, it is important to reconsider their

classification and evolutionary relationships especially by the use of gene

sequences and biological molecules in the study of variation that is inherent in

the population.

9
1.4 Statement of the Problem

Determining the type of flower (i.e., staminate or carpellate) on a plant or

branch is very important and requires careful study because some species may

show very few staminate flowers among the carpellate ones (Gobotany, 2022).

Amaranthus shows a wide variety of morphological diversity among and even

within certain species. Although the family (Amaranthaceae) is distinctive, the

genus has few distinguishing characters among the 75 species present across six

continents. This complicates taxonomy and Amaranthus has generally been

considered among systematics as a "difficult" genus and hybridize often (Gbif,

2022).

Therefore, sources that were insufficiently investigated in the past are now

utilized in revising the taxonomy of the group so as to ease adequate

identification of the various taxa due to their intrinsic values. Moreover, the

family has problems of synonymy, taxa misidentification, doubtful specific

status and grouping dissimilar taxa in the same higher taxonomic rank.

1.5 Aim and objectives

The aim of this study is to use DNA barcoding and molecular techniques to

identify and differentiate Amaranthus species (local green leaf) found in Ikot

Akpaden in Akwa Ibom State using genetic markers.

1.6 Research objectives

10
The objectives of the study are to:

a) Collect plant samples of Amaranthus hybridus from Ikot Akpaden in Akwa

Ibom State.

b) Extract DNA from the collected plant samples using standard molecular

techniques.

c) Amplify and sequence the DNA regions of interest (e.g., ITS) using PCR

sequencing.

d) Generate a molecular database of Amaranthus species found in Ikot Akpaden

for future reference and conservation efforts.

1.7 Significance of the study

The aim of the study is to identify the Amaranthus species present in the study

area and to provide a molecular basis for their identification. The study is

significant because the accurate identification of plant species is essential for

the development of effective conservation and management strategies.

In addition, Amaranthus species are important sources of food and medicine in

many parts of the world. Accurate identification of these species is important

for their proper utilization and preservation. DNA barcoding is a powerful tool

for species identification, as it is based on the comparison of short DNA

sequences that are conserved within species but vary between species.

11
The study will contribute to the overall knowledge of the genetic diversity and

distribution of Amaranthus species in Nigeria. This information will be useful

for the development of sustainable agricultural practices, as well as for the

conservation of genetic resources. Furthermore, the results of the study can be

used for the development of DNA-based identification tools for Amaranthus

species, which can be used in the field to quickly and accurately identify

species.

CHAPTER TWO

LITERATURE REVIEW

2.1 Introduction

Amaranthus (pigweed) species are small-seeded, broadleaf weeds distributed

throughout the United States and Canada as well as other areas of the world

(Uva, et al., 1997). Often weedy, these plants are annuals and can be difficult to

manage in agronomic crops because of extended germination times, relatively

fast growth, high seed production, long seed viability, and difficulty in proper

identification of the individual species when herbicide applications are most

effective (Horak and Loughin, 2000). Palmer Amaranth and common

waterhemp are dioecious (separate male and female plants) species, whereas

redroot pigweed, smooth pigweed, tumble pigweed, and spiny pigweed are

monoecious (male and female flowers on the same plant) (Great Plains Flora

Association, 1986). All species grow at least 50 cm tall, with some species

12
capable of growing to nearly 3 m (Horak and Loughin 2000). Pigweed seeds

generally germinate early in the growing season, within 350 growing degree

days (base temperature of 10 C), but can emerge until fall.

Flowers vary interspecifically from the presence of 3 or 5 tepals and stamens,

whereas a 7-porate pollen grain structure remains consistent across the family.

Species across the genus contain concentric rings of vascular bundles, and fix

carbon efficiently with a C4 photosynthetic pathway. Leaves are approximately

6.5 - and of oval or elliptical shape that are either opposite or alternate across

species, although most leaves are whole and simple with entire margins.

