Morphological Variation and Discriminating Traits of Kersting’s Groundnut Accessions

54 Kersting’s (KG) is a nutritious, subterranean 55 grain legume in West and Central Africa. Only limited information is available on the morphological traits that 56 can discriminate accessions; without such information, appropriate breeding strategies cannot be devised. This 57 study aimed to identify discriminating traits and assess the diversity among accessions of Kersting ’s groundnut. 58 Eighty-one KG accessions from Benin and Burkina Faso were evaluated based on 29 qualitative and quantitative 59 traits. An experiment was conducted using an Alpha lattice design with three replications. Standardized Shannon- 60 Weaver index (H') and descriptive statistics were calculated for qualitative traits. Pearson correlation coefficients, 61 stepwise discriminant analysis, principal component analysis, cluster analysis and canonical discriminant analysis 62 were conducted. Results showed that accessions varied greatly based on growth habit (H'= 0.68), flower color (H' 63 = 0.50), seed-eye shape (H' = 0.47), and stem pigmentation (H' = 0.41). Eight quantitative traits, viz ., seed width, 64 seed thickness, number of branches per plant, petiole length, days to 50% flowering, number of seeds per pod, pod 65 width, and pod length, were found to significantly discriminate the accessions. Accessions were grouped into three 66 clusters based on quantitative traits. Cluster 1 had accessions with late flowering and good vegetative growth, 67 Cluster 2 contained accessions with high germination percentage and Cluster 3 had accessions with high yield 68 performance. Seed length varied greatly among accessions, thus indicating the potential for improving yield via 69 seed size. 70

The seed is a rich source of crude protein, with high levels of essential amino acids, such as 91 phenylalanine (3.2/100 g), histidine (2.1/100 g), lysine and methionine (Ajayi and Oyetayo, 92 2009). Seeds have high vitamins contents (Leung et al., 1968). According to Adazebra (2013), 93 Kersting's groundnut is one of the less-known leguminous crops, but it contributes significantly 94 toward rural nutrition, livelihoods and sustainable development. The present study aimed at filling the above-mentioned gaps by i) assessing diversity among 114 accessions in a regional germplasm collection, obtained from Benin and Burkina Faso, and ii) 115 identifying discriminating traits that could be included in a list of descriptors to be used for 116 morphological characterization of Kersting's groundnut. 117

Study area 119
The study was carried out at the Regional Center of Agricultural Research (CRA-CF) in Djidja, 120 village of Djegbatin (7°19'04.362'' N and 1°54'58.914'' E). The climate of Djidja is sub-121 equatorial and the rainfall is generally bimodal but can also be unimodal. The soils are ferralitic, 122 ferruginous and hydromorphic. Rainfall, temperature, sunshine, and relative humidity during 123 the period of experimentation are presented in Table 1. 124 Table 1. Monthly average climatic data recorded on study site during experiment. 125

Plant material and experimental design 126
Genetic material consisted of a collection of 81 accessions, of which 70 were from Benin and 127 11 from Burkina Faso (Table 2). Planting was done on 23 August 2018. The experiment was 128 conducted using an Alpha lattice design, with 9 plots per block × 9 blocks and three replications. 129 Each plot consisted of three rows, each 4.5 m in length. The rows were spaced 0.75 m apart. 130 Plant-to-plant spacing was 0.30 m, giving a plant population of 44500 plants per hectare. 131 Distance between plots was 1 m. One seed was sown per hill at a depth of 5 cm. No fertilizer 132 was applied and weeding was done manually 3 weeks, 7 weeks and 12 weeks after sowing. 133 Table 2. Name, seed color and origin of Kersting's groundnut accessions used in this study. 134

Data collection and analysis 135
The quantitative traits evaluated were: germination percentage, number of leaves per plant, 136 number of flowers per plant, number of pods per plant, yield, number of branches per plant, 137 days to 50% flowering, 100-seed weight, seed length, seed width, seed thickness, leaf length, 138 leaf width, petiole length, pod length, pod width, and number of seeds per pod. In addition, data 139 were collected on 12 qualitative traits (Table 3). 140 Table 3 Pearson's correlation was used to examine the relationship between yield and other quantitative 174 traits. Further, quantitative trait data were subjected to stepwise discriminant analysis to 175 determine the traits that best discriminated the accessions. Canonical discriminant analysis was 176 performed to describe relationship between seed and flower color based on discriminating traits. 177 Principal component analysis (PCA) was performed to determine the patterns of agro-178 morphological variation. Hierarchical classification was done to group the accessions. 179 Thereafter, descriptive statistics and analysis of variance (ANOVA) were used to describe the 180 clusters. All analyses were performed in R software 3.5.2. (R Core Team, 2019). 181

