Abstract
This dataset illustrates the genetic diversity and evolutionary linkages of five underutilized Nigerian native legume species: Parkia biglobosa, Albizia lebbeck, Cassia fistula, Leucaena leucocephala, and Senna alata. These species are vital sources of sustenance, nutrition, revenue, and natural fertilizer, significantly contributing to sustainable agriculture. Nevertheless, limited knowledge exists regarding their genomes and their genetic diversity. We examined 22 high-quality chloroplast matK (Maturase K) gene sequences to assess the genetic divergence among the species and their phylogenetic relationships. The analysis of sequence alignment and diversity recorded 888 nucleotide positions, identified 10 segregating sites, 11 mutations, and 3 distinct haplotypes. Gene diversity was 0.654, whereas nucleotide diversity was negligible at 0.028. The analysis of molecular variance (AMOVA) indicated that 88.04% (p < 0.001) of genetic variation was shared among populations, whereas 11.96% was detected within populations. Employing the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm for phylogenetic analysis, the species examined were classified into unique evolutionary lineages along species lines with a minimal exception. This information serves as a valuable genetic resource for enhancing conservation, breeding initiatives, and the sustainable utilization of underutilized native legumes in tropical settings. It impacts food security, agroforestry, and environmental restoration.
Keywords: Genetic diversity, Phylogeny, Sustainable agriculture, Underutilized legumes
Specifications Table
| Subject | Biology |
| Specific subject area | Genetic Diversity, Phylogeny in underutilized legumes, Conservation, Legume Breeding and utilization. |
| Type of data | Raw analyzed, Tables, Charts, aures |
| Data collection | Fresh leaves samples of the five selected underutilized Nigerian native legumes were collected from the campus of Bowen University and its surroundings, located in Iwo, Osun State, Nigeria. The samples were immediately stored in air-tight zip-lock bags under cool conditions. Genomic DNA was amplified using PCR and Sanger sequencing. All accessions' matK gene sequences were analyzed using MEGAX, DnaSP v6.12.03, and Geneious Prime version 2024.2.1. |
| Data source location | Bowen University Campus and surroundings, Iwo, Osun State, Nigeria. Table 1 provides a summary of the collection data and GenBank information of the sequences. |
| Data accessibility | The following accession numbers correspond to the sequencing data that has been deposited in the NCBI GenBank database. Repository: GenBank, the NIH genetic sequence database. Data identification number: PV691468.1, PV691469.1, PV691470.1, PV691471.1, PV691472.1, PV691473.1, PV691474.1, PV691475.1, PV691476.1, PV691477.1, PV691478.1, PV691479.1, PV691480.1, PV691481.1, PV691482.1, PV691483.1, PV691484.1, PV691485.1, PV691486.1, PV691487.1, PV691488.1, PV691489.1 Direct URL to data: https://www.ncbi.nlm.nih.gov/nuccore/PV691468.1,PV691469.1,PV691470.1,PV691471.1,PV691472.1,PV691473.1,PV691474.1,PV691475.1,PV691476.1,PV691477.1,PV691478.1,PV691479.1,PV691480.1,PV691481.1,PV691482.1,PV691483.1,PV691484.1,PV691485.1,PV691486.1,PV691487.1,PV691488.1,PV691489.1 Popset Members: https://www.ncbi.nlm.nih.gov/nuccore?term=popset+representative+uid+2999182786[word] Guidelines for gaining access to these data: To find sequence identifiers and annotations using Entrez Nucleotide, click on the links above or conduct a GenBank search. |
| Related research article | None |
1. Value of the Data
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The dataset provides a reliable DNA barcoding library for precise species identification from every tissue sample.
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The dataset offers a comprehensive matK gene-based genetic diversity and phylogenetic assessment of five underutilized tropical legume species including Albizia lebbeck, Cassia fistula, Leucaena leucocephala, Parkia biglobosa, and Senna alata. The information generated can be used for research, improvement, utilization, and conservation of the species.
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Gene flow and genetic differentiation data suggest a high genetic divergence (Gst/Fst = 1) and no shared polymorphism (Hs = 0) for most species’ pairs, indicating that the taxa are genetically distinct.
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Comparisons including Parkia biglobosa, on the other hand, reveal common polymorphism and moderate divergence (Fst ∼ 0.2 – 0.5), suggesting either inadequate lineage sorting or closer evolutionary ties within this group of Fabaceae.
