Genetic Inconsistency Assessment and Selection Criteria of Bambara groundnut ( Vigna 1 subterranea L.Verdc.) Landraces for High Yield Revealed by Qualitative and Quantitative traits

As a crop for the new millennium Bambara groundnut ( Vigna subterranea L. Verdc.) considered as leading 16 legumes in the tropical regions due to its versatile advantages. The main intent of this study was to find out 17 the high yielding potential genotypes and considering these genotypes to develop pure lines for commercial 18 cultivation in Malaysia. Considering the 14 qualitative and 27 quantitative traits of fifteen landraces the 19 variation and genetic parameters namely, variability, heritability, genetic advance, characters association, 20 and cluster matrix were determined. ANOVA revealed significant variation for all the agronomic traits 21 (except plant height). Among the accessions, highly significant differences (P ≤ 0.01) were found for 22 almost all the traits excluding fifty percent flowering date, seed length, seed width. The 16 traits out of the 23 27 quantitative traits had a coefficient of variation (CV) ≥ 20%. A positive and intermediate to perfect 24 highly significant association (r = 0.23 to 1.00; P < 0.00) was found between yield and its related traits. 25 The trait dry seed weight per plant (g) had the highest GCV = 59.91% and PCV = 59.57% whereas the trait 26 fresh pod weight (99.55%), dry seed weight (98.86%), and yield (98.10%) were highly heritable. The 27 genetic advance recorded the highest for dry seed weight (122.01%) and lowest (3.97%) for plant height. 28 To validate the genetic disparity, an unweighted pair-group produce with arithmetic mean (UPGMA), 29 principal component analysis (PCA), and Hˊ-index was performed considering 27 quantitative traits. The 30 constructed dendrogram showed five distinct groups of accessions. Genotypes G2, G3, and G9 from Group 31 IV consider as promising lines which gave 70.05% higher mean yield compared to grand mean yield (1180 32 kg ha -1 ) with desirable traits. Group II had a maximum number of accessions while group III and group V 33 had one of each. However, findings declared that the availability of genetic variance will be beneficial for this crop improvement and plant breeders to prefer desirable traits in V. subterranea L. Verdc. for further 35 breeding purposes.

almost all the traits excluding fifty percent flowering date, seed length, seed width. The 16 traits out of the 23 27 quantitative traits had a coefficient of variation (CV) ≥ 20%. A positive and intermediate to perfect 24 highly significant association (r = 0.23 to 1.00; P < 0.00) was found between yield and its related traits. 25 The trait dry seed weight per plant (g) had the highest GCV = 59.91% and PCV = 59.57% whereas the trait 26 fresh pod weight (99.55%), dry seed weight (98.86%), and yield (98.10%) were highly heritable. The 27 genetic advance recorded the highest for dry seed weight (122.01%) and lowest (3.97%) for plant height. 28 To validate the genetic disparity, an unweighted pair-group produce with arithmetic mean (UPGMA), 29 principal component analysis (PCA), and Hˊ-index was performed considering 27 quantitative traits. The 30 constructed dendrogram showed five distinct groups of accessions. Genotypes G2, G3, and G9 from Group 31 IV consider as promising lines which gave 70.05% higher mean yield compared to grand mean yield (1180 32 43 Bambara groundnut is a future emerging legume grown in Africa and Asia, is usually known as a poor 44 man's crop or as "Women´s Crop" [1] and newly noted as Crop for New Millennium [2]. The present 45 binomial name Vigna subterranea (L.) Verdc was suggested by Verdcourt, 1980 [3] and chromosome 46 number is 2n=2x=22 [4]. This crop occupied 3 rd position after Arachis hypogea and Vigna unguiculate in 47 K2O /ha) and all portion of Phosphorus and Potassium were applied during field preparation 111 hence, 70% N was applied at 5 weeks after sowing [27]. 112 113 Twenty-seven quantitative and 14 qualitative characters (Table 2) were considered during the 114 morphological characterization. For comfort description, quantitative traits were categorized as 1) 115 Phenological traits; 2) Growth and vegetative traits; 3) Yield traits. Following the Bambara 116 groundnut description and descriptors states by IPGRI, IITA, BAMNET [28] data were recorded 117 at several growth stages in the field and post-harvest data in the physiology lab. Pods texture (PT) 8 Number of leaves per plant NL 10 Seeds shape (SS) 9 Number of nodes per stem NNS 11 Seeds colour (SC) 10 Internode length IL(cm) 12 Eyes color (EC) 11 Biomass fresh weight per plant BFW(g) 13 Testa pattern (TP) 12 Biomass dry weight per plant BDW(g) 14 Testa color with eye pattern around hilum.