Amaranth has a primary root with deeper spreading secondary fibrous root

structures. Inflorescences are in the form a large panicle that varies from

terminal to axial, color, and sex. The tassel of fluorescence is either erect or bent

and varies in width and length between species. Flowers are radially symmetric

and either bisexual or unisexual with very small, bristly perianth and pointy

bracts. Species in this genus are either monecious (e.g., A. hybridus,) or

dioecious (e.g., A. palmeri). Fruits are in the form of capsules referred to as a

unilocular pixdio that opens at maturity. The top (operculum) of the unilocular

pixdio releases the urn that contains the seed. Seeds are circular form from 1 to

1.5 millimeters in diameter and range in color with a shiny, smooth seed coat.

The panicle is harvested 200 days after cultivation with approximately 1,000 to

3,000 seeds harvested per gram (GBIF, 2020).

13
2.2 Ecology

Amaranth weed species have an extended period of germination, rapid growth,

and high rates of seed production, and have been causing problems for farmers

since the mid-1990s. This is partially due to the reduction in tillage, reduction in

herbicidal use and the evolution of herbicidal resistance in several species

where herbicides have been applied more often. (Wetzel, et al., 1999). A new

herbicide-resistant strain of A. palmeri has appeared; it is glyphosate-resistant

and so cannot be killed by herbicides using the chemical. Also, this plant can

survive in tough conditions. The species Amaranthus palmeri (Palmer

Amaranth) causes the greatest reduction in soybean yields and has the potential

to reduce yields by 17-68% in field experiments. Palmer Amaranth is among the

"top five most troublesome weeds" in the southeast of the United States and has

already evolved resistances to dinitroaniline herbicides and acetolactate

synthase inhibitors (Culpepper, 2006). This makes the proper identification of

Amaranthus species at the seedling stage essential for agriculturalists. Proper

weed control needs to be applied before the species successfully colonizes in the

crop field and causes significant yield reductions. An evolutionary lineage of

around 90 species within the genus has acquired the carbon fixation pathway,

which increases their photosynthetic efficiency.

14
2.2 Taxonomy

Amaranthus shows a wide variety of morphological diversity among and even

within certain species. Amaranthus is part of the Amaranthaceae that is part of

the larger grouping of the Carophyllales. Although the family (Amaranthaceae)

is distinctive, the genus has few distinguishing characters among the 75 species

present across six continents. This complicates taxonomy and Amaranthus has

generally been considered among systematists as a "difficult" genus and

hybridize often. In 1955, Sauer classified the genus into two subgenera,

differentiating only between monoecious and dioecious species: Acnida (L.)

Aellen ex K.R. Robertson and Amaranthus. Although this classification was

widely accepted, further infrageneric classification was (and still is) needed to

differentiate this widely diverse group. Mosyakin and Robertson 1996 later

divided into three subgenera: Acnida, Amaranthus, and Albersia. The support

for the addition of the subdivision Albersia because of its indehiscent fruits

coupled with three elliptic to linear tepals to be exclusive characters to members

of this subgenus. The classification of these groups is further supported with a

combination of floral characters, reproductive strategies, geographic

distribution, and molecular evidence. The phylogenies of Amaranthus using

maximum parsimony and Bayesian analysis of nuclear and chloroplast genes

suggest five clades within the genus: Diecious / Pumilus, Hybris, Galapagos,

Eurasian/ South African, Australian (ESA), ESA + South American.

15
Amaranthus includes three recognized subgenera and 75 species, although

species numbers are questionable due to hybridisation and species concepts.

Infrageneric classification focuses on inflorescence, flower characters and

whether a species is monoecious/dioecious, as in the (Sauer, 1955) suggested

classification. Bracteole morphology present on the stem is used for taxonomic

classification of Amaranth. Wild species have longer bracteoles compared to

cultivated species. A modified infrageneric classification of Amaranthus

includes three subgenera: Acnida, Amaranthus, and Albersia, with the taxonomy

further differentiated by sections within each of the subgenera. There is near

certainty that A. hypochondriacus is the common ancestor to the cultivated grain

species, however the later series of domestication to follow remains unclear.