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The data can serve as a valuable reference for breeding programs and in agroforestry for initiatives seeking to take advantage of genetic variation and the ecological resilience of these selected underutilized legumes.
2. Background
The Fabaceae family consists of over 21,000 species and is among the most ecologically and economically significant plant families worldwide [1,2]. While widely studied legumes such as soybean (Glycine max) and common bean (Phaseolus vulgaris) have garnered significant attention, many underutilized species remain genetically underexplored, despite their potential to enhance food security, agroforestry, and environmental remediation [[3], [4], [5]]. Among these underutilized species are five important species, Albizia lebbeck, Cassia fistula, Leucaena leucocephala, Parkia biglobosa and Senna alata. These five species play critical roles in tropical ecosystems, including nitrogen fixation, soil improvement, and the provision of food and medicinal products [6,7]. These species, however, face increasing threats from habitat loss, overexploitation, and climate change, leading to genetic erosion [8]. The efforts to conserve these species are hindered by the scarcity of genomic resources. The maturase K (matK) gene which is a chloroplast gene is a valuable gene for studying genetic diversity due to its high evolutionary rate and ability to resolve closely related species [9]. The dataset's objectives are to discover intra and inter genetic diversity, gene flow and genetic differentiation and phylogenetic linkages, and accurately identify the selected five underutilized legumes using the matK gene towards genetic improvement, sustainable utilization and conservation.
3. Data Description
The dataset comprises 22 nucleotide sequences of five underutilized legume species, including Cassia fistula (4), Senna alata (5), Albizia lebbeck (5), Parkia biglobosa (4), and Leucaena leucocephala (4). Each sequence entry is represented by species name, accession number, collection sites, GenBank accession number, sequence length (bp), matched organism and percentage identity (Table 1). The complete set of sequences has been deposited in GenBank under accession numbers PV691468.1 – PV691489.1 and as presented in Table 1. Table 2 shows the general nucleotide compositions derived from the analyzed underutilized legume sequences. The statistics include the percentage T(U), C, A, G, total and mean sequence length. Table 3 shows genetic diversity parameters of the 22 sequences of underutilized legumes studied generated from the aligned dataset. These include polymorphic sites (S), total mutations (Eta), number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (Pi), Theta-W (per site), Theta (per site) from Eta, Theta (per site) from Pi (Finite Sites) and average nucleotide differences (k). Table 4 represent the within-population genetic diversity metrics for each of the five species. It further details sequence count, segregating sites, number of haplotypes, haplotype diversity, mean number of nucleotide differences, and nucleotide diversity. Table 5 summarizes the gene flow and genetic differentiation among five populations of the 22 sequences of underutilized legumes. Key parameters such as haplotype-based diversity (Hs), nucleotide sequence variation (Ks), average percentage of nucleotide changes between populations (Kxy), coefficient level of population divergence (GammaSt), haplotype frequency differences (Gst) among others were evaluated (Table 5). Table 6 provides the Analysis of Molecular Variance (AMOVA) results, listing the degrees of freedom, sum of squares, variance components, and percentage contributions of variation among and within populations. Fig. 2 shows the phylogenetic tree constructed from all 22 sequences with the clustering pattern and branch relationships among the five underutilized legume species analyzed. Table 7 provides the matrix from the test of homogeneity of substitution patterns between all pairs of sequences. Sequence identifiers (1–22) in Table 7 correspond to the GenBank accession numbers and species designations provided in Table 1.
Table 1.
Accession number, Collection sites, GenBank number, Sequence Length, matched organisms and % identity of the underutilized legumes used for this study.