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(TCEP) 13 Total no. of pod per plant TNP 14 Mature pods number per plant NMP 15 Immature pods number per plant NIP 16 Fresh pod weight FPW (g) 17 Dry pod weight DPW(g) 18 Length of pod PL (mm) 19 Width of pod PW (mm) 20 Number of seed per NSP 21 Dry seed weight per plant DSW(g) 22 Length of seed SL (mm) 23 Width of seed SW (mm) 24 Hundred seed weight HSW (g) 25 Shelling percentage (%) Shell% 26 Harvest Index HI (%) 27 Yield Kg per hectare Yld(Kg/ha) 121 The SAS (statistical analysis software) version 9.3 was followed to test the significant differences 122 using the analysis of variance (ANOVA) procedure at the level of LSD; P ≤ 0.05 and to compare 123 among the mean of significant of traits. The correlations between the quantitative variables were 124 determined using Pearson [29] correlation coefficient formula. The genotypic and phenotypic 125 variation were calculated as per following the formula given by Singh and Choudhary` [30]. The 126 coefficient variation of phenotypic (PCV) and genotypic (GCV): were estimated as per formula 127 given by Shabanimofrad et al. [31] also relative differences was estimated using the formula (RD) =

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Relative difference between PCV and GCV. The estimated values of PCV and GCV were categorized by 129 Shabanimofrad et al. [31], Sohrabi, et al. [32] and Robinson et al. [33] like as between 0% -10% for low, 130 10% -20% for intermediate and greater than (≥20%) for high. Broad sense heritability (ℎ 2 ) was estimated 131 using the formula given by Oladosu et al [34], Usman et al. [35] and Falconer [36]. In accordance with 132 Johnson et al. [37] and Assefa et al. [38], the heritability grade was ordered between 0% -30% for 133 low, 30% -60% for intermediate and greater than 60% as high. Genetic Advance (GA) (as a 134 percentage of mean): was calculated with 5% selection intensity (K) following the method of [37]. 135 Genetic advance is categorized as between 0% to 10% for low, 10% to 20% for intermediate and 136 more (>20%) than for high, following the formula given by Juangsamoot et al. [39]. K for constant 137 also indicates the intensity of selection. According to Adewale et al. [40]   The frequency of distribution of some qualitative variables are summarized in Table 3 and Figure   154 1. After two weeks later, we observed 46 (Table 7).     Legend: "**" correlation is significant at the 0.01 level; "*" correlation is significant at the 0.   23 24 The output of genetic components analysis was compiled in Table 8. Apparently, the phenotypic 25 variance ( 2 ) is higher than the genotypic variance ( 2 ) regarding all the traits evaluated. The      83 In this study, the homogenized data was used to calculate the Euclidean distances among the 15 84 Bambara groundnut accessions and a UPGMA dendrogram was designed (Figure 3).    (Table 10).

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The Shannon-Weaver diversity index was used to assess the phenotypic diversity for each trait.

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The estimation of the Shannon-Weaver diversity index (H) and Evenness (EH) for the twenty-152 seven traits shown in

.1. Qualitative disparity
The existence of a significant qualitative variation was found for all the qualitative traits, supported by Gbaguidi et al. [46] he found significant variation among all the qualitative traits.
We recorded three types of growth habit and similar observation were noticed by Ntundu et al. [1] in Tanzania and Azam-Ali et al. [47] in Cameroon. We categorized the vegetative growth of Bambara groundnut namely; bunches type, semi bunches type and spreading type which is matched by the result of Doku [48] and highly significant difference among the qualitative trait was noted by Egbadzor et al. [49].

