American Journal of Plant Sciences, 2019, 10, 179-207
http://www.scirp.org/journal/ajps
ISSN Online: 2158-2750
ISSN Print: 2158-2742
Genetic Diversity and DNA Barcoding of Yam
Accessions from Southern Nigeria
George N. Ude1*, David O. Igwe1,2, Julian McCormick1, Onyinye Ozokonkwo-Alor3,
Jonathan Harper1, Daniel Ballah1, Cecille Aninweze3, Obih Chosen3, Michael Okoro3,4,
Christabel Ene3, Venatus Chieze3, Mariam Unachukwu3, Christie Onyia3, George Acquaah1,
James Ogbonna3,5, Aditi Das1
1
Department of Natural Sciences, Bowie State University, Bowie, USA
Department of Biotechnology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria
3
Godfrey Okoye University, Enugu, Nigeria
4
DNA Learning Center, Cold Spring Harbor, NYC, USA
5
Department of Microbiology, University of Nigeria, Nsukka, Nigeria
2
How to cite this paper: Ude, G.N., Igwe,
D.O., McCormick, J., Ozokonkwo-Alor, O.,
Harper, J., Ballah, D., Aninweze, C., Chosen, O., Okoro, M., Ene, C., Chieze, V.,
Unachukwu, M., Onyia, C., Acquaah, G.,
Ogbonna, J. and Das, A. (2019) Genetic
Diversity and DNA Barcoding of Yam
Accessions from Southern Nigeria. American Journal of Plant Sciences, 10, 179-207.
https://doi.org/10.4236/ajps.2019.101015
Received: December 21, 2018
Accepted: January 19, 2019
Published: January 22, 2019
Copyright © 2019 by author(s) and
Scientific Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
Open Access
DOI: 10.4236/ajps.2019.101015
Abstract
Knowledge of genetic diversity and barcoding of yam is lacking in Enugu and
Ebonyi States of southern Nigeria. Therefore, DNA barcoding was used to facilitate identification and biodiversity studies of yam species from Southern
Nigeria. Seventy five yam accessions were collected from Enugu and Ebonyi
States, including International Institute of Tropical Agriculture for DNA extraction and amplification using a chloroplast DNA (cpDNA) ribulose-1,5bisphosphate carboxylase (rbcL) marker. There was high level of similarity
among the accessions and presence of 534 conserved and 7 variable sites. A
transversional mutation of G/T at a consensus position of 335 was identified
followed by transitions at 362 (A/G), 368 (A/G), 371 (C/T) and 391 (C/T)
within the accessions. Phylogeny resolved the yam accessions into ten major
groups with their bootstrap values ranging from 0 - 100. Phylogenetic diversity was highest in group X, followed by VII, VI and IX. The inter-group genetic distance based on Kimura 2-parameter model ranged from 0.5000 ±
0.4770 - 5.0560 ± 2.5760, while the intra-group had 0.5250 ± 0.5000 - 2.0103
± 1.2579. The mean genetic diversity within the entire population was 0.7970
± 0.06910. BLAST analysis of total bit score, query coverage, and percentage
identity were in the ranges of 411 - 1011, 99% - 100% and 97% - 100%, respectively. However, the rbcL could not resolve the yam accessions well following the comparative assessment of some discrepancies in the detected
number of species from phylogenetic groupings, genetic diversity indices and
NCBI BLAST hits, thereby, exposing the inefficiency of this marker in dis-
Jan. 22, 2019
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criminating the yam accessions. It was demonstrated that rbcL is not an effective marker; therefore, it should not be recommended as a standard-alone
marker of choice for DNA barcoding of yam accessions, especially, when accurate identification, discrimination and estimation of genetic diversity of
this vital crop are of paramount importance for crop improvement and
germplasm conservation.
Keywords
BLAST, Kimura 2-Parameter, Phylogenetic Diversity, rbcL, Transitional
Mutation
1. Introduction
Yam (Dioscorea spp.) is a monocotyledonous, an annual or perennial stem tuber
belonging to the family Dioscoreaceae of flowering plants. Dioscorea has been
described as the largest genus with an estimated 600 species, 10 of which are cultivated and of economic importance [1] [2] [3]. It is the second most important
crop after cassava in West Africa [4] [5] [6]. Important and cultivatable species
of this vital crop include D. cayenensis Lam., D. alata L., D. rotundata Poir., D.
trifida L. f., D. bulbifera L., D. pentaphylla L., D. opposita Thunb., D. transversa
R. Br., D. nummularia Lam. and D. esculenta (Lour.) Burkill. [7]. Within Africa,
the common species cultivated include D. rotundata (white yam), D. alata (water
yam) and D. cayenensis (yellow yam), some of which have been reported to
possess medicinal and ornamental values [8].
The crop ranks fourth after potato, sweet potato and cassava as the most important food tuber crop in the world [6]. Yam is important in the economic and
social life of people in West Africa [9] [10]. As a starchy food, it provides a major source of cheap caloric energy food for millions of people in the tropical and
sub-tropical regions of the world particularly in West Africa, the Caribbean,
parts of Asia, South and Central America and the Pacific [8] [11]. Yam tubers
are rich sources of energy, vitamin C, musin (glycoprotein), minerals (K, P, Ca,
Mg, Fe, Cu, Co), phytosterols and steroidal saponins [12]. They are converted
into different types of food products such as pounded yam, boiled yam, roasted
yam, fried yam slices, yam balls, mashed yams, yam chips, and yam flakes [13].
Fresh yam tubers are also peeled, chipped, dried, and milled into flour that is
used to prepare dough called “amala” or “telibowo” [14].
Yams are widespread in the tropics and subtropics. Nigeria is the leading
producer of yam with 71% of the world production [15] [16] [17]. West Africa
accounts for over 92% of the world’s production (54.2 million tonnes) [6]. In
Ghana and Nigeria, 26.2% and 31.8% of people, respectively rely on yam species
for income generation and food security [4]. Despite the increasing demand for
local consumption and export of yams, there has been a marginal decline in its
production due to lack of proper identification of unique species for biodiversity
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G. N. Ude et al.
diversification for resistance to drastic changes in climate, introgression, cross
hybridization and conservation processes to reduce genetic erosion [18]-[24]. To
have adequate knowledge of these yam accessions, characterization to the species
level, genetic richness and assessment of phylogenetic diversity (PD) are of utmost importance following the genetic resource preservation roles of PD in crop
extinction [25], functionality in ecosystems [26] [27] and abiotic variability [28]
and these can be achievable with accurate, sensitive and reliable methods.
Morphotaxonomy, the use of morphological characters to identify and classify
plants, is currently the most widely used in yams in Nigeria. It entails using traits
such as size, form and number of tubers per plant, bulbil formation, presence of
spines on the stem, twining direction, fruit shape, and aerial bulbils, which could
lead to misidentification of yam species [1] [3] [29]. Further, morphotaxonomy-based method requires cumbersome assessment of whole plants and the
importance of this approach declines when specimens/tissue materials are utilized [30]. Use of molecular markers has become significant for accurate identification of these yams to the species level and to harness the genetic diversity inherent in them. Different markers including Restriction fragment length polymorphism (RFLP) [31], Random amplified polymorphic DNA (RAPD) [32],
Simple sequence repeat (SSR) [32], Inter-simple repeat (ISSR) [12] and Amplified fragment length polymorphism (AFLP) [32] and gene sequencing [33] [34]
have been applied in the characterization of yam species. The use of molecular
tools to support morphotaxonomy-based identification is important to clear
ambiguous species classification.
