Assessment of Genetic Diversity of a Collection of Senna obtusifolia (L.) Irwin and Barneby Using SSRs Markers in Burkina Faso

Sennaobtusifolia (L.) is a plant in the genus Senna that contributes to improving nutritional quality, food security, and better health protection for rural populations. However, very few studies have been devoted to it in Burkina Faso. Consequently, its genetic diversity remains poorly known. Such neglect would lead to the erosion of its genetic resource. The general objective of this study is to contribute to a better knowledge of the genetic diversity of the species in order to be able to issue scientific bases for its conservation, valorization, and genetic improvement. Sixty (60) accessions of Senna obtusifolia were collected in the wild from five provinces of three climatic zones of Burkina Faso. Molecular characterization was carried out using 18 SSR markers. Fifteen were polymorphic microsatellite markers leading one hundred and one (101) alleles in total, with an average of seven (7) alleles per locus. The number of effective alleles was 2.33. Expected heterozygosity, Shannon diversity index, and polymorphism information content averaged 0.47, 1.05, and 0.47. Molecular characterization revealed the existence of genetic diversity within the collection. This diversity has been structured into three genetic groups. Genetic group 3 presents the highest genetic diversity parameters.


Introduction
Senna obtusifolia (L.), also known as Foetid Cassia in French or Katr-nanguri in Mooré, is an annual or perennial herb with an erect, branched stem that can reach an average height of 2.5 m and has alternate, pinnate leaves [1,2]. According to Irwin and Barneby [3], Senna obtusifolia occurs in two forms: one that originates from the Caribbean which has an extraforal uniglandular nectary on the upper surface of the rachis located between the two lower leafets and with a chromosomal formula of 2n � 28. Te diameter of the pods varies between 3.5 and 6 mm and the other originates from British Guiana, Surinam, and Venezuela which has two extraforal nectaries. Its chromosomal formula is 2n � 26, and its pods are relatively narrower with a diameter varying between 2 and 3.5 mm.
It is a plant that contributes to the improvement of nutritional quality, food security, and better health protection of rural populations [4]. Indeed, its leaves are used as a vegetable in Africa and Asia [5,6]. In addition, according to the chemical composition reported by Busson [7], the plant contains more iron (6 mg), calcium (608 mg), phosphorus (95 mg), vitamin C (120 mg), and protein (5.66 mg) than Amaranthus hybridus and Hibiscus sabdarifa and more vitamin A than Hibiscus sabdarifa per 100 grams of fresh matter, which, however, are already domesticated and widely consumed species. High levels of iron and vitamins A and C represent particularly important health issues in countries plagued by numerous cases of anemia caused mainly by malaria [8]. Te leaves, seeds, and roots of Senna obtusifolia (L.) are used to cure diseases such as night blindness, hepatitis, ulcers, stomach ulcers (leaves), skin diseases, conjunctivitis, and poisonous insect bites (seeds); diseases caused by helminths, against herpes and the expulsion of intestinal worms (roots) [9]. However, very few studies have been devoted to it in Burkina Faso, and its diversity remains poorly known. Several studies have been conducted mainly on their medicinal role [9]; biochemical composition [10]; ethnobotanical study [4]; agromorphological characterization [11], and competitiveness evaluation of it as an invasive species [12].
Agromorphological characterization of 70 accessions including the present collection of Senna obtusifolia was conducted in 2018 by Nacambo et al. [11] using morphological markers. Tis study showed the existence of variability within the Senna obtusifolia. Te morphological markers used are the number of days to emergence, number of days to fowering, number of primary branches, leaf biomass, length of the fruit, fruit width, number of fruits per plant, number of seeds per fruit, and number of seeds per plant.
According to Banerjee et al. [13], agromorphological markers although indispensable for the evaluation of variability are often under the infuence of the environment, thus do not allow a better appreciation of genetic variability. Tus, since 1980, molecular markers that ofer a better approach to diversity have been developed [14]. Knowledge of the intraspecifc variability of a plant is important in the process of conservation and development of its genetic resources [15]. It is therefore necessary to better appreciate the variability observed during the agromorphological evaluation using molecular markers. Work carried out by Mohanty et al. [16] revealed a total of 158 alleles, 109 alleles, and 78 alleles for seventeen (17) RAPD markers, eleven (11) ISSR markers, and seven (7) SSR markers on Senna obtusifolia (Cassia tora).
Many molecular markers exist; however, simple sequence repeat (SSR) markers are excellent tools for cultivar identifcation, pedigree analysis, and assessment of genetic distances within many plant species. Tey are also excellent tools for the assessment of genetic diversity because of their codominant nature and their ability to be analyzed with quality [17,18]. As molecular markers, SSRs combine many properties desirable for markers, including high levels of polymorphism and information content and unambiguous designation of alleles [19]. Indeed, according to Sorrells [20], they are considered a class of markers that contribute to the direct selection of alleles. Tis is how this activity comes into play, which aims at a better knowledge of the genetic diversity within a collection of the species on the basis of SSR markers. Te specifc objectives were (i) to determine the level of genetic diversity within the collection and (ii) to determine the population structure within the collection of Senna obtusifolia.