| S/N | Species | Accession Number | Latitude | Longitude | GenBank Accession Number | Sequence Length (bp) | Matched Organism | % Identity |
|---|---|---|---|---|---|---|---|---|
| 1 | Cassia fistula | CF001 | 7.621865 | 4.207808 | PV691468.1 | 777 | Cassia fistula | 100 % |
| 2 | CF002 | 7.622114 | 4.190884 | PV691469.1 | 249 | Cassia fistula | 100 % | |
| 3 | CF003 | 7.619423 | 4.206458 | PV691470.1 | 588 | Cassia fistula | 100 % | |
| 4 | CF004 | 7.61967 | 4.206312 | PV691471.1 | 885 | Cassia fistula | 100 % | |
| 5 | Senna alata | SA001 | 7.620416 | 4.206403 | PV691472.1 | 843 | Senna alata | 100 % |
| 6 | SA002 | 7.619888 | 4.206447 | PV691473.1 | 791 | Senna alata | 100 % | |
| 7 | SA003 | 7.622114 | 4.190884 | PV691474.1 | 876 | Senna alata | 100 % | |
| 8 | SA004 | 7.622975 | 4.195276 | PV691475.1 | 705 | Senna alata | 100 % | |
| 9 | SA005 | 7.624211 | 4.194525 | PV691476.1 | 834 | Senna alata | 100 % | |
| 10 | Albizia lebbeck | AL001 | 7.624752 | 4.191486 | PV691477.1 | 857 | Albizia lebbeck | 100 % |
| 11 | AL002 | 7.623579 | 4.191949 | PV691478.1 | 882 | Albizia lebbeck | 100 % | |
| 12 | AL003 | 7.623826 | 4.193575 | PV691479.1 | 597 | Albizia lebbeck | 100 % | |
| 13 | AL004 | 7.622564 | 4.205882 | PV691480.1 | 848 | Albizia lebbeck | 100 % | |
| 14 | AL005 | 7.622957 | 4.202318 | PV691481.1 | 747 | Albizia lebbeck | 100 % | |
| 15 | Parkia biglobosa | PB001 | 7.62286 | 4.191115 | PV691482.1 | 844 | Parkia biglobosa | 100 % |
| 16 | PB002 | 7.619501 | 4.197404 | PV691483.1 | 606 | Parkia biglobosa | 100 % | |
| 17 | PB003 | 7.618655 | 4.197922 | PV691484.1 | 870 | Parkia biglobosa | 100 % | |
| 18 | PB004 | 7.622768 | 4.207621 | PV691485.1 | 876 | Parkia biglobosa | 100 % | |
| 19 | Leucaena leucocephala | LL001 | 7.623733 | 4.190481 | PV691486.1 | 854 | Leucaena leucocephala | 100 % |
| 20 | LL002 | 7.619926 | 4.196999 | PV691487.1 | 860 | Leucaena leucocephala | 100 % | |
| 21 | LL003 | 7.619426 | 4.197878 | PV691488.1 | 846 | Leucaena leucocephala | 100 % | |
| 22 | LL005 | 7.6187 | 4.198007 | PV691489.1 | 866 | Leucaena leucocephala | 100 % |
Table 2.
Nucleotide Composition of the 22 sequences of the underutilized legumes.
| Sequence | T(U) | C | A | G | Total |
|---|---|---|---|---|---|
| PV691468.1CF001 | 37.32 | 17.63 | 29.73 | 15.32 | 777 |
| PV691469.1CF002 | 40.56 | 16.47 | 28.51 | 14.46 | 249 |
| PV691470.1CF003 | 39.29 | 17.69 | 29.25 | 13.78 | 588 |
| PV691471.1CF004 | 37.06 | 17.29 | 30.28 | 15.37 | 885 |
| PV691472.1SA001 | 37.01 | 17.67 | 29.89 | 15.42 | 843 |
| PV691473.1SA002 | 37.17 | 17.83 | 29.08 | 15.93 | 791 |
| PV691474.1SA003 | 36.99 | 17.69 | 29.91 | 15.41 | 876 |
| PV691475.1SA004 | 37.87 | 17.73 | 29.79 | 14.61 | 705 |
| PV691476.1SA005 | 36.81 | 17.87 | 29.74 | 15.59 | 834 |
| PV691477.1AL001 | 36.17 | 17.85 | 31.16 | 14.82 | 857 |
| PV691478.1AL002 | 36.51 | 17.91 | 31.07 | 14.51 | 882 |
| PV691479.1AL003 | 37.69 | 17.09 | 30.49 | 14.74 | 597 |
| PV691480.1AL004 | 36.17 | 17.97 | 30.85 | 15.01 | 846 |
| PV691481.1AL005 | 36.55 | 18.21 | 30.52 | 14.73 | 747 |
| PV691482.1PB001 | 36.37 | 18.25 | 30.57 | 14.81 | 844 |
| PV691483.1PB002 | 37.79 | 17.82 | 29.7 | 14.69 | 606 |
| PV691484.1PB003 | 36.78 | 17.59 | 30.11 | 15.52 | 870 |
| PV691485.1PB004 | 36.76 | 17.58 | 30.02 | 15.64 | 876 |
| PV691486.1LL001 | 36.18 | 18.03 | 30.8 | 14.99 | 854 |
| PV691487.1LL002 | 36.28 | 17.91 | 30.93 | 14.88 | 860 |
| PV691488.1LL003 | 36.17 | 18.09 | 30.61 | 15.13 | 846 |
| PV691489.1LL005 | 36.49 | 17.78 | 30.95 | 14.78 | 866 |
| Average. | 36.91 | 17.77 | 30.26 | 15.05 | 777.23 |
T(U) – Thyamine/Uracil, C – Cytosine, A – Adenine, G – Guanine.