Quantitative traits
The estimated 27 quantitative traits showed a massive genetic variation and similar variation was confirmed by Ntundu et al. [1] and Aliyu et al. [50] in Vigna subterranean (L.) Verdc and the cowpea (Vigna unguiculata L) [51]. The estimated high coefficients of variation (CV) in our study is the indication of vast scale of heterogeneity confirmed by Goli et al. [52] in Bambara groundnut. We found D50%F close to 39 days but [53] noticed close to 68 days in Ghana. The indeterminate [54] nature of flower bearing make it vital issue for adjustment mechanism to an environment [55]. The inconsistency of flowering time was reported by [52]  structure as well as genotypic nature is responsible to bearing flower in Bambara groundnut [24] and reported flowering happened between 36 to 53 days. In our study, genotype G1, G2, G3, G8, G9 and G10 identified as early flowering lines; early flowering ensures early maturity [57]. A significant difference (P ≤ 0.01) was recorded for maturity (119.67 to 141.33) days is supported by Goli et al. [52] and Masindeni [56] and due to diverse cultivar along with multienvironmental factors maturation time varied from 90 to180 days [58]. Plant hight had no significant variation, supported by Ntundu et al. [1] in Tanzania and Shegro et al. [24] in south  [60] in west Africa whereas we calculated from 588.98 to 2991.77 kgha -1 . Typically, FAO [19] estimated average yield Bambara groundnut is lower than our estimated yield 1180 kgha -1 .

Correlation coefficient
In plant breeding correlation matrix is a prominent approach for the judgement of degree of the association between two or more variables, is supported by Mohammed [53]. For superior genotype's selection programme consideration of correlation matrix can be a great scale of measurement [61]. Strong and positive significant correlation for total number of pods (TNP) was identified with the traits NMP, DPW, NSP, DSW and Yield this result is consistent with the study of Karikari [42] and Jonah et al. [63]. We got moderate and positively high significant association of plant hight (PH) with TNP, NMP, FPW and yield can be proposed the selection based on these traits may be beneficial for yield enhancement of this crop as well as fodder production for animal feeding. Similar recommendation was stated by Mohammed [53] in Cote d'Ivoire and [64] in Cameroon.

Genetic components
For the selection program variation presents among the traits was taken into consideration which depends on the degree of heritability. To know the projected gain from selection, valuation of genetic advance with heritability can be a significant approach of crop improvement. Various research findings reported that the selection may be effective for a specific trait improvement using available genetic variation with the degree of heritability [25], [65]. Consideration of both heritability and genetic advance is more effective over the uniquely use of heritability [66,67].
Like the previous reporters Adebola et al. [68] findings, we disclose higher phenotypic variance values than genotypic variance for all traits, indicates the trait expression govern by the environment. The obtained GCV and PCV value was categorized based on the suggested index of 0%-10% for low, 10-20% for moderate and ≥ 20% for high variation [31][32][33]69]. Intermediate to strong genetic advance with heritability was found for all yield related traits except seed width and shelling% is the indication of the traits have significant potential in the selection process due to low environmental influences, supported by Meena et al. [70] and Oladosu et al. [34]. The improvement of the traits with low heritability and genetic advance can be boost over heterosis breeding this is supported by Bijalwan & Madhvi [71]. The value of relative differences between GCV and PCV had higher for the trait plant hight, seed width, days to 50% flowering, and seed length is the sign of higher environmental effect and the improvement of these traits are tough via direct selection whereas the trait with lower difference is the symbol of lower influence by the environment which may give desirable strong and significant output in crop improvement program, is supported by Umar et al. [25] and Usman et al. [35]. Direct selection can be effective considering the traits having low relative differences [72]. Considering the heritability and genetic advance index [37,38] [65,73]. Low to moderate heritability and genetic advance values may hindrance in the trait's betterment due to high environmental effects over the genetic effects on its stated by Ridzuan [74]. So, only an effective selection can be gained picking the traits with higher GCV, PCV, HB, and GA meaning that effect of additive genes is sufficiently robust than environmental effect [35].