There has been opposing hypotheses of a single as opposed to multiple

domestication events of the three grain species. There is evidence of

phylogenetic and geographical support for clear groupings that indicate separate

domestication events in South America and Central America. A. hybridus may

derive from South America, whereas A. caudatus, A. hypochondriacus, and

A. quentiensis are native to Central and North America (GBIF, 2020).

2.3 DNA Barcoding

The DNA barcode technology is a novel molecular recognition technology that

uses short and standard DNA fragments for species identification (Feng, 2015;

De-Boer, 2017). DNA barcodes were originally utilized to identify

16
microorganisms (CBOL Plant Working Group, 2009), but now it is able to

quickly and accurately identify species at the level of species with unlimited

reasons for development stage, internal morphological diversity, environmental

factors and user's professional level (Kim, et al, 2014; Feng, 2015; Asahina, et

al., 2010; Echen, et al., 2014). Thus, the DNA barcoding technology has been

rapidly applied in species identification, biosystematics, biodiversity, ecological

community evolution, species protection, archaeological sample identification

and other aspects.

DNA barcoding involves the production of PCR amplicons from particular

regions to sequence them and these sequence data are used to identify or

“barcode” that organism to make a distinction from other species ( (John-James,

et al., 2019). Recently, DNA barcoding has been used and proposed as a useful

technique for identifications of cyanobacterial communities in environmental

samples (Amaral, et al., 2019). Marjorie, (2013) defined DNA barcoding as a

method of identifying organisms based on a short, standardized fragment of

genomic DNA. This method he said in his research has been developed for use

by taxonomist, ecologist, conversation biologists, regulatory agencies and

others. He further claimed that once the DNA is sequenced, it can be analyzed,

it would allow identification of all animal and plant species. It can also be

useful for surveying biodiversity, may allow rapid identification of species. de-

Vere, Rich, Trinder, and Long, (2015) opined that DNA barcoding usies specific

17
regions of DNA in order to identify species. This has seen different intiatives

taking place around the world to generated DNA barcoding for all groups of

living organisms and to make data publicly available in order to help

understand, conserve and utilize the world’s biodiversity. For land plants the

core DNA barcode markers are two sections of coding regions within the

chloroplast, part of the genes, rbcL and matK. In order to create high quality

databases, each plant that is DNA barcoded needs to have an herbarium voucher

that accompanies the rbcL and matK DNA sequences. Li, et al., (2021) carried

out research on DNA barcoding of four (4) chloroplast genes (matK, rbcL, ndhF

and ycf1) and the result was used to provide theoretical basis for species

identification, germplasm conservation and innovative utilization of orchids. Li,

et al., (2021)) used four chloroplast gene sequences (matK, rbcL, ndhF and

ycf1) and three combined sequences including matK + rbcL, matK + ycf1, ndhF

+ ycf1 of Orchidaceae species to develop unique identification fragments of a

certain species of Orchidaceae based on phylogenetic analyses and SNP site

analyses. The most commonly used DNA barcodes for the plant species are ITS,

rbcL, psbA-trnH and matK. DNA barcodes has several applications for the

detection of plants species by providing specific information about the taxa

(Muhammad, et al., 2020).

The potency of DNA barcoding was directly linked to availability of data in the

libraries of barcode which was also helpful in the formation of absolute DNA

18
barcoding database (Goldstein & DeSalle, 2019). To fulfill all the requirements

of barcoding there is the need of PCR conditions with stranded rang and also the

set of PCR primers for every gene that act as a marker of barcode. Recently

many combinations of DNA region have been proposed by (Andújar, et al.,

2018). Possibly in future the combination of DNA region for barcode surely will

contain trnH-psbA that is noncoding intergenic spacer and also matK which is a

plastidial coding sequence but at present there is no any concurrency on a

certain gene candidate that is best for the plant barcoding (Chase, et al., 2007).