A DNA barcode facilitates taxonomic identification through the use of a
standardized short genomic segment that is generally found in target lineages
with adequate variations capable of discriminating living animals to the species
level [35]. DNA barcoding techniques are useful tools in characterization as they
allow more objective and rapid specimen identification, which can be
cost-effective in providing a central catalog of species diversity. In general, DNA
barcoding can improve biodiversity and genetic resource databases [36] [37].
Also, a phylogenetic diversity (PD) method possesses the merits of ease of reconstruction of phylogenetic relationships of species and as such it has resultant
potential to enlighten effective taxonomic challenges [38]. MatK and rbcL which
are the two plant barcode loci have been chosen for phylogenetic studies of Dio-
scorea [1] [39]. In this study, a barcoding marker of rbcL was used for identification and genetic characterization of Dioscorea accessions cultivated in southern
Nigeria.
2. Materials and Methods
2.1. Sample Collection
Different yams were sampled from different locations across Eastern and Western Nigerian, including the ones in the germplasm collection at the International
Institute of Tropical Agriculture (IITA), Ibadan, Nigeria (Table 1). A total of
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Table 1. List of yam samples collected from different locations and used for DNA
barcoding.
Sample IDs
DOI: 10.4236/ajps.2019.101015
Location
LGA
State
1_TDa85.00250
IITA
Akinyele
Oyo
3_TDa3050
IITA
Akinyele
Oyo
4_TDb3050
IITA
Akinyele
Oyo
5_TDb3044
IITA
Akinyele
Oyo
6_TDb2857
IITA
Akinyele
Oyo
7_TDb3058
IITA
Akinyele
Oyo
8_TDb3690
IITA
Akinyele
Oyo
9_TDd3101
IITA
Akinyele
Oyo
10_TDd3829
IITA
Akinyele
Oyo
11_TDd3935
IITA
Akinyele
Oyo
12_TDd08-38-53
IITA
Akinyele
Oyo
13_TDdYellow
IITA
Akinyele
Oyo
14_TDd3100
IITA
Akinyele
Oyo
15_TDc0471-2
IITA
Akinyele
Oyo
16_TDc0497-4
IITA
Akinyele
Oyo
17_TDc2813
IITA
Akinyele
Oyo
18_TDc2796
IITA
Akinyele
Oyo
19_TDc2792
IITA
Akinyele
Oyo
20_TDc03-5
IITA
Akinyele
Oyo
21_TDc04-71-2
IITA
Akinyele
Oyo
22_TDm2938
IITA
Akinyele
Oyo
23_TDm3053
IITA
Akinyele
Oyo
24_TDm3052
IITA
Akinyele
Oyo
25_TDm3055
IITA
Akinyele
Oyo
27_TDes3035
IITA
Akinyele
Oyo
28_TDes3033
IITA
Akinyele
Oyo
29_TDes 3027
IITA
Akinyele
Oyo
30_TDes 3030
IITA
Akinyele
Oyo
31_TDesculenta
IITA
Akinyele
Oyo
33_TDaNwokporo
IITA
Akinyele
Oyo
34_Adakavariety
IITA
Akinyele
Oyo
35_Pepa
IITA
Akinyele
Oyo
36_Ke-emi
IITA
Akinyele
Oyo
37_Ame
IITA
Akinyele
Oyo
38_TDr 89.002665
IITA
Akinyele
Oyo
39_AlataTda 98.01176
IITA
Akinyele
Oyo
40_TDa00.00.94 41_Alata
IITA
Akinyele
Oyo
41_Tda00.00600
IITA
Akinyele
Oyo
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G. N. Ude et al.
Continued
42_OgojaVariety.1
IITA
Akinyele
Oyo
43_Gbangu_Variety.1
IITA
Akinyele
Oyo
44_ObioturuguVariety.1
IITA
Akinyele
Oyo
45_AmolaVariety .1
IITA
Akinyele
Oyo
46_OginiVariety
IITA
Akinyele
Oyo
47_Damieha
IITA
Akinyele
Oyo
48_Aloshivariety.1
IITA
Akinyele
Oyo
49_IghuUna
Osonu
Ezeagu
Enugu
51_Alata2
Osonu
Ezeagu
Enugu
52_Ighu_Dumenturum
Osonu
Ezeagu
Enugu
53_Ighu
Osonu
Ezeagu
Enugu
54_IghuUna.2
Osonu
Ezeagu
Enugu
Ukaka Ngwo
Enugu North
Enugu
59_D10WhiteNwopoko-Adaka
Agbalenyi Nachi
Oji-River
Enugu
60_D1Water-Nbana1
Agbalenyi Nachi
Oji-River
Enugu
61-6-EDO
Ukaka Ngwo
Enugu North
Enugu
62_3LeavedYam-Ono
Ukaka Ngwo
Enugu North
Enugu
65_WaterYam.Nbana
Ukaka Ngwo
Enugu North
Enugu
Ndibinagu Umuaga
Udi
Enugu
71_D1WaterYam-Nbana2
Agbalenyi Nachi
Oji-River
Enugu
72_1-Water_Yam-_ Nbana
Ndibinagu Umuaga
Udi
Enugu
Nkalagu
Ishielu
Ebonyi
76_OnaTDd
Ezzamgbo
Ohaukwu
Ebonyi
78_Obella
Ezzamgbo
Ohaukwu
Ebonyi
80_UtekpeVariety_2
Ezzamgbo
Ohaukwu
Ebonyi
81_WhiteYam-Nw-opoko
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
82_Yellowyam_Akpukpu
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
83_WaterYam-Mbuna
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
84_BitterYam-Iwu_obe
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
85_AerialYam_Edugbe
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
86_3LeavedYam_Ona
Ede Oballa
Nsukka
Enugu
87_WaterYam-Mbana
Nru
Nsukka
Enugu
89_WhiteYam_Nwopoko
Ibagwa Aka
Igboeze South
Enugu
90_Yellowyam_Oku
Ihe Owerre
Nsukka
Enugu
91_TrifoliateYam_TDb
Ukana
Udi
Enugu
92_ChineseYam_TDes
Ukana
Udi
Enugu
93_YellowYam_TDes
Ukana
Udi
Enugu
57_2-WhiteYam-Iyo
68_9ENEGBE
73_Water yamji_mbala
IITA = International Institute of Tropical Agriculture; LGA = Local Government Area.
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eleven Local Government Areas (LGAs), cutting across three States including
Oyo (where IITA, Ibadan is located), Enugu and Ebonyi States were used for the
yam collection (Figure 1). The IITA, Ibadan, has Genetic Resources Unit that
contains many yam species from other parts of Nigeria.
2.2. DNA Extraction
Fresh young leaves of yam species weighing from 0.1 - 0.2 g were collected for
DNA extraction using Silica resin method standardized by the DNA Learning
Center (http://www.dnabarcoding101.org/lab/protocol-2.html) [40] In brief,
fresh young yam leaf samples were weighed and homogenized in 300 µL of lysis
solution using sterile mortar and pestle followed by incubation in a heat block at
65˚C for 10 minutes. Next, samples were centrifuged in a balanced configuration
at maximum speed (13,000 rev/min) for 1 min to pellet debris. A 150 μL sample
of the supernatant was transferred to fresh micro centrifuge tubes, being careful
not to disturb the debris pellet. A 3 μL silica resin, was subsequently added to the
respective supernatants, mixed well by pipetting up and down, and placed for 5
minutes in a heat block at 57˚C. The silica resin is a DNA binding matrix which
in the presence of lysis solution binds readily to nucleic acids. After incubation,
tubes were subject to centrifugation, with cap hinges pointing outward, for 30
seconds at maximum speed to pellet the silica resin, which was now bound to
nucleic acid. Using a micropipette with fresh tip the supernatant was removed
Figure 1. Map of Nigeria showing geographical areas for collection of yam accessions.