Table 3.
Genetic diversity parameters of the 22 sequences of underutilized legumes studied.
| Parameter | Value |
|---|---|
| Number of sequences | 22 |
| Total sites (excluding gaps) | 177 |
| Polymorphic sites (S) | 10 |
| Total mutations (Eta) | 11 |
| Number of haplotypes (h) | 3 |
| Haplotype diversity (Hd) | 0.654 |
| Nucleotide diversity (Pi) | 0.0282 |
| Theta-W (per site) | 0.0155 |
| Theta (per site) from Eta | 0.01705 |
| Theta (per site) from Pi (Finite Sites) | 0.0293 |
| Average nucleotide differences (k) | 4.996 |
Table 4.
Within-Population Genetic Diversity Statistics of the underutilized legumes studied.
| Population | No. of Sequences | Segregating Sites (S) | No. of Haplotypes (h) | Haplotype Diversity (Hd) | Avg. No. of Differences (K) | Nucleotide Diversity (Pi) | Nucleotide Diversity (PiJC) |
|---|---|---|---|---|---|---|---|
| Cassia fistula | 4 | 0 | 1 | 0 | 0 | 0 | 0 |
| Senna alata | 5 | 0 | 1 | 0 | 0 | 0 | 0 |
| Albizia lebbeck | 5 | 0 | 1 | 0 | 0 | 0 | 0 |
| Parkia biglobosa | 4 | 9 | 2 | 0.667 | 6 | 0.034 | 0.035 |
| Leucaena leucocephala | 4 | 0 | 1 | 0 | 0 | 0 | 0 |
| Total | 22 | 10 | 3 | 0.654 | 4.996 | 0.028 | 0.035 |
Table 5.
Gene flow and genetic differentiation among five populations of the 22 sequences of underutilized legumes.
| POPULATION 1 | POPULATION 2 | Hs | Ks | Kxy | Gst | DeltaSt | GammaSt | Nst | Fst | Dxy | Da |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Cassia fistula | Senna alata | 0.000 | 0.000 | 4.000 | 1.000 | 0.011 | 1.000 | 1.000 | 1.000 | 0.023 | 0.023 |
| Cassia fistula | Albizia lebbeck | 0.000 | 0.000 | 9.000 | 1.000 | 0.025 | 1.000 | 1.000 | 1.000 | 0.051 | 0.051 |
| Cassia_fistula | Parkia biglobosa | 0.333 | 3.000 | 4.500 | 0.200 | 0.006 | 0.333 | 0.333 | 0.333 | 0.025 | 0.008 |
| Cassia_fistula | Leucaena leucocephala | 0.000 | 0.000 | 9.000 | 1.000 | 0.025 | 1.000 | 1.000 | 1.000 | 0.051 | 0.051 |
| Senna alata | Albizia lebbeck | 0.000 | 0.000 | 8.000 | 1.000 | 0.023 | 1.000 | 1.000 | 1.000 | 0.045 | 0.045 |
| Senna alata | Parkia biglobosa | 0.267 | 2.667 | 6.000 | 0.527 | 0.010 | 0.481 | 0.495 | 0.500 | 0.034 | 0.017 |
| Senna alata | Leucaena leucocephala | 0.000 | 0.000 | 8.000 | 1.000 | 0.022 | 1.000 | 1.000 | 1.000 | 0.045 | 0.045 |
| Albizia lebbeck | Parkia biglobosa | 0.267 | 2.667 | 4.500 | 0.218 | 0.006 | 0.357 | 0.333 | 0.333 | 0.025 | 0.008 |
| Albizia lebbeck | Leucaena leucocephala | 0.000 | 0.000 | 0.000 | 1.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| Parkia biglobosa | Leucaena leucocephala | 0.333 | 3.000 | 4.500 | 0.200 | 0.006 | 0.333 | 0.333 | 0.333 | 0.025 | 0.008 |
Hs = haplotype-based diversity; Ks = nucleotide sequence variation; Kxy = average percentage of nucleotide changes between populations; GammaSt = coefficient level of population divergence; Gst = haplotype frequency differences; Fst = fixation index; Da = net nucleotide changes per site that distinguish populations; Dxy = average number of nucleotide substitutions per site between populations.