The certain species that are related to each other cannot be differentiated

morphologically at the early stages of life (Andersen, et al., 2012)

2.4 Internal Transcribed Spacer (ITS)

The ITS or internal transcribed spacer belong to the nuclear genome. It is a

nonfunctional RNA sequence that is located between the coding region of 18S

and 25S rRNA coding region. The ITS1 located between the 18S and 5.8S

rRNA and ITS2 present between 5.8S and 25S rRNA. During rRNA maturation

the ITS that is a transcriptional subunit present between the structural ribosomal

RNA. As these ITS spacers are actually the product of maturation that are

nonfunctional so they are readily degraded. When studies were conducted on

yeast it was shown that if deletion in certain regions of ITS1 was promoted it

19
cause the inhibition of production of mature small and large subunits of rRNAs,

while on the other hand the mutations in the ITS2 effect the processing of larger

subunit of RRNA. In all flowering plants the length of ITS1 and ITS2 is

variable but it is always less than 300bp for ITS1 and about 250 bp for ITS2.

While the total length of ITS region is about 700 bp that includes the region of

5.8S rRNA, which has the constant length of 163 or164 bp (Gonzalez, et al.,

1990).

In multiple chromosomal loci the nuclear region of ITS, occurs as tandem

repeats (Li, et al., 2015). The high copy number of the region of ITS encourage

the cloning, detection, sequencing and amplification of nr DNA. As compared

to other barcode candidate the ITS region gives better and clear result in PCR,

that’s why it can further be put into restriction digestion which results into

diagnostic bands. These bands are helpful in identification of plants at their

specie level (Selvaraj, et al., 2013). The purpose of DNA barcoding research

was to identify the better candidate genes to identify all the species of plants by

utilizing both the coding and noncoding regions (Telfer, et al., 2015). With the

help of DNA barcoding a person who even don’t have enough taxonomic

training can easily identify the plant specimen. DNA barcode also have

important role in the evolutionary studies. In the differentiation of plant species

of family fabaceae ITS2 was proven very helpful. By using ITS2 as a barcode

gene about 893 species in 96 diverse genera from family Rosaceae were easily

20
evaluated. ITS have specie discrimination success rate of 78% and 100% at the

specie and genus level respectively.

To know the authentication of Chinese herbal medicines ITS2 region is most

commonly used barcode (Song, et al., 2009). To discriminate between plant

species of Asteraceae ITS2 was used as barcode and got the success rate of

about 80%. ITS2 was also utilized in the identification of TEO morphologically

same species such as Swartzia grandifolia and S. longicarpa and also to classify

the species Caranga rosea and C. Sinica of the plant Fabaceae.

The important feature of ITS2 is that it can also be used for the identification of

system Paneukaryote and Eukaryota, that’s why it is used as a universal barcode

for both plants and animals. The chloroplast inter-generic region psbA-trnH was

also reported as the excellent candidate of DNA barcode because it is utilized

for the identification of Dendrobium species (Kress, et al., 2015). DNA

barcoding was applied to identify the distribution of cryptic species in India

Velliangiri hills, this provides useful information in both traditional and

scientific fields. Similar technique was utilized to identify the Berberis species

in India and also for the endangered species of Paphiopedilum (Parveen, et al.,

2012).

The morphologically similar species has also been distinguished by using ITS2

barcode. For the identification of contaminants present in the North American

herbal products was done through the ITS2 when it is used in combination with

21
rbcL (Ceriaco, et al., 2016). The phylogenetic analysis and also studies of

molecular evolution of species of Panax was done by using chloroplast

intergenic spacer (IGS) like trnEtrnT, trnT-psbD, ndhF-rpl32 and rpl14-rpl16.

To discriminate the species of Korean Orchidaceae the IGS like atpF-atpH,

psbK-psbI and trnH-psbA in addition to the coding region of rbcL and matK

were used as barcodes (Kim, et al, 2014).

2.5 Use of barcode genes for species identification

Miller, (2007), associated DNA barcoding with the renaissance of taxonomy,

because it could lead to more and faster species-identification services and

better information about biodiversity. Barcoding was seen as having the

potential to “accelerate our discovery of new species, improve the quality of

taxonomic information and make this information readily available to non-

taxonomists and researchers outside of major collection centers”.