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and 500 μL of ice cold wash buffer added to the pellet. The silica resin bound to
nucleic acid was re-suspended by vortexing and centrifuged to repeat the wash
procedure. The wash buffer removes contaminants from the samples while nucleic acids remain bound to the resin. A dry spin step after wash was performed
to remove any remnant drops of supernatant with a micropipette. Finally, 100
μL of distilled water was added to the silica resin, mixed well by vortexing and
incubated at 57˚C for 5 minutes. Samples were then centrifuged for 30 seconds
at maximum speed to pellet the resin. This time 90 μL of the supernatant was
transferred to fresh tubes as the nucleic acids eluate from the resin. The eluted
DNA was stored to proceed to PCR step.
2.3. Polymerase Chain Reaction, Agarose Gel Electrophoresis and
DNA Sequencing
PCR amplification was performed using Ready-To-Go PCR beads in a total
volume of 25 µL: 2 µL of ~100 ng DNA and 23 µL of primer/loading dye mix for
plant cocktail with rbcL primers (rbcLaf:
5'-TGTAAAACGACGGCCAGTATGTCACCACAAACAGAGACTAAAGC-3'
and rbcLa-revM13:
5'-CAGGAAACAGCTATGACGTAAAATCAAGTCCACCRCG-3'). The PCR
tubes were placed in a thermal cycler that had been programmed with the appropriate PCR protocol with initial step at 94˚C for 1 min., 35 cycles of 94˚C for
15 sec, 54˚C for 15 sec, and 72˚C for 30 sec., and 8 min final extension at 72˚C
was maintained. The PCR products or amplicons were electrophoresed in a 1.5%
agarose gel containing 0.5 mg/ml ethidium bromide and photographed on Transilluminator UV light (Omega G). The generated PCR amplicons sent to
Genewiz LLC, New Jersey, USA, for DNA sequencing.
2.4. Data Analysis
The sequencing results generated from the Applied Biosystems Genetic automated sequencer (ABI Prism 3130X1, Froster City, CA 94404, USA) at Genewiz
LLC were uploaded in the blue line of DNA Subway
(https://dnasubway.cyverse.org/), which is an intuitive interface for analysing
DNA barcodes. Using the Blue Line, the assembled sequences were end-trimmed,
paired in their respective forward and reverse sequences to build consensus sequences. The consensus sequences from DNA subway were further edited, filtered and assembled for polymorphism detection using BioEdit software
(BioEdit sequence aligner editor, version 7.6.2.1). Sequence alignment and percentage similarity searches were compared with GeneBank databases using
NCBI web-based site, BLAST. Multiple alignments were done using the
ClustalW [41] [42]. Phylogenetic tree reconstruction was performed using
MEGA 6 software [43]. Phylogenies were constructed using the Maximum Parsimony and Maximum Likelihood options [44] [45] and the effectiveness of the
trees was determined by bootstrapping up to 1000 replicates [46].
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3. Results
3.1. Sequence Alignment of Sequences Generated from Dioscorea
Spp. Using rbcL Barcoding Marker
A total length of sequence alignment, conserved sites, and variable sites of 525,
534 and 7 were respectively identified among the sequenced yam species. Different regions of polymorphisms and conserved regions at nucleotide level
across the sequences exhibited variations among them. At a position of 335,
62_3LeavedYam_Ono and 76_Ona_TDd possessed a transversional mutation by
having G nucleotide, while other samples had a T nucleotide (Additional file 1:
Figure S1). At a consensus position of 362, yam species such as 3_TDa3050,
4_TDb3050, 5_TDb3044, 6_TDb2857, 7_TDb3058, 8_TDb3690, 61_6-EDO,
83_WaterYam-_Mbuna and 85_AerialYam_Edugbe showed a transitional mutation of A nucleotide, while the rest of the accessions had a G nucleotide. At a position of 368, accessions such as 1_TDa85.00250, 3_TDa3050, 4_TDb3050,
5_TDb3044, 6_TDb2857, 7_TDb3058, 8_TDb3690, 61_B-6-EDO,
83_WaterYam-_Mbuna, 85_AerialYam_Edugbe had a transitional mutation of
A in place of G nucleotide possessed by other accessions at the same consensus
position.. Also at 371 position, accessions including 35_Pepa, 36_Ke-emi,
37_Ame, 38_TDr.89.002665, 39_AlataTda98.01176, 40_TDa00.00.94,
42_OgojaVariety.1, 43_Gbangu_Variety.1, 44_ObioturuguVariety,
45_AmolaVariety.1,
46_OginiVariety,
47_Damieha,
48_Aloshivariety.1,
57_2-WhiteYam_Iyo, 61_6-EDO, 68_9ENEGBE, 78_Obella, 80_UtekpeVariety_2,
81_WhiteYam-Nwoopoko, 82_Yellowyam_Akpukpu, 83_WaterYam-Mbuna,
85_AerialYam_Edugbe, 89_WhiteYam_Nwoopoko and 93_YellowYam_TDes
exhibited a transitional mutation by possessing a C nucleotide, while the remaining species had a T nucleotide. Also at a position of 391, 76_Ona_TDd
possessed C, while other remaining yam species had T nucleotide.
3.2. Phylogenetic Tree Reconstruction (PTR) and Phylogenetic
Diversity (PD)
Out of the 75 nucleotide sequences used for the analyses, a total of 270 codon
positions including 1st, 2nd, 3rd, and non-coding regions as well as 4.3582% invariable (monomorphic) sites were found in the final dataset. From the phylogenetic tree analysis, the yam accessions were resolved into ten groups with
variable phylogenetic diversities (PDs) (Figure 2). Group I with PD in the range
of 0-27 consisted of twenty five accessions including 43_Gbangu_variety,
82_Yellowyam-Akpukpu, 81_Whiteyam-Nwopoko, 89_WhiteYam-Nwopoko,
24_TDm3052, 23_TDm3053, 20_TDc03-5, 19_TDc2792, 80_Utekpevariety,
17_TDc2813, 21_TDc04-71-2, 93_Yellowyam-TDes, 18_TDc2796, 68_9ENEGBE,
25_TDm3055, 15_TDc0471-2, 46_Oginivariety, 57_2-Whiteyam-Iyo,
45_Amolavariety, 40_TDa00.00.94, 38_TDr89.002665, 16_TDc0497-4, 78_Obella,
37_Ame and 35_Pepa grouping with D. rotundata obtained from NCBI data
with a reference sequence accession of KR072483. The yam accessions were
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Figure 2. Phylogenetic tree of different yam species as revealed by rbCL barcoding
marker.
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collected from different locations including Enugu, Ebonyi and International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Group II (with PD of
2-49) contained four accessions such as 47_Damieha, 48_Aloshivariety, 39_Alata
TDa98-01176, 44_Obioturuguvariety grouped together KJ629251-D. abyssinica,
KJ629254-D. cayenensis and KJ629260-D. praehensillis. Group III (with PD of
1) contained only 42_Ogojavariety.