Table 6.
Analysis of Molecular Variance (AMOVA) for Genetic Variation.
| Source of Variation | d.f. | Sum of Squares | Variance Components | Percentage of Variation |
|---|---|---|---|---|
| Among Populations | 4 | 117.455 | 6.49314 (Va) | 88.04 % |
| Within Populations | 17 | 15 | 0.88235 (Vb) | 11.96 % |
| Total | 21 | 132.455 | 7.3755 | 100.00 % |
Fig. 2.
The evolutionary history of the 22 sequences of five underutilized legume species inferred using unweighted pair group method with arithmetic mean (UPGMA) method.
Table 7.
Test of the Homogeneity of Substitution Patterns Between Sequences.
| Taxa | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PV691468.1 | ||||||||||||||||||||||
| PV691469.1 | 1.00 | |||||||||||||||||||||
| PV691470.1 | 1.00 | 1.00 | ||||||||||||||||||||
| PV691471.1 | 1.00 | 1.00 | 1.00 | |||||||||||||||||||
| PV691472.1 | 1.00 | 0.32 | 1.00 | 1.00 | ||||||||||||||||||
| PV691473.1 | 1.00 | 0.29 | 1.00 | 1.00 | 1.00 | |||||||||||||||||
| PV691474.1 | 1.00 | 0.31 | 1.00 | 1.00 | 1.00 | 1.00 | ||||||||||||||||
| PV691475.1 | 1.00 | 0.32 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |||||||||||||||
| PV691476.1 | 1.00 | 0.32 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | ||||||||||||||
| PV691477.1 | 0.33 | 0.15 | 0.36 | 0.13 | 0.19 | 0.10 | 0.17 | 0.19 | 0.22 | |||||||||||||
| PV691478.1 | 0.36 | 0.13 | 0.31 | 0.13 | 0.21 | 0.11 | 0.13 | 0.19 | 0.19 | 1.00 | ||||||||||||
| PV691479.1 | 1.00 | 0.24 | 1.00 | 1.00 | 0.28 | 0.32 | 0.26 | 0.29 | 0.30 | 1.00 | 1.00 | |||||||||||
| PV691480.1 | 0.35 | 0.13 | 0.28 | 0.13 | 0.16 | 0.11 | 0.15 | 0.20 | 0.20 | 1.00 | 1.00 | 1.00 | ||||||||||
| PV691481.1 | 0.30 | 0.13 | 0.28 | 0.28 | 0.14 | 0.14 | 0.12 | 0.19 | 0.14 | 1.00 | 1.00 | 1.00 | 1.00 | |||||||||
| PV691482.1 | 0.36 | 0.09 | 0.14 | 0.26 | 0.33 | 0.18 | 0.32 | 0.35 | 0.37 | 0.24 | 0.25 | 1.00 | 0.27 | 0.36 | ||||||||
| PV691483.1 | 0.35 | 0.25 | 0.27 | 0.38 | 0.35 | 0.34 | 0.33 | 0.34 | 0.36 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |||||||
| PV691484.1 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.12 | 0.11 | 1.00 | 0.12 | 0.27 | 0.24 | 0.39 | ||||||
| PV691485.1 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.11 | 0.10 | 1.00 | 0.13 | 0.30 | 0.29 | 0.40 | 1.00 | |||||
| PV691486.1 | 0.33 | 0.19 | 0.12 | 0.28 | 0.21 | 0.15 | 0.32 | 0.22 | 0.16 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.38 | 0.27 | 0.25 | ||||
| PV691487.1 | 0.31 | 0.21 | 0.11 | 0.27 | 0.21 | 0.17 | 0.26 | 0.27 | 0.20 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.38 | 0.23 | 0.28 | 1.00 | |||
| PV691488.1 | 0.33 | 0.20 | 0.15 | 0.15 | 0.20 | 0.19 | 0.20 | 0.25 | 0.17 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.37 | 0.18 | 0.18 | 1.00 | 1.00 | ||
| PV691489.1 | 0.34 | 0.21 | 0.12 | 0.26 | 0.18 | 0.19 | 0.28 | 0.24 | 0.21 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 0.43 | 0.29 | 0.25 | 1.00 | 1.00 | 1.00 |
1–22 represents the species-Genbank Sequence Number as depicted under the taxa.