Plant species identification is the basis of botanical research and application. In

plant taxonomy, applying plant DNA barcoding can aid in the identification of

some cryptic species, as well as new species (Besse, et al, 2021). Liu, et al.,

(2011) analyzed Taxus from Eurasia using four chloroplast gene fragments and

one nuclear gene fragment and identified eleven (11) species and four new taxa.

DNA barcoding has also been extensively applied to identify plant germplasm

resources. Barcode genes to differentiate species within the same species. For

example, three DNA barcodes were used to identify six species and seven easily

22
confused plants of the genus Sabia, and the sequence difference rate between

the Sabia species and the easily confused plants was as high as 24.5% (Sui, et

al., 2011). For instance, by analyzing the DNA barcodes of 274 plant species

belonging to 87 genera, 77 plant species were found to be (China Plant BOL

Group, Li., et al, 2011). Druzhinina, et al., (2005) presented a

DNA oligonucleotide barcode method for the species identification

of Hypocrea and Trichoderma, based on the ITS1-2 regions. (Peter, et al.,

2016), suggested that a graded continum of intra- and interspecies distances,

with barcode commonly shared among realted species. This can result into

standard plant barcodes which are best suited to being molecular augmentations

to existing classification and plant barcodes which required attention at the

outset to ensure a match between the resolving power of the technique. In the

discovery of species several unexpected sequece divergence has led to re-

examination of morphological/ecological variation and ultimately resulted in the

recognition of new species.

Plants’ DNA barcoding has emerged as a scientific breakthrough and is often

used to help with species identification or as a taxonomical tool decades ago.

For identification and authentication of 61 medicinal plants from 30 families in

Indonesia, a pair of ITS2, matK, rbcL, and trnL primers were used for a DNA

barcoding study consisting of molecular and sequence analyses (Cahyaningsih,

et al., 2022). Their study was to analyze how the four identification and

23
conversation and to investigate their effectiveness of DNA barcoding for species

under investigation. At the end their research resulted in 212 DNA barcoding

sequences and identification of new ones for the observed medicinal plant

species. (Cahyaningsih, et al., 2022), recommended the matK as the main

region for their DNA barcoding with ITS2 and rbcL as alternatives. Examined

the effectiveness of DNA barcoding in their specic organisms. They use

mitochondrial cytochrome C oxidase (COI) gene of 83 specimens belonging to

40 species of 18 genera in their research and obtain a result which showed that

there was a barcode gap between species of Naiddiae and the intraspecific

genetic distance of each species.

24
 CHAPTER THREE

MATERIALS AND METHODOLOGY

3.1 Study site

This study was carried out at Genomics Training Centre and Laboratory

Limited, No. 1, Atiku Abubakar Way, Opposite the African Church, Uyo, Akwa

Ibom State.

3.2 Materials

The following materials were used to successfully carry out experiment:

 Fresh plant samples of Amaranthus species

 Liquid nitrogen

 CTAB buffer

 Chloroform

 Isoamyl alcohol

 70% and 100% ethanol

 TE buffer

 Agarose gel

 Gel extraction kit

 ITS primers

 PCR reagents (Taq polymerase, dNTPs, MgCl2, buffer)

 DNA sequencing service

25
3.3 Sample collection

A sample specie of Amaranthus were used in the study, which is the

Amaranthus hybridus (Plate 3.1). Fresh leaves of the Amaranthus hybridus was

collected from environs of Akpaden and were taken to Herbarium of the

Department of Botany for Identification (Table 3.1), before taken to Genomic

Training Center and Laboratory for molecular analysis. While ITS2 gene was

amplified for Amaranthus hybridus, the DNA extraction from the Amaranthus

hybridus was carried out using Genomic DNA extraction kit (Genomic DNA

mini kit, Geneaid).

Plate 3.1: Amaranthus hybridus plant

Table 3.1: Amaranthus hybridus and its Herbarium Identification Number

S/N Amaranthus Herbarium Identification Number

1 Amaranthus hybridus AKSUH/P0068

26
3.4 DNA Extraction

Amaranthus young leaf material was gathered, ground in a mortar and pestle

with 400 l of extraction buffer, and then transferred to 1.5 microcentrifuge tubes

for vortex mixing. After 10 minutes of incubation at 60 degrees Celsius, 100 l of

GP2 buffer was added, mixed by vortex, and placed in the tubes for 3 minutes

of incubation on ice.