Groups IV (PD = 6) and V (PD = 31) had 36_Ke-emi and 22_TDm2938, respectively. Group VI (PD = 20 - 79) consisted fourteen including
59_10-Whiteyam-Nwopoko-Adaka, 90_YellowYam-Oku, 33_TDaNwopoko,
41_TDa00.00600, 71_D1WaterYam-Nbana2, 1_TDa85.00250, 73_WaterYam-Mbala,
72_1WaterYam-_Nbana, 87_WaterYam-Mbana, 60_D1WaterYam-Nbana 1,
92_ChineseYam-TDes, 51_Alata2, 34_AdakavarietyIITA, and 65_WaterYam-Nbana
that grouped together with D. alata retrieved from NCBI database with an accession number of HQ637868. Group VII with PD value in the range of 18-86, had
nine
accessions
including
6_TDb2857,
4_TDb3050,
5_TDb3044,
83_WaterYam-_Mbana, 85_AerialYam- Edugbe, 8_TDb3690, 61_6-Edo, 3_TDa3050
and TDb3058 grouped together with a known D. bulbifera species (with an accession No: KR072458) that was retrieved from NCBI database. Yam accessions
28_TDes3033, 30_TDes3030, 31_TDesculenta, 27_TDes3035 and 29_TDes3027
were in the same group VIII (PD = 17 - 51) identified as D. esculenta using a
reference of D. esculenta (KR072467) obtained from the NCBI database. In
group IX (PD = 2 - 60), 86_3leavedYam-Ona, 91_TrifoliateYam-TDd, 53_Ighu,
52_Ighu-Dumenturum, 9_TDd3101, 12_TDd08-38-53, 14_TDd3100, 49_IghuUna,
84_BitterYam-Iwu-obe, 11_TDd3935, 13_TDd-yellow, 10_TDd3829 and
54_Ighu-Una-2 were found grouping with D. hispicia (HQ637815), D. dregeana
(JQ025042) and D. dumenturum (JF705531). Group X with PD of 88 had only
62_3leavedYam-Ono and 76_Ona-TDd, while outgroups (PD = 89 - 100) contained two Ipomoea triloba (trilobed (white potatoes), Colocasia esculenta (taro)
(cocoyam) and Coccinia quinqueloba (96_unknown_sample) grouped together
with Solanum vermiculata and S. lycopersicum with NCBI accession numbers
KR057204 and KM008705, respectively.
3.3. Genetic Diversity Analysis
The analysis involved 75 nucleotide sequences between different groups. The
highest inter-group genetic distance calculated based on K2P was 5.0560 ±
2.5760, while the lowest was 0.5000 ± 0.4770 (Table 2). The increment in genetic
diversity started from the group combinations in ascending order: 0.5000 ±
0.4770 (groups, gps: I and II, I and III, I and IV, I and V) < 0.6700 ± 0.5500 (gps:
II and VI, III and VI, V and VI) < 0.7510 ± 0.4240 (gps: II and IX) < 0.8100 ±
0.5500 (gps: III and IX, V and IX) < 0.8820 ± 0.5550 (gps: I and VI) < 0.9210 ±
0.4970 (gps: I and IX) < 1.0090 ± 0.9870 (gps: IV and VI) < 1.1390 ± 0.9170 (gps:
IV and IX) < 1.2540 ± 0.6540 (gps: II and VIII) < 1.2770 ± 0.6800 (gps: III and
VIII) < 1.3810 ± 0.8090 (gps: IV and VIII) < 1.5090 ± 1.4360 (gps: VII and VIII)
< 1.5350 ± 0.8200 (gps: I and VIII) < 1.5820 ± 0.8660 (gps: VI and VIII) < 1.9060
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Table 2. Genetic distances based on Kimura 2-parameter (K2P) between different groups
of yam species.
DOI: 10.4236/ajps.2019.101015
Species 1
Species 2
Distance
Standard error
Group I
Group II
0.500
0.477
Group I
Group III
0.500
0.477
Group I
Group IV
0.500
0.477
Group I
Group V
0.500
0.477
Group I
Group VI
0.882
0.555
Group I
Group VII
3.020
1.704
Group I
Group VIII
1.535
0.820
Group I
Group IX
0.921
0.497
Group II
Group III
n/c
NC
Group II
Group IV
n/c
NC
Group II
Group V
n/c
NC
Group II
Group VI
0.670
0.550
Group II
Group VII
3.020
1.700
Group II
Group VIII
1.254
0.654
Group II
Group IX
0.751
0.424
Group III
Group IV
n/c
NC
Group III
Group V
n/c
NC
Group III
Group VI
0.670
0.550
Group III
Group VII
3.020
1.700
Group III
Group VIII
1.277
0.680
Group III
Group IX
0.810
0.550
Group IV
Group V
n/c
NC
Group IV
Group VI
1.009
0.987
Group IV
Group VII
3.020
1.700
Group IV
Group VIII
1.381
0.809
Group IV
Group IX
1.139
0.917
Group V
Group VI
0.670
0.550
Group V
Group VII
3.020
1.700
Group V
Group VIII
1.277
0.680
Group V
Group IX
0.810
0.550
Group VI
Group VII
5.056
2.576
Group VI
Group VIII
1.582
0.866
Group VI
Group IX
1.906
0.814
Group VI
Group X
2.276
1.792
Group VII
Group VIII
1.509
1.436
Group VII
Group IX
3.020
1.676
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Continued
Group IX
Group VIII
2.456
1.458
Group VII
Group X
3.107
2.390
Group IX
Group X
1.746
1.479
Group I
Group X
2.718
2.076
Group V
Group X
2.790
2.130
Group VIII
Group X
1.664
1.491
Group IV
Group X
2.790
2.130
Group II
Group X
2.790
2.130
Group III
Group X
2.790
2.130
Group I = 43_Gbangu_variety, 82_Yellowyam-Akpukpu, 81_Whiteyam-Nwopoko, 89_Whiteyam-Nwopoko,
24_TDm3052, 23_TDm3053, 20_TDc03-5, 19_TDc2792, 80_Utekpevariety, 17_TDc2813, 21_TDc04-71-2,
93_Yellowyam-TDes, 18_TDc2796, 68_9ENEGBE, 25_TDm3055, 15_TDc0471-2, 46_Oginivariety,
57_2-Whiteyam-Iyo, 45_Amolavariety, 40_TDa00.00.94, 38_TDr89.002665, 16_TDc0497-4, 78_Obella,
37_Ame and 35_Pepa; Group II = 47_Damieha, 48_Aloshivariety, 39_Alata TDa98-01176,
44_Obioturuguvariety; Group III = 42_Ogojavariety; Group IV = 36_Ke-emi; Group V = 22_TDm2938;
Group VI = 59_10-Whiteyam-Nwopoko-Adaka, 90_YellowYam-Oku, 33_TDaNwopoko, 41_TDa00.00600,
71_D1WaterYam-Nbana2, 1_TDa85.00250, 73_WaterYam-Mbala, 72_1WaterYam-_Nbana,
87_WaterYam-Mbana, 60_D1WaterYam-Nbana 1, 92_ChineseYam-TDes, 51_Alata2, 34_AdakavarietyIITA,
and 65_YaterYam-Nbana; Group VII = 6_TDb2857, 4_TDb3050, 5_TDb3044, 83_WaterYam-_Mbana,
85_AerialYam-Edugbe, 8_TDb3690, 61_6-Edo, 3_TDa3050 and TDb3058; Group VIII = 28_TDes3033,
30_TDes3030, 31_TDesculenta, 27_TDes3035 and 29_TDes3027; Group IX = 86_3leavedYam-Ona,
91_TrifoliateYam-TDd, 53_Ighu, 52_Ighu-Dumenturum, 9_TDd3101, 12_TDd08-38-53, 14_TDd3100,
49_IghuUna, 84_BitterYam-Iwu-obe, 11_TDd3935, 13_TDd-yellow, 10_TDd3829 and 54_Ighu-Una-2; and
Group X = 62_3leavedYam-Ono and 76_Ona-TDd, N/C = Not computable.
