Genetic and cytogenetic analysis of Moringa oleifera short and long capsule phenotypes

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In the case of moringa, such studies are scarce. Therefore, the objective of this research was to determine the chromosomal number and genetic diversity parameters in two phenotypes of Moringa oleifera (short and long capsule) using chromosomal analysis and ISSR markers, respectively. Cytogenetic analyses were conducted using the "Somatic chromosomes in root apices" technique, with acetocarmine and papain for staining. It was possible to identify 2N = 28 chromosomes in a single cell of the long capsule phenotype, ranging in size from 0.05 to 0.10 µm. Cells in both moringa phenotypes were observed in prophase, metaphase, anaphase, and telophase. High genetic variability was found in both phenotypes of moringa, as indicated by a Shannon index of 0.81. Additionally, the principal component analysis and UPGMA groups revealed genetic isolation between the studied moringa phenotypes. The significant polymorphism obtained with most primers suggests that the tested ISSR markers are highly useful for studying genetic diversity in moringa. Chromosomal number ISSR Genetic diversity diploid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The genus Moringa is a monophyletic group comprising 13 species (AbdAlla et al., 2023 ), whose research has focused on their nutritional, antioxidant, pharmacological and pharmacokinetic properties. At present, chromosomal analysis is of utmost importance in plant breeding programs, as well as in phylogenetic and evolutionary plant studies, allowing to obtain multiple data such as the length of chromosomes, the presence of primary and secondary constrictions, the position of the centromere (Sánchez-García et al., 2018 ) the number of chromosomes, which is a central feature in eukaryotic genomes (Rice & Mayrose, 2021 ). The Moringa genus has been characterized mostly by its morphological characteristics (Hausiku et al., 2020a ) with few cytological studies (both in mitosis and meiosis); and presenting limitations in karyotype analysis due to the presence of too small chromosomes (Boopathi & Raveendran, 2021 ; Mohamed Anwar, 2016 ) which hinders a complete analysis. One species of scientific importance for its biological properties in the pharmaceutical and food area is M. oleifera (Hassan et al., 2021 ; Islam et al., 2021 ), whose short capsule (15 to 25 cm) and long capsule (30 to 80 cm) variants are of scientific importance (Castillo-López et al., 2017 ), and whose cytological information is limited describing the presence of 14 bivalents in diakinesis as meiotic behavior (Boopathi & Raveendran, 2021 ; Silva et al., 2011 ). On the other hand, the use of molecular genetic markers is of great importance to detect genetic variability of genotypes of interest at the DNA level, and to develop effective selection strategies for breeding programs (Alavilli et al., 2022 ; RS et al., 2022 ; Saraswathi et al., 2023 ), since detection of genetic polymorphisms can contribute to improve the accuracy and efficiency of conventional breeding programs (Darkwa et al., 2020 ; Li et al., 2020 ). The most commonly used molecular marker techniques are based on DNA amplification employing the PCR technique, which generates copies of a DNA region, providing genetic information for genetic variability analysis (Orozco-Sifuentes et al., 2023 ). M. oleifera has been studied previously using AFLP (amplified fragment length polymorphism) markers showing variations among the studied populations (Peter et al., 2023 ), likewise, studies by employing RAPD molecular markers recorded low genetic diversity in Moringa accessions from the Brazilian Germplasm Bank and a high level of polymorphism (81.5%) in Nigerian populations (Bernard et al., 2013 ). On the other hand, reports on the use of ISSR (Inter Simple Sequence Repeat) markers, are very limited (Hassanein & Al-Soqeer, 2018 ; Peter et al., 2023 ), being this information for the detection of genetic variability in multiple plant species by the its high polymorphism rate, co-dominance, high reproducibility, wide variability and economic feasibility (Cardoso et al., 2019 ; Tamboli et al., 2018 ). ISSR markers are based on the amplification of intermediate zones between microsatellites, presenting the favorable characteristics of markers such as microsatellite (SSR), AFLP and RAPD (Aydin et al., 2023 ). Due to the limited information on chromosome number and molecular characterization of moringa diversity, the objectives of this research were to determine the cytological behavior, chromosome number and estimate genetic diversity by ISSR molecular markers of two phenotypes of M. oleifera (short capsule and long capsule). Materials and Methods Material vegetal To obtain meristems (cytogenetic analysis) and leaf tissue (DNA extraction), seeds of two phenotypes of M. oleifera were sown: short capsule (15 to 25 cm) and long capsule (30 to 80 cm) (Castillo-López et al., 2017 ). Moringa seeds were purchased from the Autonomous University of Nuevo León, San Nicolás, Nuevo León, México. Cytogenetic analysis Chromosome number determination For this section, 10 moringa seeds of each phenotype (short and long