The mixture was transferred to a filter column in a 2 ml collection tube. The

supernatant was then transferred from the 2ml collection tube to a fresh 1.5 ml

microcentrifuge tube. The 700ul of the mixture was transferred to the GD

column, which was then centrifuged at 14-16µg for 2 minutes, the flow-through

was removed, and the GD column was then placed back into the 2ml collecting

tube.

The remaining mixture was added to the GD column, which was then

centrifuged at 14–16,000 g for 2 minutes, flow-through was discarded, and the

GD column was put back in a 2ml collection tube. Next, 40µl of W Buffer was

added to the GD column, which was then centrifuged at 14 -16,000 g for 30

seconds, the flow-through was discarded, and the GD column was put back in a

2 ml collection. The dried GD column was transferred to a clean 1.5ml micro

centrifuge tube, 100𝜇l of preheated elution buffer or TE buffer was added to the

center of the GD column matrix and I waited for about 3-5 minutes to ensure

27
the elution buffer is completely absorbed and was centrifuge at 14-16µg for

30seconds to elite the purified DNA.

3.5 Qualitative Analysis - Agarose Gel Electrophoresis

Agarose gel electrophoresis was also used to assess the quantity and quality of

the DNA. 1.5% agarose gel electrophoresis was used to test the DNA quality; it

was done at 120V for 20 minutes in a 1 TAE (Tris-base acetate, EDTA) buffer.

The gel was stained with 8µl of safe view dye, subjected to a 20-minute gel

electrophoresis, had the TAE buffer drained from it, and was then examined

with a UV transilluminator.

3.5 DNA Quantification

A spectrophotometer was employed to determine the concentration of DNA

(Gene Quant Pro). By measuring optical density (OD) at 260 and 280 nm, the

absorbance of total genomic DNA (gDNA) was measured.

3.6 Gene sequencing

With the aid of a PCR purification and concentrator kit from Zymo Research,

the amplified PCR product that displayed only single bands was purified before

being eluted with 20µl of GIBCO water (Invitrogen Corporation). In order to

achieve the requirements necessary for effective DNA sequencing procedures,

purified PCR products were sufficiently quantified with 1.5% agarose gel

electrophoresis (Sigma Life Science). The same PCR primers were used to

sequence the PCR product obtained after the purification stage using an ABI

28
system 3130 x1 genetic automated sequencer (Applied Biosystems) and the

BigDye terminator 3.1 kit (Applied biosystems). The sequencing reaction was

set up in 10.0 liters with 1.0μl of 1ml BigDye terminator 3.1 (Applied

Biosystems), 1.54μl of BigDye buffer (5x), 1.5μl of 2.5pmole of either the

forward or reverse primer, 0.1μl of purified template DNA produced from the

sequencing reaction product, and 5.0μl of 500 ml GIBCO water (Invitrogen

Corporation).

The PCR profile for sequencing starts with a 96°C denaturation for 1 min,

followed by 25 cycles of the following program: 96°C for 10 sec, 50°C for 5

sec, 60°C for 4 min, and executed quick thermal ramp for 4°C and kept forever.

Before adding 9μl of Hi-Di formamide (Applied Biosystems) to each of the

purified sequencing PCR products, the reaction products were cleaned up using

an ethanol purification technique that included 1.0μl 125 mM EDTA,

1.0μl 3m NaOAc2, and 25.0μl 100% ethanol. Prior to sample analysis using an

ABI Prism 3130X1 Genetic automated sequencer (Applied Biosystems), the

mixtures were denatured for 5 min at 95oC.

The obtained raw sequences were modified by deleting undesirable

chromatograms and trimming both ends. By using ClustalW, nucleotide

sequences were aligned (Thompson et al., 1994). To determine whether the

sequenced samples had mutations or nucleotide polymorphisms, multiple

sequence alignment was done in BioEdit ver. 7.2. To ascertain the sequence's

29
identification and degree of sequence similarity, the BLAST tool was used using

matched sequences from NCBI.