± 0.8140 (gps: VI and IX) < 1.6640 ± 1.4910 (gps: VIII and X) < 1.7460 ± 1.4790
(gps: 2.2760 ± 1.7920 (gps: VI and X) < 2.4560 ± 1.4580 (gps: VIII and IX) <
2.7180 ± 2.0760 (gps: I and X) < 2.7900 ± 2.1300 (gps: II and X, III and X, IV and
X, V and X) < 3.0200 ± 1.7000 (gps: I and VII, II and VII, III and VII, IV and
VII, V and VII, VII and IX) < 3.1070 ± 2.390 (gps: VII and X) < 5.0560 ± 2.5760
(gps: VI and VII). The intra-group genetic distance ranged from 0.5250 ± 0.5000
- 2.0103 ± 1.2579 and some intra-group genetic distances were not computable
which were denoted by n/c (Table 3). Groups I, VI and X were found computable with their respective values of 0.5250 ± 0.5000, 0.5616 ± 0.4788, and 2.0103
± 1.2579. The mean genetic diversity within entire population was 0.7970 ±
0.06910, while the transitional to transversional distances per site from mean interpopulational diversity calculations was 2.1478 × 108 ± 4.5300. Also, the coefficient of differentiation of transitional to transversional distances per site was
1.1947 × 108 ± 6.9419 × 107.
3.4. BLAST Analysis of the Sequences Generated from the Yam
Accessions Using rbcL Barcoding Gene
The output of the BLAST computations of the grouped sequences produced significant hits and some of the previously unknown sequences were fully identified
(Table 4). The analysis identified ten putative species of yams including Diosco-
rea alata, D. bulbifera, D. cayenensis, D. rotundata, D. wallichii, D. aspersa,
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Table 3. Genetic distances based on Kimura 2-parameter (K2P) within different groups of yam species.
Group name
Distance
Standard error
Group I
0.5250
0.5000
Group II
n/c
n/c
Group III
n/c
n/c
Group IV
n/c
n/c
Group V
n/c
n/c
Group VI
0.5616
0.4788
Group VII
n/c
n/c
Group II
n/c
n/c
Group III
n/c
n/c
Group X
2.0103
1.2579
Group I = 43_Gbangu_variety, 82_Yellowyam-Akpukpu, 81_Whiteyam-Nwopoko, 89_Whiteyam-Nwopoko, 24_TDm3052, 23_TDm3053, 20_TDc03-5,
19_TDc2792, 80_Utekpevariety, 17_TDc2813, 21_TDc04-71-2, 93_Yellowyam-TDes, 18_TDc2796, 68_9ENEGBE, 25_TDm3055, 15_TDc0471-2,
46_Oginivariety, 57_2-Whiteyam-Iyo, 45_Amolavariety, 40_TDa00.00.94, 38_TDr89.002665, 16_TDc0497-4, 78_Obella, 37_Ame and 35_Pepa; Group II =
47_Damieha, 48_Aloshivariety, 39_Alata TDa98-01176, 44_Obioturuguvariety; Group III = 42_Ogojavariety; Group IV = 36_Ke-emi; Group V =
22_TDm2938; Group VI = 59_10-Whiteyam-Nwopoko-Adaka, 90_YellowYam-Oku, 33_TDaNwopoko, 41_TDa00.00600, 71_D1WaterYam-Nbana2,
1_TDa85.00250, 73_WaterYam-Mbala, 72_1WaterYam-_Nbana, 87_WaterYam-Mbana, 60_D1WaterYam-Nbana 1, 92_ChineseYam-TDes, 51_Alata2,
34_AdakavarietyIITA, and 65_YaterYam-Nbana; Group VII = 6_TDb2857, 4_TDb3050, 5_TDb3044, 83_WaterYam-_Mbana, 85_AerialYam-Edugbe,
8_TDb3690, 61_6-Edo, 3_TDa3050 and TDb3058; Group VIII = 28_TDes3033, 30_TDes3030, 31_TDesculenta, 27_TDes3035 and 29_TDes3027; Group IX
= 86_3leavedYam-Ona, 91_TrifoliateYam-TDd, 53_Ighu, 52_Ighu-Dumenturum, 9_TDd3101, 12_TDd08-38-53, 14_TDd3100, 49_IghuUna,
84_BitterYam-Iwu-obe, 11_TDd3935, 13_TDd-yellow, 10_TDd3829 and 54_Ighu-Una-2; and Group X = 62_3leavedYam-Ono and 76_Ona-TDd.
Table 4. BLAST outputs of total score, query coverage, e-value, percentage identity and accession number obtained from different
yam accessions.
Sequence name
Hit in NCBI database
Total score Query coverage
E-value
%Identity
Accession No
Dioscorea alata
852
852
0
100
KY710782
3_TDa3050
D. bulbifera
736
736
0
100
KR087030
4_TDb3050
D. bulbifera
771
100
0
100
KR087030
5_TDb3044
D. bulbifera
756
100
0
100
KR087030
6_TDb2857
D. bulbifera
839
100
0
100
KR087030
7_TDb3058
D. bulbifera
826
100
0
99
KR087030
8_TDb3690
D. bulbifera
737
100
0
100
KR087030
9_TDd3101
D. dregeana
1009
100
0
100
KR087039
10_TDd3829
D. dregeana
985
100
0
100
KR087039.1
11_TDd3935
D. dregeana
996
100
0
100
KR087039.1
12_TDd08-38-53
D. dregeana
1005
100
0
100
KR087039.1
13_TDdYellow
D. dregeana
996
100
0
100
KR087039.1
14_TDd3100
D. dregeana
1003
100
0
100
KR087039.1
15_TDc04-71-2
D. wallichii
835
100
0
100
MF142259.1
16_TDc04-97-4
D. cayenensis/rotundata
1005
100
0
100
KJ629254.1/
KJ490011.1
D. rotundata
743
100
0
100
KY679568.1
1_TDa85.00250
17_TDc2813
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Continued
18_TDc2796
D. rotundata
715
100
0
100
KY679568.1
19_TDc2792
D. wallichii/rotundata
739
100
0
100
MF142259.1/
KY679568.1
20_TDc03-5
D. rotundata
739
100
0
100
KY679568.1
21_TDc04-71-2
D. rotundata
758
100
0
100
KY679568.1
22_TDm2938
D. cayenensis/rotundata
1002
100
0
100
KJ629254.1/
KJ490011.1
23_TDm3053
D. rotundata
739
100
0
100
KY679568.1
24_TDm3052
D. rotundata
739
100
0
100
KY679568.1
25_TDm3055
D. wallichii/rotundata
824
100
0
100
MF142259.1/
KY679568.1
27_TDes3035
D. esculenta
941
100
0
100
KJ956696.1
28_TDes3033
D. esculenta
828
100
0
100
KJ956696.1
29_TDes3027
D. esculenta
998
100
0
100
KJ956696.1
30_TDes3030
D. esculenta
736
100
0
100
KJ956696.1
31_TDesculenta
D. esculenta
734
100
0
100
KJ956696.1
33_TDaNwopoko
D. alata
1003
100
0
100
KY710782.1
34_Adakavariety.IITA
D. alata
1000
100
0
100
KY710782.1
35_Pepa
D. cayenensis/rotundata
1003
100
0
100
KJ629254.1/
KJ490011.1
36_Ke-emi
D. cayenensis/rotundata
981
100
0
100
KJ629254.1/
KJ490011.1
37_Ame
D. cayenensis/rotundata
1000
100
0
100
KJ629254.1/
KJ490011.1
38_TDr89.002665
D. cayenensis/rotundata
1005
100
0
100
KJ629254.1/
KJ490011.1
39_AlataTda_98.01176
D. cayenensis/rotundata
1007
100
0
100
KJ629254.1/
KJ490011.1
40_TDa00.00.94
D. cayenensis/rotundata
1005
100
0
100
KJ629254.1/
KJ490011.1
41_Tda00.00600
D. rotundata
992
100
0
100
KY710782.1
D. cayenensis/rotundata
998
100
0
100
KJ629254.1/
KJ490011.1
D. praehensilis/cayennensis/
rotundata
955
100
0
99
KR072476.1/
KJ629254.1/
KJ490011.1
44_ObioturuguVariety.1
D. cayenensis/rotundata
1011
100
0
100
KJ629254.1/
KJ490011.1
45_AmolaVariety.1
D. cayenensis/rotundata
1005
100
0
100
KJ629254.1/
KJ490011.1
D. wallichii/rotundata
857
100
0
100
MF142259.1/
KY679568.1
D. cayenensis/rotundata
1005
100
0
100
KJ629254.1/
KJ490011.1
42_OgojaVariety.1
43_GbanguVariety.1
46_OginiVariety
47_Damieha
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Continued
D. cayenensis/rotundata
1007
100
0
100
KJ629254.1/
KJ490011.1
D. dregeana/hispida