4. Experimental Design, Materials and Methods
4.1. Plant material
Fresh, young leaf samples were collected from five underutilized legume species: Albizia lebbeck, Cassia fistula, Leucaena leucocephala, Parkia biglobosa, and Senna alata. Collections were performed during the rainy season from open-field sites within the rainforest region of Bowen University, and its surrounding environment, Iwo, Osun State, Nigeria. For each species, five replicate samples were collected, resulting in a total of 25 samples. Sources of leaf samples were shown in Fig. 1. All samples were harvested at the flowering stage to ensure physiological uniformity. Immediately upon collection, leaves were placed in air-tight zip-lock bags, stored under cool conditions to prevent degradation, and transported to the laboratory for processing.
Fig. 1.
Sources of leaf samples for DNA extraction. A. Senna alata. B. Cassia fistula. C. Leucaena leucocephala. D. Parkia biglobosa. E. Albizia lebbeck.
4.2. Genomic DNA extraction
Genomic DNA was extracted from approximately 150 mg of fresh leaf tissue using the Zymo Research Quick-DNA Plant/Seed Miniprep Kit (Zymo Research Corp., Irvine, CA, USA), following the manufacturer's instructions. Tissue homogenization was performed in ZR BashingBead™ Lysis Tubes (2.0 mm) using a FastPrep® bead beater (MP Biomedicals, Santa Ana, CA, USA) for 1–2 min. The resulting lysate was centrifuged at 10,000 × g for 1 min, and the supernatant was subsequently filtered through a Zymo-Spin™ III-F column. DNA was purified by binding to a Zymo-Spin™ IC Column, washed with DNA Wash Buffer, and eluted in 100 µL of DNA Elution Buffer. DNA quality and concentration were assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Samples exhibiting 260/280 absorbance ratios between 1.8 and 2.0 were considered suitable for downstream molecular applications. Three samples, one each from Cassia fistula, Parkia biglobosa, and Leucaena leucocephala, yielded insufficient DNA quantity or quality for further analysis and were excluded from the analysis. Table 1 shows the resulting 22 samples, their accession numbers, GenBank number, sequence length, matched organisms and percentage identity of the underutilized legumes used for this study.
4.3. PCR amplification, purification and visualization
A final volume of 25 µL was used for PCR amplification of the matK regions, which included 12.5 µL of NEB OneTaq 2X Master Mix with Standard Buffer, 2 µL of genomic DNA (10–30 ng/µl), 0.5 µL of matK390_F (5-CGATCTATTCATTCAATATTTC-3 (Tm = 50 °C), matK132_R (5- TCTAGCACACGAAAGTCGAAGT-3 (Tm = 50 °C) (10 µM), and 9.5 µL of nuclease-free water. and 9.5 µL of nuclease-free water. The purity of the PCR samples and the parameters of the PCR reaction followed established methods [9]. The PCR results were evident after being detected and analyzed on an agarose gel electrophoresis using 1 % agarose and a 1 kbp DNA ladder.
4.4. DNA sequencing and processing
Following PCR amplification, all amplicons were enzymatically purified using ExoSAP-IT reagent (Applied Biosystems, Foster City, CA, USA) to eliminate excess primers and unincorporated nucleotides. The purified PCR products were then subjected to Sanger sequencing (chain termination method) by Inqaba Biotechnical Industries (Pty) Ltd. (Ibadan, Nigeria). Initial visual inspection of the raw. abi trace files was performed using FinchTV version 1.4.0 (Geospiza Inc.). For stringent quality control, sequences were subsequently processed with Chromas Pro software (Technelysium Pty Ltd.), where low-quality regions were precisely trimmed from both 5' and 3' ends, and ambiguous base calls (Ns) were resolved or removed. The resulting high-quality, cleaned sequences were exported in FASTA format for downstream analysis. These processed sequences (22 in total) have been deposited in the NCBI GenBank database and are accessible under accession numbers PV691468.1 to PV691489.1 (Table 1).