Clustering patterns
Five clusters were constructed based on the 27 quantitative traits at 1.16 of the distant that indicates a degree of diversity among the genotypes. The cluster V considered as potential group of genotypes for the crop betterment associated with high yielding capacity. The findings of previous researchers [26,46,75,76] stated that they constructed same type of cluster and found significant variation regarding morphological traits in Bambara groundnut. The study of Unigwe et al. [26] explored the four distinct groups of Bambara groundnut genotypes in south Africa using UPGMA model. The timing of flowering duration is a motivational factor for the final yield also play a positive role to the best yield of the group and selection could be effective from this class noted by Tourél et al. [77]. Flowering in Bambara groundnut is indeterminate up harvesting stage explained by Kumaga et al. [78]. However, early flowering has been considered as a well agronomic trait of crops to quick maturity, uniform yield as well as generally crop production [78] thus, accessions that have early flowering criteria should be treated as best to production of Bambara groundnut [79]. The groups achieved from the cluster analysis of quantitative characteristics illustrate the performances of Bambara groundnut accession cultivated in Benin would be the future guideline for this crop improvement [80,81].
The clustering and characterization of accessions considering their agro-morphological traits and genetic similarity would be the crucial issue to identification and selection of the best parents for hybridisation [82]. Additionally, cluster IV produced 70.05% higher mean yield than the average grand mean yield of 1180 kgha -1 while the other groups gave lower yield and this finding were supported by Onwubiko et al. [73]. Therefore, current research represents significant information to the plant breeders based on their similarity and grouping of accessions through univariate and multivariate methods.

Principal Component Analysis.
The principal component analysis (PCA) is the re-validation instrument of cluster analysis. To estimate the total variation, exist in a set of characters, PCA is effective noted by Johnson [41].
The first axes (PC1) elucidate utmost portion of total variation in any PCA [83].  [24] grouped the 20 Bambara groundnut accessions by PCA analysis using quantitative traits. For yield improvement the selection PC1 was revealed as the most powerful criterion concluded by the work of Adéoti et al. [87] and Mih et al. [88]. In my research total pod numbers, mature pods number, seed number, dry seed weight and yield kg/ha occupied high values in PC1. This finding supported by Stoilova & Pereira [81] described that the most significant components for yield are the pods number and seeds number per plant. The cluster analysis together with principal component analysis explored the common association among landraces in terms of seed yield and related agronomic traits.

Shannon diversity index (H) and Evenness (E)
Shannon's diversity index (H) is another index that is generally used to categorize the species diversity in a certain community. Shannon's diversity index is an account for both richness and evenness present in the species also used for a wide diversity of fields. The estimated Hˊ Index varied from 2.57 for dry seed weight per plant to 2.71 for plant height, maturity date and shelling percent among the phenotypic traits. In our study the observed diversity index value was more than 2.50 for most of the traits evaluated. Olukolu et al. [80] reported Hˊ Index of nineteen qualitative traits (0.1 to 0.15) and twenty-eight numerical traits (0.09 to 0.16) of Bambara groundnut that supported our findings. Bonny et. al. [76] evaluated the diversity in qualitative traits of Bambara groundnut landraces of similar findings with our result.     genetic advance (as percentage mean) with high heritable values. Additionally, it was declared that the landraces were diverse and depending on the similarity issues landraces are grouped into five cluster.
Moderate to perfect significant association was noted between the yield and its related traits. The landraces G2, G3, G8 and G9 identified as high yielding promising lines and suggested that further research can be conducted to gain the homogeny by providing emphasis on this potentially high yielding lines, following conventional breeding together with molecular approaches.

Acknowledgments and Funding
The

Conflict of Interests
The authors declare no conflict of interests.