992
100
0
99
KR087039/
HQ637815.1
D. alata
736
100
0
100
KY710782.1
D. hispida
774
100
0
100
KY710783.1
53_Ighu
D. dumetorum/hispida
830
100
0
100
KY710783.1
54_IghuUna.2
D. hispida/dumetorum
756
100
0
100
KY710783.1
D. rotundata
872
100
0
100
KR072483.1
48_Aloshivariety.1
49_IghuUna
51_Alata.2
52_Ighu_Dumenturum
57_2-WhiteYam-_Iyo
D. spicata/intermedia/wallichii/
rotundata/oppositiflia
534
100
4.00E−148
100
KY457460.1/
KY457459.1/
KY679569.1/
KY679568.1/
KY679566.1
D. alata
778
100
0
100
KY710782.1
D. bulbifera
737
100
0
100
KR087030.1
62_3LeavedYam-Ono
D. aspersa/petelotii/daunea
665
99
0
97
HQ637816.1/
AY904802.1/
AY904793.1
65_WaterYam.Nbana
D. alata
730
100
0
100
KY710782.1
D. rotundata
822
100
0
100
KR072483.1
71_D1WaterYam-Nbana2
D. alata
989
100
0
100
KY710782.1
72_1-WaterYam-_Nbana
D. alata
798
100
0
100
KY710782.1
73_Wateryamji_mbala
D. alata
852
100
0
100
KY710782.1
D. aspersa
612
100
2.00E−171
97
HQ637816.1
D. cayenensis/rotundata
992
100
0
100
KJ629254.1/
KJ490011.1
D. wallichii/rotundata
741
100
0
100
MF142259.1/
KY679568.1
81_WhiteYam-Nwoopoko
D. rotundata
737
100
0
100
KR072483.1
82_Yellowyam_Akpukpu
D. rotundata
806
100
0
100
KR072483.1
83_WaterYam-_Mbuna
D. bulbifera
750
100
0
100
KR087030.1
84_BitterYam-Iwu_obe
D. dregeana/hispida
998
100
0
100
KR087039/
HQ637815.1
85_AerialYam_Edugbe
D. bulbifera
739
100
0
100
KR087030.1
86_3LeavedYam_Ona
D. hispida
861
100
0
100
KU865503.1
87_WaterYam-Mbana
D. alata
782
100
0
100
KY710782.1
D. rotundata
739
100
0
100
KR072483.1
D. alata
963
100
0
100
KY710782.1
91_TrifoliateYam_TDd
D. hispida
837
100
0
100
KU865503.1
92_ChineseYam_TDes
D. alata
739
100
0
100
KY710782.1
93_YellowYam_TDes
D. rotundata
697
100
0
100
KY710782.1
59_10-WhiteNwopoko-Adaka
60_D1Water-Nbana1
61_6-EDO
68_9ENEGBE
76_OnaTDd
78_Obella
80_UtekpeVariety_2
89_WhiteYam_Nwoopoko
90_Yellowyam_Oku
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D. trifida, D. dregeana, and D. mangenotiana. The total bit score obtained in all
ranged from 411 - 1011. The query coverage spanned between 99 and 100%,
while the expected values (e-values) were 9e-111 or less. The percentage sequence
identity ranged from 97% - 100%. Some accessions with acronyms including
TDa, TDc and TDm denoting D. alata, D. cayenensis and D. manganotiana were
found to be D. bulbifera, D. rotundata or cayenensis, respectively. Some of the
sequences had NCBI hits ranging from two to four sequences with synonymous
values of total bit score, query coverage, e-value, percentage identity but different accession numbers. For instance, 16_TDc04-97-4, 22_TDm2938, 35_Pepa,
36_Ke-emi, 37_Ame, 38_TDr.89.002665 and many others in this category had
hits of D. cayenensis and D. rotundata. For accessions of 19_TDc2792,
25_TDm3055, 46_OginiVariety and 80_UtekpeVariety_2 had D. wallichii and D.
rotundata as their hits with similar values in all the BLAST indices. Also, three
species of yam including D. praehensilis, D. cayenensis and D. rotundata were
obtained with a yam accession of 43_Gbangu_Variety.1 in the process of BLAST
analysis, while 62_3LeavedYam-Ono produced D. aspersa, D. petelotii and D.
daunea that had same values of total bit score, query coverage, e-value, percentage identity but different accession numbers. The yam accession, 59_D10
White-Nwopoko-Adaka, had five different NCBI hits of D. spicata, D. intermedia, D. wallichii, D. rotundata and D. oppositifolia with three having similar accession number, while the remaining two had a separate accession number as
revealed by BLAST analysis.
4. Discussion
DNA barcoding has become an effective method for species discrimination of
flowering plants in the Polygonaceae [35] [47] and Fabaceae families [39], and
other land plant species [35] [42] [48] [49]. While mitochondrial cytochrome
oxidase 1 (CO1) has proven a standardized animal DNA barcoding for necessary
discrimination, no single barcode sequence works across all plants [49]. In the
present work, the candidate barcoding marker, rbcL satisfied the DNA barcoding process, regarding the ease of amplification and sequencing Hollingsworth et
al. [49]. However, this barcoding marker, rbcL, was not able to achieve the basic
quality of discriminating different yam species in this study. Sequence alignment
showed low degree of polymorphisms among the sequences. This study of genetic diversity in yam accessions is also dependent on the nucleotide variations
occurring within the genome that are informative for the identification of different species. The discriminatory level of the rbcL marker has been linked to
other researches, which contradict its potential for use as a universal DNA barcode for plants [50] [51] [52]. This low resolution of different accessions of yams
into their respective species level could be attributed to the poor efficiency of
rbcL marker when not jointly applied with other plastid markers. It has been
reported that the joint application of rbcL+matK as a marker of choice in species
resolution was based on clear recovery of the region of rbcL and discriminatory
efficiency of fast evolving coding region of matK [53].
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In this study, 525 bp distinct total lengths of sequence alignment, 534 conserved sites, and variable sites of 7 were identified in the sequenced yam species.
The alignment of 525 bp out of the total lengths of 568 bp, followed by the existence of similar regions (conserved sites) and low points of variations (variable
areas) among the sequences demonstrate the low level of informativeness of rbcL
in DNA barcoding of yam species. These findings are not in complete agreement
with a previous report on yam species [30], where 568 bp, 538, and 30 as total
lengths of sequence alignment, conserved sites and variable sites were identified
among accessions. Also, the sequence alignment length, conserved sites excluding the variable sites detected in this work correlate with the findings of Sun et
al. [54] in which 553 bp, 522 bp and 31 of alignment length, conserved sites and
variable sites were found among the accessions of Dioscorea species. The difference in the variable sites could have emanated from the number of samples
studied.