3.7 Phylogenetic Relationship among the Genes under Study

Using the neighbor joining approach, a neighbor-joining tree was created to

infer the relationships between the Amaranthus species (Saitou & Nei, 1987).

Codon positions that were included were 1st + 2nd + 3rd + Noncoding, and all

alignment gaps were classified as missing data. Positions with holes and

incomplete data were all removed. Software known as MEGA 7 was used to

conduct the neighbor-joining tree (Kumar, et al., 2016).

30
 CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 DNA Extraction and Quantification

An example of the Amaranthus hybridus DNA extracted and stored in a 1.5 ml

micro centrifuge tube can be seen in (Plate 4.1). A spectrophotometer analysis

of the DNA concentration showed good quality DNA of 367µg/l and a purity of

1.9. The DNA appeared to be of acceptable standard with distinct bands when

run on a 1% agarose gel and seen with a UV transilluminator (Plate 4.1).

Plate 4.1: Extracted DNA from Amaranthus hybridus

31
4.2 Polymerase Chain Reaction (PCR) Analysis

The ITS genes were used as the forward and reverse primers for PCR in this

study. Amplicons were separated using electrophoresis in 1.5% agarose gel. The

molecular weight benchmark employed was a DNA ladder of 100 bp. The PCR

product for the ITS2 genes was visible on the UV transilluminator as an

electrophoresis result at about 500 (Plate 4.3).

The polymerase Chain Reaction (PCR) is a technique that is frequently used to

quickly create millions to billions of copies (complete copies or partial copies)

of a specific DNA sample, enabling researchers to take a very small DNA

sample and amplify it (or a portion of it) to a large enough amount to study it in

detail. In a series of temperature-changing cycles, copies of very small amounts

of DNA sequences are quickly amplified using PCR.

Plate 4.2: Agarose gel electrophoresis amplification of PCR product and DNA of the

Amaranthus hybridus.

32
 M 1

500pb

* M=100bp DNA ladder

* 1 = ITS2
Plate 4.3: Amplified PCR product of about 500bp for ITS gene compared against a

100bp DNA ladder.

4.3 Sequence Editing and BLASTn Analysis

ITS genes had acceptable quality alignments longer than 200 nucleotides in the

top 100 BLASTn hits in the genebank. This is the cause of these queries' high

query coverage (100%) (Table 4.1). The most important alignment of both

genes for the Amaranthus hybridus under study had a high alignment score with

no gaps (Figures 4.2 and 4.3). Amaranthus hybridus was identified with 100%

query coverage and 100% percentage identity, coverage and 99% percentage

identity using the ITS2 gene. Between a query sequence and the subject

sequences of the database searches, BLAST computes a pairwise analysis

(Figures 4.2 and 4.3). Based on the alignment of those (database) sequences to

the query sequence, an implicit alignment between the database sequences was

created.

33
They deduced from the research that the low sequence quality of ITS2 made it

untrustworthy. As a result of their findings, they hypothesized that although

ITS2 can be utilized as a barcode for portulaca species, the issue of ITS2

sequence quality needs to be fixed before it can be advised for further usage.

Table 4.1: ITS2 BLAST Hits with Highest Identities in the Gene Bank

Description Scientific Max Total Query E- Ident Acc. Accession

Name Score Score Cover value % Len

Amaranthus Amaranthus 710 710 100% 0.0 100.00 151 CM025916.1

hybridus hybridus
isolate AhG2s
chromosome

Figure 4.4: Distribution of the top 100 BLAST hits in the genebank showed
good quality alignments greater than 200 nucleotides.

34
Figure 4.6: Alignment for Amaranthus hybridus to isolate AMDU_a_E.R_2of3.5.8S
ribosomal RNA gene, partial sequence, internal transcribed spacer 2, complete
sequence, and large subunit ribosomal RNA gene partial sequence.

