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-1,5-bisphosphate carboxylase (<i> rbc</i> L) 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 <i>rbc</i>L 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 discriminating the yam accessions. It was demonstrated that <i>rbc</i>L 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.
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 [
The crop ranks fourth after potato, sweet potato and cassava as the most important food tuber crop in the world [
Yams are widespread in the tropics and subtropics. Nigeria is the leading producer of yam with 71% of the world production [
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 [
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 [
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 (
Sample IDs | 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 |
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 |
57_2-WhiteYam-Iyo | 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 |
68_9ENEGBE | Ndibinagu Umuaga | Udi | Enugu |
71_D1WaterYam-Nbana2 | Agbalenyi Nachi | Oji-River | Enugu |
72_1-Water_Yam-_ Nbana | Ndibinagu Umuaga | Udi | Enugu |
73_Water yamji_mbala | 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 |
IITA = International Institute of Tropical Agriculture; LGA = Local Government Area.
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 (
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) [
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.
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.
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 [
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:
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) (
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.
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 (
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 |
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 (
The output of the BLAST computations of the grouped sequences produced significant hits and some of the previously unknown sequences were fully identified (
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.
Sequence name | Hit in NCBI database | Total score | Query coverage | E-value | %Identity | Accession No |
---|---|---|---|---|---|---|
1_TDa85.00250 | 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 |
17_TDc2813 | D. rotundata | 743 | 100 | 0 | 100 | KY679568.1 |
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 |
42_OgojaVariety.1 | D. cayenensis/rotundata | 998 | 100 | 0 | 100 | KJ629254.1/ KJ490011.1 |
43_GbanguVariety.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 |
46_OginiVariety | D. wallichii/rotundata | 857 | 100 | 0 | 100 | MF142259.1/ KY679568.1 |
47_Damieha | D. cayenensis/rotundata | 1005 | 100 | 0 | 100 | KJ629254.1/ KJ490011.1 |
48_Aloshivariety.1 | D. cayenensis/rotundata | 1007 | 100 | 0 | 100 | KJ629254.1/ KJ490011.1 |
---|---|---|---|---|---|---|
49_IghuUna | D. dregeana/hispida | 992 | 100 | 0 | 99 | KR087039/ HQ637815.1 |
51_Alata.2 | D. alata | 736 | 100 | 0 | 100 | KY710782.1 |
52_Ighu_Dumenturum | 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 |
57_2-WhiteYam-_Iyo | D. rotundata | 872 | 100 | 0 | 100 | KR072483.1 |
59_10-WhiteNwopoko-Adaka | D. spicata/intermedia/wallichii/ rotundata/oppositiflia | 534 | 100 | 4.00E−148 | 100 | KY457460.1/ KY457459.1/ KY679569.1/ KY679568.1/ KY679566.1 |
60_D1Water-Nbana1 | D. alata | 778 | 100 | 0 | 100 | KY710782.1 |
61_6-EDO | 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 |
68_9ENEGBE | 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 |
76_OnaTDd | D. aspersa | 612 | 100 | 2.00E−171 | 97 | HQ637816.1 |
78_Obella | D. cayenensis/rotundata | 992 | 100 | 0 | 100 | KJ629254.1/ KJ490011.1 |
80_UtekpeVariety_2 | 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 |
89_WhiteYam_Nwoopoko | D. rotundata | 739 | 100 | 0 | 100 | KR072483.1 |
90_Yellowyam_Oku | 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 |
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.
DNA barcoding has become an effective method for species discrimination of flowering plants in the Polygonaceae [
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 [
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. [
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 [
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, 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 [
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 accessions. The marker should be used in combination with other chloroplast markers for accurate DNA barcoding of yams for their improvement and germplasm conservation.
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.
National Science Foundation (NSF) for the Targeted Infusion HBCU_UP funding was received to conduct this study
Consent was obtained from farmers before using their individual farms for sample collection.
Not applicable.
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
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.
The authors declare that they have no competing interests.
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
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 |
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 |
57_2-WhiteYam- Iyo | Ukaka Ngwo | Enugu North | Enugu | MH078148 |
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 |
68_9ENEGBE | Ndibinagu Umuaga | Udi | Enugu | MH078149 |
71_D1WaterYam-Nbana2 | Agbalenyi Nachi | Oji-River | Enugu | MH078121 |
72_1-Water_Yam-_Nbana | Ndibinagu Umuaga | Udi | Enugu | MH078122 |
73_Water yamji_mbala | 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 |
IITA = International Institute of Tropical Agriculture; LGA = Local Government Authority.