Results and discussion 182
The standardized Shannon Weaver diversity index (Hʹ) values ranged from 0.16 to 0.68 (Table  183   4). There was a high level of phenotypic variation among accessions for plant growth habit 184 (H'=0.68) and flower color (H'=0.50). Moderate variation was observed for seed-eye shape, 185 easy pod detachment, stem pigmentation and seed coat color. Pod color, terminal leaflet shape, 186 terminal leaflet color, pod shape and pod texture exhibited a relatively low level of variation 187 (Table 4). Growth habit and flower color could be used as key qualitative descriptors for 188 Kersting's groundnut. Stepwise discriminant analysis (SDA) performed on quantitative traits revealed 8 traits, viz., 270 seed width, seed thickness, number of branches per plant, petiole length, days to 50% flowering, 271 number of seeds per pod, pod width, and pod length, which discriminated the accessions (Table  272 8). These discriminating traits could be used as descriptors for describing Kersting's groundnut (49.03±0.21 mm). Accessions belonging to C1 also had low 100-seed weight (12.54 ± 0.09 g), 352 small seed length (8.11 ± 0.03 mm) and low number of flowers per plant (14.7 ± 0.39) ( Table  353 9). Overall, C1 was characterized by accessions with high vegetative growth and late flowering. 354 C2 was characterized by a significant (p<0.001) germination percentage (60.47 ± 4.72), high 355 number of flowers per plant (25.8±0.6) and wide petiole length (165.82 ± 2.53 mm), and fewer 356 days to 50% flowering (46.11 ± 0.11). In addition, accessions in C2 had medium number of 357 branches per plant (7.57 ± 1.17) and medium seed length (8.30 ± 0.12 mm) (Table 9) compared 358 to accessions in clusters 1 and 3. C3 was characterized by high 100-seed weight (15.18 ± 0.29g), 359 high seed length (8.88 ± 0.12 mm), high pod width (7.94± 0.06), long leaflet (69.4 ± 0.71) and 360 wide seed width (6 ± 0.06 mm) but low number of branches per plant (6.56 ± 0.37). Overall, 361 C3 was characterized by accessions with high performance for yield components (Table 9). In 362 Benin, accessions with cream seed coat and eye color (pure cream) are preferred the most. 363 Accessions in C3 showed high performance for yield-related traits, such as seed weight, seed 364 length and seed width. These accessions were Zhla 2, Gbo 4, BUR 3, BUR 7, BUR 8, BUR 9, 365 BUR 14, BUR 16 and BUR 18, all having black or brown seed coat or black-eyed seeds; none 366 of these accessions had pure cream color. Breeding efforts could therefore perform backcross 367 between pure cream accessions and accessions from C3 to obtain improved pure cream varieties 368 with high yield performance. 369 370 Table 9. Mean values and standard errors of discriminating traits in Kersting's groundnut 371 accessions 372 373

374
The evaluation of 81 KG accessions based on the 29 traits revealed high diversity, both for 375 qualitative and quantitative traits. Three diversity groups were identified based on the 376 quantitative traits, clusters were characterized by late flowering, good vegetative growth, high 377 germination percentage and high yield performance. Besides, the study identified seed width, 378 seed thickness, number of branches per plant, petiole length, days to 50% flowering, number of 379 seeds per pod, pod width, and pod length as the quantitative traits that best discriminated the 380 accessions. This could be a starting point for the establishment of a list of descriptors to be 381 measured while studying the crop. 382 (RUFORUM) for funding part of the experiments through Carnegie Cooperation of New York. 386 We are grateful to The World Academy of Science (TWAS) for its support. We are grateful to 387 the Benin Institute of Agricultural Research (INRAB) that has provided site and technical 388 support for the experiments. 389

Conflict of Interest Statement 390
The authors declare that the research was conducted in the absence of any commercial or 391 financial relationships that could be construed as a potential conflict of interest.  Fig 1 f) Black coat seed, Fig 1g) brown coat seed, Fig 1h) butter y shape eyes, Fig 1i) triangular eyes, Fig 1j) irregular eyes.   Hierarchical clustering of Kersting's groundnut accessions based on quantitative traits.