4.5. Sequence alignment and data analysis
4.5.1. Sequence alignment and phylogenetic analysis
The matK gene sequences were aligned using Geneious Prime® 2025.1.3. Summary statistics for nucleotide variation were also generated within Geneious Prime. The phylogenetic relationships among the 22 matK sequences, representing five underutilized legume species, were inferred using the Geneious Tree Builder. Tree construction employed the Tamura-Nei genetic distance model and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm. To validate the use of a single evolutionary model, the homogeneity of substitution patterns across all sequence pairs (PV691468.1 to PV691489.1) was assessed in MEGA v11.0.13 [9].
4.5.2. Population genetic diversity and gene flow
Genetic diversity parameters, within-population divergence statistics, gene flow and genetic differentiation among the five populations of the 22 sequences of the underutilized legumes were estimated using DnaSP v6.0 [10]. Key genetic diversity parameters evaluated include polymorphic sites (S), total mutations (Eta), number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (Pi), Theta-W (per site), Theta (per site) from Eta, Theta (per site) from Pi (Finite Sites) and average nucleotide differences (k). Several common molecular statistics were used to evaluate population divergence. Hs was used to assess haplotype-based diversity, whereas Ks was used to quantify nucleotide sequence variation. Kxy was used to calculate the average percentage of nucleotide changes between populations. GammaSt, a coefficient that indicates the level of population divergence, and Gst, which is based on haplotype frequency differences, were examples of measures of genetic differentiation. To measure total genetic divergence between populations, the fixation index Fst was also calculated. Furthermore, Da was computed to find the net nucleotide changes per site that distinguish populations, and Dxy was estimated to get the average number of nucleotide substitutions per site between populations. All the above parameters were also generated using in DnaSP v6.0 [10]. Further, genetic differentiation and partitioning of molecular variance, an Analysis of Molecular Variance (AMOVA) was conducted using Arlequin v3.5 [11]. Genetic variation was partitioned within and among species. Statistical significance for AMOVA components was determined through 10,000 permutations.
Limitations
The sample collection was restricted to Bowen University and its adjacent environment, despite the studied species being prevalent across Nigeria's major ecological zones. This geographical limitation may have resulted in the comparatively low genetic diversity observed. Subsequent studies should encompass a more comprehensive sampling that reflects the entire geographical distribution of these underutilized legume species to obtain a wider range of genetic variation. Nevertheless, the matK gene efficiently barcoded and identified the species, clearly distinguishing them as genetically distinct, in agreement with known sequences from other legume species [2]. The dataset indicates that the matK sequences of these underutilized legume species have evolved under similar patterns of nucleotide substitution, a finding that is further supported by the results of the phylogenetic analysis.
Ethics Statement
A total of 22 matK sequences were generated from genomic DNA isolated from fresh leaf samples collected within Bowen University and its surrounding environment in Iwo, Osun State, Nigeria. All sequences, as presented in Table 1, were submitted to the National Center for Biotechnology Information (NCBI) GenBank database, where accession numbers were subsequently assigned. The corresponding GenBank accession numbers are provided in Table 1. No data was collected through social media.
Credit Author Statement
Popoola Jacob Olagbenro: conceptualize the project, performed bioinformatics analyses, drafted the original manuscript and supervised the project. Olaoluwa Favour Daniel: drafted the original manuscript, performed data analyses. Obisesan Idowu Arinola: review manuscript. All authors contributed to the manuscript writing and approved the final manuscript.
Acknowledgments
The authors thank the Directorate of Research and Strategic Partnerships of Bowen University, Iwo, Nigeria for providing part of the funding for sequencing services with a grant number (BURG202417). We extend thanks to Inqaba Biotec West Africa Ltd laboratory in Ibadan, Nigeria and Babalola Favour Olanrewaju for technical support during the submission of the sequences to the NCBI GenBank.
Declaration of Competing Interest
The authors declare no competing interests.
Data Availability
NCBI-Genebank Database22 nucleotides MatK (Original data).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
NCBI-Genebank Database22 nucleotides MatK (Original data).