Plant Material.
Te plant material consists of sixty (60) accessions of Senna obtusifolia (L.) collected during a survey. Tey come from ten (10) villages distributed in fve (5) provinces of the three climatic zones surveyed and sector 14 of the city of Bobo-Dioulasso ( Figure 1). Tese included 9 (15%) accessions from the Sahelian zone, 33 (55%) from the Sudano-Sahelian zone, and 18 (30%) from the Sudanian zone. Tese accessions were all collected from spontaneous populations, tied up in sachets, and stored in bags ( Figure 2).

Microsatellite Markers Used for Genetic Diversity
Study. A total of eighteen (18) microsatellite markers were used in our study; they were selected from those used by Li et al. [19] on Vigna unguiculata (cowpea) ( Table 1). Tese markers consist of tandem repeats of single, di, tri, or tetra nucleotide motifs and are abundant in the genome of eukaryotes [21]. Tese primers were used because they do not have species-specifc markers. Terefore, we tested for markers of the legume family since Caesalpiniaceae is a subfamily of Leguminosae according to Satish et al. [22].

Extraction of Genomic DNA.
For each accession, 0.1 g of fresh leaves from 14-day-old plants was fnely ground in 750 μl of ultrapure water using a mortar and pestle. Te grindings from each sample collected in a 1.5 ml Eppendorf tube were centrifuged at 10,000 rotations per minute (RPM) for 10 min at 4°C. At the end of the centrifugation, the supernatant was removed, and 200 μl of DNAzol was added to the pellet and homogenized. Te tubes were then incubated at 65°C in a water bath for 2 h and then centrifuged for 15 min at 4°C. At the end of the centrifugation, the supernatant was collected and stored. Te amount of DNA was determined using NanoDrop 2000. Te DNA extraction took place in November 2020 in the Molecular Biology Unit of the Plant Genetics and Improvement Research Team of the Biosciences Laboratory of the Joseph KI-ZERBO University.

PCR Amplifcation.
For each DNA sample, a reaction volume of 25 μl was prepared in an Eppendorf tube. Te reaction mixture consisted of 4.5 μl of 40 ng/μl genomic DNA; 2 μl of 10 μM primer; 16 μl of ultrapure water; and 2.5 μl of the PCR Mix consisting of Taq polymerase (1U), dNTP (10 μM), and bufer (10X) (10 mM Tris-HCL pH 9, 50 mM KCl, 1.5 mM MgCl 2 ). After homogenization, amplifcation was performed in a thermal cycler following a program consisting of an initial DNA denaturation phase at 94°C for 3 minutes, followed by a series of 70 PCR cycles with denaturation at 94°C for 1 minute, hybridization at 48°C for 2 minutes, extension at 72°C for 2 minutes, and a fnal extension at 72°C for 15 minutes.