Phylogenetic reconstruction of the generated Dioscorea species using rbcL
marker resolved them into ten groups and this indicates different existing isolated groups inherent in the accessions. The existence of these different accessions among the collections could be attributable to lack of exchange of yam tubers by farmers among villages thereby resulting in a stronger heterozygosity
among species compared to wild ones as reported by Ngo Ngwe et al. [24]. A
contribution of evolutionary biology regarding conservation is the knowledge of
diverse phylogenetic diversities, defined by the sums of branch lengths of the
evolutionary trees connecting a set of taxa or individuals [55]. In this present
work, group X had the highest PD value of 88, followed by groups VII, VI, IX,
and VIII with their respective PDs of 86, 79, 60 and 51. The highest PD was
identified in a group containing wild species of D. aspersa and this is in agreement with a previous report though in a different wild species wild called D.
praehensilis [24]. When compared with other unrelated crops, the highest was
observed in Cocoyam and other crops which were deliberately included to access
the accuracy of this marker. The group with the lowest PD value D. rotundata
clustered with other species and they were collected from a given single region.
In this way, a given set of taxa will have a greater PD if they are widely spread
out on a phylogenetic tree. Lack of or total loss of PD is generally assumed as a
declining signal in the degree of biodiversity [56]. Furthermore, PD is associated
with functional diversity since it is a measure of features also due to the fact that
evolutionarily distant species are more likely to possess variable molecular functions in an ecosystem [28] [57] [58]. Also in group I, some accessions including
45_Amolavariety, 40_TDa00.00.94, 38_TDr89.002665, 16_TDc0497.4, 78_Obella,
37_Ame, and 35_Pepa had a PD value of 0 and this could be attributed to lack of
sequence divergence. It could also be attributable to occurrence of common ancestral sequence homology [59] or poor resolving power of the rbcL DNA barcoding marker in yams [54]. Most of the accessions were accurately grouped according their species. For instance in group VIII, all the D. esculenta species was
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grouped with a known reference sequence from NCBI database. Also, group VII
had all the accessions classified as D. bulbifera thereby identifying correctly an
accession, 3_TDa3050, which was regarded as D. alata. A particular yam species
was given different names as Ighu or Una (Ona) at a village in Enugu State but it
was found to be just one species called D. dumetorum through the use of rbcL
thereby resolving the issue of multiple names for the plant. Group IX had three
reference sequences as D. dregeana, D. hispida and D. dumetorum but most of
the accessions in the group are D. dumetorum and this could be as a result of
their genetic relatedness [59]. However, accessions in group I of the trees were
not correctly resolved following the existence of different yam species in various
distinct subclades and non-grouping of any of the retrieved yam sequences from
NCBI database. This may possibly be linked to sample contamination or a deficiency on the part of the rbcL resolution.
The identified genetic distances (0.5000 ± 0.4770 - 5.0560 ± 2.5760) based on
K2P model regarding the inter-groups were in agreement with the previous
works of other researchers in yams [24] [54] and in authentication of native
plants [60]. High genetic diversity indices were obtained from between group
calculations, producing 5.0560 ± 2.5760 with the highest in two combined
groups (groups VI and VII) and this demonstrates higher interspecific diversity
than intraspecific one within the yam accessions as obtained in an earlier report
involving ornamental plants with interspecific value of 3.080 [61]. Assessment of
genetic diversity within the groups (intra-group genetic diversity) could not be
computed in most of the groups except three groups (groups I, VI and X), where
group I had the lowest value of 0.5250 ± 0.5000, while X had the highest value of
2.0103 ± 1.2579. These values are higher than the ones obtained by Sun et al.
[54]. The mean genetic diversity within entire population was 0.7970 ± 0.06910
and this is higher than the one (0.00266 ± 0.0044) obtained by Sun et al. [54].
BLAST hits obtained in this study showed some degrees of similarity matches
to the ones already annotated and deposited in NCBI database and some were
not purely specific. The percentage sequence identity ranged from 97% - 100%,
demonstrating low efficiency of this tool in identification of unknowns in yam
species. However, some of the yams sampled from different regions were differently identified from what they were previously known to be using this method,
indicating the potential of rbcL barcoding marker to resolve misclassification
encountered via morphotaxonomy based approach despite the low discriminatory power. For instance, yam accessions with acronyms including TDa, TDc
and TDm denoting D. alata, D. cayenensis and D. manganotiana were found to
be D. bulbifera, D. rotundata or cayenensis, respectively. Furthermore,
62_3LeavedYam-Ono and 76_Ona_TDd sequences were correctly identified as
D. aspersa. In the community where the two species (D. aspersa and D. dumetorum) were collected, they were misclassified by the villagers who generally
called them D. dumetorum due to their similar morphological features. According to the villagers, the ones in group X which were later identified as D. aspersa
are normally boiled and eaten directly, while the other ones (D. dumetorum,
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which had similar values of NCBI hits with D. hispida) are usually boiled, processed to remove bitterness in them before they are consumed. The discriminatory level of the rbcL marker in plants as a potential universal DNA barcode is
demonstrated in this study as reported in other researches [50] [51]. However,
some of the yam sequences had two, three or five NCBI hits of different species
of yams with synonymous values of total bit score, query coverage, -value and
percentage identity with different accession numbers except in one that had five
BLAST outputs with three having similar accession numbers and two with different accession numbers. For instance, the yam accession, 59_D-10-WhiteNwopoko-Adaka, had five different NCBI hits of D. spicata, D. intermedia, D.
wallichii, D. rotundata and D. oppositifolia with three (D. wallichii, D. rotundata, D. oppositifolia) having similar accession number (KY679569), while the remaining two (D. spicata and D. intermedia) had separate accession numbers of
KY457460 and KY457459, respectively, after the BLAST analysis. Also, sequences generated from accessions 45_Amolavariety, 40_TDa00.00.94,
38_TDr89.002665, 16_TDc0497.4, 78_Obella, 37_Ame, and 35_Pepa hit two (D.
cayenensis and D. rotundata) sequences with similar values of query coverage,
e-value and percentage identity, while total bit score ranged from 1000-1005.
This is possible due to existence of common ancestral homology as opined by
Pearson [59] or due to redundancy, which in bioinformatics is observed when
one or more homologous or synonymous sequences are found in the same set of
data [62]. It could also be attributable to the low discriminatory potency of rbcL
marker to correctly resolve species as previously reported in yams [54] and ornamental plants [61].
5. Conclusion
The candidate barcoding marker, rbcL, was found to be ambiguously discriminatory in DNA barcoding process of yam accessions. Some of the accessions
were not correctly identified to the species level and low polymorphisms were
detected and this further demonstrates the low distinguishing potency of rbcL
barcoding marker. The use of phylogenetic diversity (PD), which is associated
with functionality in biodiversity and which was applied in the computational
processes for the estimation of phylogenetic groups with lowest and largest collections in terms of diversity was of great potential. The highest phylogenetic diversity was in D. aspersa, while some were not computable due to the low efficacy of the marker. The group with the lowest PD value, D. rotundata clustered
with other indistinguishable species and they were collected from a given single
region. The accessions with high PD within the yam accessions should be considered for use in breeding programme to enhance biodiversity of Dioscorea
species within the studied region. However, the rbcL could not resolve the yam
accessions well following some noted discrepancies in the detected number of
species from phylogenetic groupings and NCBI BLAST hits possibly due to inefficiency of the marker. Therefore, the rbcL may not be a marker of choice for
species identification, discrimination and estimation of genetic diversity of yam
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accessions. The marker should be used in combination with other chloroplast
markers for accurate DNA barcoding of yams for their improvement and germplasm conservation.
Acknowledgements
The authors are grateful to International Institute of Tropical Agriculture
(IITA), Ibadan for providing part of the accessions used in the study. We thank
National Science Foundation (NSF) for the Targeted Infusion HBCU_UP funding that supported this undergraduate student’s research project. We are also
grateful to Dr. Dave Micklos of the Cold Spring Harbor Laboratory, DNA
Learning Centre, for the technical and research assistance offered to us.