4.4 Phylogenetic Analysis

With the MEGA 7 program, a neighbor joining tree was built using the

sequences of the Amaranthus hybridus understudy and the most important hits

from the genebank. The UPGMA algorithm was used to infer the relationship

between the Amaranthus species using a neighbor-joining tree. For the ITS2

genes, the best tree with the sum of branch length = 1.32941890 is displayed.

Using branch lengths in the same units as the evolutionary distances used to

estimate the phylogenetic tree, the tree was drawn to scale. The evolutionary

distances, which are measured in base substitutions per site, were calculated

using the Maximum Composite Likelihood technique.

35
In molecular biology, phylogenetic analysis has emerged as a crucial technique

for comparing data on genes, individuals, populations, and species.

Phylogenetic analysis is used to estimate the historical link of genes or species

and to represent this relationship in the form of a branching diagram known as a

phylogenetic tree. The ITS2 gene are grouped Amaranthus hybridus, bitum,

caudatus, spinosus, etc.

12 Amaranthus sp.

8 KY700218.1:69-469 Amaranthus dubius

60
KU310615.1:389-692 Amaranthus hypochondriacus
10 KY968887.1:465-767 Amaranthus cruentus
96
KF493807.1:387-681 Amaranthus palmeri
MG256201.1:49-364 Amaranthus hybridus
87
62
MH711405.1:440-741 Amaranthus retroflexus
26 OM021392.1:252-546 Amaranthus powellii
MH547548.1:69-469 Amaranthus hybridus

96 MG235146.1:21-419 Amaranthus tuberculatus

KY968934.1:463-765 Amaranthus tuberculatus

29 MG237199.1:20-418 Amaranthus albus

MK256242.1:16-419 Amaranthus viridis
54

70
MH547497.1:66-466 Maerua crassifolia isolate
75 MH547487.1:69-469 Ephedra alata
MH547511.1:71-471 Amaranthus graecizans

Figure 4.7: Evolutionary Relationships of Taxa for ITS2

36
 CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

Genes or nucleotide sequences on chromosomes that may identify between

different types of cells, individuals, or species are called molecular markers. In

comparison to other marker systems, molecular markers are indisputably

higher-level indicators and have an advantage since they are unaffected by

aging, the environment, or physiological factors. These markers can also be

identified at any stage of a plant's growth since they are tissue-neutral. Although

it has been shown that some barcode genes have high success rates for

identifying species and other genes have poor success rates, employing

molecular tools for plant identification has been considered to be the most

reliable method.

During this investigation, the ITS2 gene was amplified in Amaranthus. All

Amaranthus variants were identified by the markers as hybridus. For the ITS2

genes, the Amaranthus specie under investigation showed 100% query

coverage.

As shown in table 4.1, the output shows the significant alignments of a query

sequence from the genome of Amaranthus hybridus isolate AhG2s against a

sequence database. The output shows both chromosome sequences and protein

37
sequences that match the query sequence, along with information about the

alignment, such as the score, coverage, identity, and E-value.

The chromosome sequences match with 100% coverage and identity, indicating

that they are highly similar to the query sequence. The protein sequences also

have a high similarity with the query sequence, but they have a lower coverage

and identity than the chromosome sequences.

The E-value is a measure of the significance of the alignment. The lower the E-

value, the more significant the alignment. In this case, all the alignments have

E-values of zero (0), indicating that they are highly significant.

5.2 Recommendation

According to the findings of this research, the hybridus was identified as

belonging to the Amaranthus species and other barcode markers like matK.

Based on the findings of this research project, the following recommendations

can be made:

a) To give a more reliable phylogenetic study of the evolutionary connections

between species, more investigations on a larger sample size of Amaranthus

species should be conducted.

38
b) Due to the matK gene's excellent resolution and accuracy in identifying

species, its usage as a DNA barcode marker for Amaranthus species

identification is advised.

c) As many Amaranthus species face extinction owing to habitat loss and

climate change, more attention should be paid to their preservation.

d) To comprehend the ecological and agricultural significance of Amaranthus

hybridus and its tight evolutionary relationship with other Amaranthus

species, as well as their potential for use as a source of food and medicine,

further research on these plants is necessary.

39
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 APPENDICES

Appendix 1: Blastn Result

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