Electrophoretic Migration and Band
Reading. PCR products were subjected to electrophoretic migration on a 3% agarose gel prepared with a 1X TBE solution. A fuorescent developer, ethidium bromide (BET) 5%, was added to the agarose gel. Deposition of the amplifcation products was performed in the presence of a molecular weight marker ranging in size from 50 to 500 bp, and migration was performed at 90 V for 2 h in 0.5X TBE bufer. Te reading of the amplifcation products was done under ultraviolet light from a transilluminator with a Canon EOS 1300D camera of 18 mega pixels. Te bands were identifed on the basis of their position on the gel. Tus, the molecular weight of each observed allele was noted for each accession and for each primer tested according to the weight marker.
PCR amplifcation, electrophoretic migration, and band reading were performed in the Laboratory of Plant Genetics and Biotechnology of the Department of Plant Production of the Institute of Environment and Agricultural Research (INERA).

Data Analysis.
Markers that gave clear bands were retained for data analysis. Genetic diversity within the S. obtusifolia population was analyzed at two levels: intrapopulation diversity and interpopulation diversity. Tus, the genetic parameters were calculated with the GenALEx software version 6.501 in order to evaluate the diversity of the whole collection and the diversity according to the climatic zones. A structure analysis was carried out using the DARwin V6.0 software. Tis software was frst used to generate the dissimilarity matrix between accessions using the "simple matching" procedure. Ten, from this    (1) Intrapopulation Diversity. Several genetic parameters were estimated: (i) Te total number of alleles (At) or allelic richness which is the total number of alleles counted in a population. Its measure At � n−1, where n is the number of all the alleles provided by all the primers so that in a monomorphic population, its value is equal to 0; (ii) Te number of diferent alleles per locus (Na) which is the sum of the alleles found per locus; (iii) Te efective number of alleles (Ne) which is the inverse of the probability that, for a given locus, two randomly taken alleles are identical. Ae � 1/(1-h) � 1/Σpi2 where pi � the frequency of allele i of the locus considered and h � heterozygosity; (iv) Te Shannon diversity index (I) was calculated according to the formula of [23] where Pi is the frequency of allele i; (v) Te expected heterozygosity (He) and Nei's gene diversity index (D) which is the probability that at a given locus any two randomly selected alleles in the population are diferent. Te expected heterozygosity rate (He) was calculated from the allele frequencies determined for each locus using the formula He � 1/N [n/n-1 (1-Σpi2)], where N is the number of loci, n is the number of accessions, and pi is the frequency of allele i of the locus considered.
(vi) Te polymorphism information content (PIC) which is a parameter that gives an estimate of the discriminatory power of a locus. It takes into account both the number of alleles expressed and the relative frequency of each allele [24]. It is calculated according to the formula PIC � 1-Σf 2 , with f being the frequency of each allele. Te PIC varies from 0 for a monomorphic locus to 1 for a highly discriminating locus, with several alleles each in low and equal frequency. (vii) Te polymorphism rate (P) which is the number of polymorphic loci (npj) divided by the total number of loci (n total). Its formula is P � npj/n total.
(2) Interpopulation Diversity. Te accessions were subdivided into subpopulations according to the climatic zone factor. Genetic diversity parameters were estimated for each of these subpopulations. Two parameters were estimated in the description of the genetic diversity between the defned subpopulations. Tese are the indices of genetic diferentiation between populations (Fst) and the minimum distance of Nei between pairs of genetic groups.
(i) Te index of genetic diferentiation between populations (Fst): its value is between 0 and 1. It is 0 in case of strong genetic similarity between subpopulations and 1 in case of fxation of diferent alleles in the subpopulations. Its formula is Fst � 1−HS/H T , where HS is the average of the expected heterozygosity of the subpopulations and H T is the expected heterozygosity of the total population; (ii) Te minimum distance of Nei between pairs of genetic groups: its value also varies from 0, for identical populations, to 1, for totally diferent populations.

Level of Diversity of Microsatellite Markers Tested.
Among the eighteen (18) microsatellite markers tested, ffteen (15) were polymorphic ( Figure 3). For the remaining three primers tested, two failed to obtain bands (VM36 and VM38) and one (VM39) yielded unreadable bands. Allele sizes ranged from 50 bp to 500 bp. Polymorphic markers revealed a total of 101 alleles (At), with an average of 6.733 alleles per locus. Te number of diferent alleles per locus (Na) ranged from three (3) (Table 2).