Funding
National Science Foundation (NSF) for the Targeted Infusion HBCU_UP funding was received to conduct this study
Ethics Approval and Consent to Participate
Consent was obtained from farmers before using their individual farms for sample collection.
Consent for Publication
Not applicable.
Availability of Data and Materials
All data generated during this study are included in this published article. Sequence data were deposited in NCBI GenBank with accession numbers ranging
from MH078114 to MH078188 to match the individual yam accessions in the list
of supplementary Table S1.
Authors’ Contributions
All authors were involved in project design. GNU, DOI, JM, OO, JH, DB, CA
and OC did the literature search process, extracted data elements, and carried
out study compilation. Data analyses were performed by DOI, MO, CE, VC, MU
and CO and reviewed by GNU, GA, JO and AD. DOI developed the first draft of
the manuscript. All authors read the manuscript and approved the final copy of it.
Conflicts of Interest
The authors declare that they have no competing interests.
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Supplementary File 1
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Figure S1. Consensus sequence of rbcL gene for Dioscorea species and its associated consensus points of polymorphisms (variations). Note that the dotted line (…) in the sequence alignment indicates similarity of nucleotide to the nucleotide of
TDa-85.00250 that serves as a reference sequence of TDa-85.00250.
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Supplementary File 2
Table S1. List of sequenced yam species collected from different locations and their GenBank accession numbers.
Sample IDs
Location
LGA
State
GenBank No
1_TDa85.00250
IITA
Akinyele
Oyo
MH078115
3_TDa3050
IITA
Akinyele
Oyo
MH078154
4_TDb3050
IITA
Akinyele
Oyo
MH078155
5_TDb3044
IITA
Akinyele
Oyo
MH078156
6_TDb2857
IITA
Akinyele
Oyo
MH078157
7_TDb3058
IITA
Akinyele
Oyo
MH078158
8_TDb3690
IITA
Akinyele
Oyo
MH078159
9_TDd3101
IITA
Akinyele
Oyo
MH078163
10_TDd3829
IITA
Akinyele
Oyo
MH078164
11_TDd3935
IITA
Akinyele
Oyo
MH078165
12_TDd08-38-53
IITA
Akinyele
Oyo
MH078166
13_TDdYellow
IITA
Akinyele
Oyo
MH078167
14_TDd3100
IITA
Akinyele
Oyo
MH078168
15_TDc0471-2
IITA
Akinyele
Oyo
MH078170
16_TDc0497-4
IITA
Akinyele
Oyo
MH078127
17_TDc2813
IITA
Akinyele
Oyo
MH078141
18_TDc2796
IITA
Akinyele
Oyo
MH078142
19_TDc2792
IITA
Akinyele
Oyo
MH078171
20_TDc03-5
IITA
Akinyele
Oyo
MH078143
21_TDc04-71-2
IITA
Akinyele
Oyo
MH078144
22_TDm2938
IITA
Akinyele
Oyo
MH078128
23_TDm3053
IITA
Akinyele
Oyo
MH078145
24_TDm3052
IITA
Akinyele
Oyo
MH078146
25_TDm3055
IITA
Akinyele
Oyo
MH078172
27_TDes3035
IITA
Akinyele
Oyo
MH078175
28_TDes3033
IITA
Akinyele
Oyo
MH078176
29_TDes 3027
IITA
Akinyele
Oyo
MH078177
30_TDes 3030
IITA
Akinyele
Oyo
MH078178
31_TDesculenta
IITA
Akinyele
Oyo
MH078179
33_TDaNwokporo
IITA
Akinyele
Oyo
MH078116
34_Adakavariety
IITA
Akinyele
Oyo
MH078117
35_Pepa
IITA
Akinyele
Oyo
MH078129
36_Ke-emi
IITA
Akinyele
Oyo
MH078130
37_Ame
IITA
Akinyele
Oyo
MH078131
38_TDr 89.002665
IITA
Akinyele
Oyo
MH078132
39_AlataTda 98.01176
IITA
Akinyele
Oyo
MH078133
40_TDa00.00.94 41_Alata
IITA
Akinyele
Oyo
MH078134
DOI: 10.4236/ajps.2019.101015
206
American Journal of Plant Sciences
G. N. Ude et al.
Continued
41_Tda00.00600
IITA
Akinyele
Oyo
MH078147
42_OgojaVariety.1
IITA
Akinyele
Oyo
MH078135
43_Gbangu_Variety.1
IITA
Akinyele
Oyo
MH078188
44_ObioturuguVariety.1
IITA
Akinyele
Oyo
MH078136
45_AmolaVariety .1
IITA
Akinyele
Oyo
MH078137
46_OginiVariety
IITA
Akinyele
Oyo
MH078173
47_Damieha
IITA
Akinyele
Oyo
MH078138
48_Aloshivariety.1
IITA
Akinyele
Oyo
MH078139
49_IghuUna
Osonu
Ezeagu
Enugu
MH078184
51_Alata2
Osonu
Ezeagu
Enugu
MH078118
52_Ighu_Dumenturum
Osonu
Ezeagu
Enugu
MH078185
53_Ighu
Osonu
Ezeagu
Enugu
MH078182
54_IghuUna.2
Osonu
Ezeagu
Enugu
MH078183
Ukaka Ngwo
Enugu North
Enugu
MH078148
57_2-WhiteYam- Iyo
59_D10WhiteNwopoko-Adaka
Agbalenyi Nachi
Oji-River
Enugu
MH078114
60_D1Water-Nbana1
Agbalenyi Nachi
Oji-River
Enugu
MH078119
61-6- EDO
Ukaka Ngwo
Enugu North
Enugu
MH078160
62_3LeavedYam-Ono
Ukaka Ngwo
Enugu North
Enugu
MH078180
65_WaterYam.Nbana
Ukaka Ngwo
Enugu North
Enugu
MH078120
Ndibinagu Umuaga
Udi
Enugu
MH078149
71_D1WaterYam-Nbana2
Agbalenyi Nachi
Oji-River
Enugu
MH078121
72_1-Water_Yam-_Nbana
Ndibinagu Umuaga
Udi
Enugu
MH078122
Nkalagu
Ishielu
Ebonyi
MH078123
76_OnaTDd
Ezzamgbo
Ohaukwu
Ebonyi
MH078181
78_Obella
Ezzamgbo
Ohaukwu
Ebonyi
MH078140
80_UtekpeVariety_2
Ezzamgbo
Ohaukwu
Ebonyi
MH078174
81_WhiteYam-Nw-opoko
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
MH078150
82_Yellowyam_Akpukpu
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
MH078151
83_WaterYam- Mbuna
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
MH078161
84_BitterYam-Iwu_obe
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
MH078169
85_AerialYam_Edugbe
Amaeke Amaigbo Ozalla
Nkanu West
Enugu
MH078162
86_3LeavedYam_Ona
Ede Oballa
Nsukka
Enugu
MH078186
87_WaterYam-Mbana
Nru
Nsukka
Enugu
MH078124
89_WhiteYam _Nwopoko
Ibagwa Aka
Igboeze South
Enugu
MH078152
90_Yellowyam_Oku
Ihe Owerre
Nsukka
Enugu
MH078125
91_TrifoliateYam_TDb
Ukana
Udi
Enugu
MH078187
92_ChineseYam_TDes
Ukana
Udi
Enugu
MH078126
93_YellowYam_TDes
Ukana
Udi
Enugu
MH078153
68_9ENEGBE
73_Water yamji_mbala
IITA = International Institute of Tropical Agriculture; LGA = Local Government Authority.
DOI: 10.4236/ajps.2019.101015
207
American Journal of Plant Sciences