Interpopulation Diversity: Diversity according to the Climatic Zone
Factor. Te analysis of molecular variance (AMOVA) revealed a nonsignifcant diference between accessions of the three climatic zones with a P value of 0.83. Te factor climatic zone would therefore have a very weak infuence on the expression of the genetic diversity of accessions (0%). Te accessions factor plays a 100% role in the expression of this diversity (Table 3). Tus, in the Sudano-Sahelian zone, the number of alleles per locus (Na) was 6.07; the Shannon diversity index (I) was 1.04; and the polymorphism information content (PIC) was 0.48 compared to 3.37 (Na), 0.88 (I), and 0.43 (PIC), respectively, for the Sahelian zone. Te values of these parameters are intermediate to those of the two previous zones in the Sudanian zone (Table 4).    Te parameters, minimum distance of Nei (0.06) and diferentiation index Fst (0.04), observed are higher between accessions from the Sahelian zone and those from the Sudanian zone. Low values of minimum Nei distance (0.03) and Fst diferentiation index (0.02) were observed between accessions from the Sudan-Sahelian zone and those from the Sudanian zone (Table 5).

Structuring of Genetic Diversity.
Te genetic diversity obtained was structured into three groups, independent of the climatic zone (Figure 4). Te AMOVA analysis of this diversity shows a signifcant diference between these three groups at the 5% threshold (P value � 0.004). In the expression of genetic diversity, the intergroup genetic variance is 2% and the intragroup variance is 98% (Table 6), so the accession factor would intervene at 98% in the expression of this diversity. Genetic group 3 includes the majority of accessions that showed better agronomic performance during agromorphological characterization, i.e., seven accessions (G-E2, S-E8, G-E4, S-E1, S-E2, Sao-E9, and P-E10) out of 13. In general, the genetic diversity parameters observed were high for group 3 and low for group 1 (Table 7). Tus, group 3 presented a Shannon diversity index (I) of 1.00 versus 0.63 for group 1, and the polymorphism information content (PIC) of group 3 was 0.47 versus 0.34 for group 1. Group 2 showed intermediate diversity parameters of the other two groups. Te values of minimum Nei distance and Fst diferentiation index were high between groups 1 and 3 (0.07 and 0.06) and low between groups 2 and 3 (0.03 and 0.02) ( Table 8).

Discussion
Sixty (60) accessions of Senna obtusifolia were characterized using 18 SSR markers. Fifteen microsatellites were polymorphic (VM) markers and revealed one hundred and one (101) alleles in total, and the number of alleles per locus ranged from three (3) to fourteen (14) with an average of 6.73 alleles per locus. According to FAO [25], an average of at least four (4) distinct alleles per locus is required and denotes average genetic polymorphism. Te average number of alleles in the present study (6.733) being higher than the average required by FAO [25], there would therefore be genetic polymorphism within the Senna obtusifolia collection. Work conducted by Mohanty et al. [16] revealed a total of 78 alleles for seven SSR markers of Senna obtusifolia. Te diference between the results of the present study and previous work could be explained by the size and nature of the plant material used. According to Kalinowski [26], large samples generally contain more alleles than small samples. Furthermore, according to Ben Naceur et al. [27], the number of alleles per locus is infuenced by several factors such as genotype and primer sequences as well as minor variations in the amplifcation protocol. Indeed, with the study [27] on the same VM markers, the number of alleles per locus varied from two to seven with an average of 4.7 in 90 cowpea accessions [19]. Te low average number of alleles per locus observed for cowpea accessions could be explained by the nature of the accessions (90 cowpea lines) that originated from the International Institute of Tropical Agriculture breeding program and one wild cowpea and that their genetic base would therefore be relatively narrow. Te rate of polymorphism being 100% for each of the markers would denote the ability of the markers to reveal the genetic diversity of the collection. Similar values were observed by Sawadogo [28] on forty-nine (49) accessions of Solanum aethiopicum L.
Tis very high rate of polymorphism testifes to the high level of polymorphism within the accessions and the efciency of the markers used for the analysis of the genetic diversity of the populations studied [29]. According to Shete et al. [30], the expected heterozygosity and PIC are two values that can be used to determine the level of polymorphism of markers. Te mean PIC value (0.47) in the present study was less than 0.5. According to Bostein et al. [31], markers with PIC greater than 0.5 are considered very highly informative. Eight (8) of the VM markers used have a PIC greater than 0.5 thus are very highly informative. However, it has been shown that PIC values between 0.25 and 0.5 provide reliable and usable information in characterization studies [32,33].
Tus, there would be moderate variability within the Senna obtusifolia accessions in the present collection. Te diferentiation index Fst allowed the study of diversity between subpopulations. Tus, the genetic diferentiation is weak between accessions of diferent climatic zones (average Fst of 0.03). According to Wright [34], Fst values indicate a genetic diferentiation of the populations studied, weak if they are between 0 and 0.05, moderate between 0.05 and 0.15, important between 0.15 and 0.25, and very important if the value is higher than 0.25. Te low value of the diferentiation index Fst confrms the fact that the climatic zone factor plays a weak role in the expression of the genetic diversity of the species. Moreover, the AMOVA analysis Te Scientifc World Journal revealed a nonsignifcant diference (P � 0.83) between accessions from diferent climatic zones. Tus, the genetic diversity of Senna obtusifolia would indeed be due to the nature of the plant material, as it is a plant that is always found in the wild. Indeed, according to Yuan et al. and Benor et al. [35,36], domestication causes increased homogenization within cultivated plants in contrast to spontaneous accessions that possess signifcant genetic diversity.
According to Ould Ahmed et al. [29], total genetic diversity is the sum of intrapopulation genetic diversity and interpopulation diversity. Based on FST values, only 3% of     Te Scientifc World Journal the total variability would be due to diferences between populations in diferent climatic zones. Te moderate genetic diversity in the present study could be due to the reproductive mode of Senna obtusifolia, which is cleistogamous, i.e., fertilization occurs before the opening of the fower buds. Tis phenomenon would reduce the genetic diversity within the species. Te diversity of the sixty (60) accessions was structured into three distinct genetic groups. Te accessions were grouped independent of their origins, thus forming composite groups. Te accessions that presented better agronomic performances during agromorphological characterization were mostly found in the genetic group 3. Te structuring of accessions into three genetic groups without taking into account their geographical origin confrms the low value of Fst (0.03), which refects a weak infuence of geographical origin on the total genetic diversity. Similar results were observed by Kiebre et al. [37,38]. Tis would therefore suggest that geographic origin does not necessarily refect the species characteristics and genetic diversity of Senna obtusifolia accessions in the present study. Similar results have been found by other authors such as Ambrosi et al. [39][40][41] on other species. Tis lack of correspondence between the molecular classifcation of accessions and geographic origin could refect their introduction from the same origin. Similar results were reported by Konan and Mergeai [38].
With a mean Fst value of 0.06, genetic diversity was moderate among the three genetic groups. Tis diference was confrmed by a P value (0.004) signifcant at 5% in AMOVA analysis. Genetic diversity parameters were high for group 3 and low for group 1. In addition, this group contains the majority of the best agronomic performance accessions from the agromorphological evaluation. According to Nacambo et al. [11], an agromorphological evaluation carried out on these same accessions revealed a group of accessions (group 3) that showed better agronomic performance. Te majority of the accessions in genetic group 3 are part of this group that showed better agronomic performance. Tus, the accessions of this group have a high number of primary ramifcations and leaf biomass. Tey produce lots of long but slender fruits and lots of seeds. Tey fower early.
Te results obtained in this study would therefore indicate the existence of a certain level of diversity that can be exploited by breeders.

Conclusion
Tis study revealed the existence of genetic diversity within the collection. Te genetic diversity of S. obtusifolia accessions was structured into three groups independent of geographic origin. Group 3 consisting of twenty-seven (27) accessions had the highest genetic parameters, which also includes the majority of the accessions that have better agronomic performances.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Disclosure
Tis research is part of a Ph.D. thesis called "Ethnobotanical Study and Genetic Diversity of a Collection of Senna obtusifolia (L.) Irwin and Barneby in Burkina Faso" [42]. Tis work was carried out during the author's unique PhD thesis at the Université Joseph KI-ZERBO de Ouagadougou.

Supplementary Materials
See the appendix for additional data. (Supplementary Materials) Te Scientifc World Journal 9