capsule) previously disinfected with 1% NaClO and treated with N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide fungicide (Captan 50 WP, ADAMA, México) were used. Seeds were placed in Petri dishes with a moistened filter paper base and subjected to germination conditions in a germinator (Achieva Basic Style, Seedburo, USA) at 25 ± 1°C (Garcia-Ortiz et al., 2023 ). Then, for cytological study, root tips of 1 to 2 cm were collected and pretreated with 0.04% 8-hydroxyquinoline at 26 ± 1°C for 2 h. Treated roots were fixed for 24 h with Farmer's 3:1 (ethanol:glacial acetic acid) solution (Ortiz-Curiel et al., 2018 ). Once the roots were fixed, they were washed three times with distilled water for 30 min in each rinse. Subsequently, roots were subjected to aceto-carmine staining (carmine dissolved in 45% acetic acid), then the flasks were placed in a burner flame (7 times) with 7 s of exposure and 7 s without exposure. The meristems were placed for five days in carmine dye, and finally a drop of FeCl3 ammonia was added for staining (Almejo Vazquez et al., 2022 ). Degradation with papain and microscopic observation For this section, the technique reported by (Ortiz-Curiel et al., 2018 ) was applied with some modifications. To promote alteration in the cell wall and pectin, Moringa roots were immersed in papaya extract (obtained from the filtrate of the mixture of 100 g of papaya and 20 mL of H 2 O) at room temperature for 30 min, subsequently, samples were washed with distilled water for 30 min, finally, roots were stained using the acetocarmine technique (Almejo Vazquez et al., 2022 ). Meristems were sampled and placed on a slide which had a drop of carmine dye, then the sample was heated and gently pressed on a filter paper to remove excess dye. Cells were observed and photographed at 100X using a visible light microscope (Axiostar plus, Karl Zeiss, Germany) coupled to a camera (Canon, PowerShot G5, Japan). Chromosome measurements and analysis were performed with AxioVision Rel 4.8 software. Genetic diversity with ISSR molecular markers Vegetal material To obtain leaf tissue, 25 seeds of each type of moringa (Table 1 ) were sown in biodegradable germination boxes under greenhouse conditions. Once the seedlings emerged, they were transplanted into one-liter polyurethane pots. Subsequently, young leaves were collected separately for analysis. Samples were transported on ice and in the absence of light. Briefly, samples were disinfected with 70% EtOH for 2 min and 1% NaClO for 1 min, rinsed with distilled water, weighed 200 mg of each sample and placed to dry. Finally, they were stored at -80°C in a freezer (Panasonic, MDF-V56VC-PA, USA) until genomic DNA extraction. Table 1 Identification of short and long capsule Moringa oleifera . Plant Short capsule Long capsule Plant Short capsule Long capsule 1 S 23 − 2 L135-1 14 S 46 − 2 L 95 − 1 2 S 33 − 1 L 137-1 15 S 59 − 2 L 100-1 3 S 44 − 1 L 132-1 16 S 62 − 1 L 102-2 4 S 2 − 1 L 136-1 17 S 63 − 1 L 103-1 5 S 4 − 1 L 133-1 18 S 64 − 1 L 109-2 6 S 5 − 1 L 128-2 19 S 65 − 2 L 120-1 7 S 6 − 1 L 129-2 20 S 67 − 1 L 121-1 8 S 7 − 1 L 138-1 21 S 70 − 1 L 11 − 2 9 S 8 − 1 L 139-1 22 S 71 − 2 L 13 − 1 10 S 21 − 1 L 70 − 2 23 S 72 − 1 L 15 − 2 11 S 22 − 1 L 71 − 3 24 S 73 − 1 L 24 − 3 12 S 26 − 2 L 90 − 1 25 S 18 − 1 L 28 − 1 13 S 32 − 1 L 92 − 3 DNA isolation In this case, the CTAB extraction method (Doyle & Doyle, 1987 ; Shahzad et al., 2013 ) with slight modifications was used, where samples were frozen at -80°C and ground employing a mortar with previously sterilized pistil, subsequently placed in 1.5 mL vials and 800 µL CTAB buffer solution with 20 µL of 20% bovine serum albumin were added, then sample was heated at 55°C for 20 min in a water bath (Premiere, HH-4). Then, samples were centrifuged at 13400 rfc for 5 min at 4°C in a refrigerated centrifuge (Select Bioproducts, Spin-17R). After, the supernatant was recovered and transferred to a vial with a chloroform:isoamyl alcohol solution (24:1 v/v), subsequently, it was mixed by gentle inversions for 2 min, finally, samples were centrifuged at 13400 rfc for 10 min. The aqueous phase was transferred to 50 µL of a solution of 7.5 M ammonium acetate and 800 µL of cold 96% EtOH, the solution was mixed by gentle inversions and left to stand for 12 h at -20°C. Samples were centrifuged at 13400 rfc for 5 min and the sediment was recovered. The DNA sediments were washed with 70% EtOH, after that, samples were allowed to dry at room temperature for 2 h, then resuspended in 80 µL of milli-Q water and 12 µL of RNAase enzyme and placed in a dry bath (Labnet, AccuBlock Digital, USA) at 37°C for 30 min. Upon completion of drying, samples were stored at -20°C for quantification and use in PCR reactions. DNA concentration was determined from the ratio of wavelength absorption at 260 and 280 nm obtained from Spectrophotometer (Biotek, Epoch). Finally, DNA integrity was observed by electrophoresis in 1% agarose gels using a transilluminator (Spectroline) with UV light and Launch VisionsWorks L5 software (version 6.8). PCR Amplification To determine polymorphism within and between both moringa types, 10 ISSR primers were tested (Table 2 ). DNA amplification was performed in a total volume per reaction of 23 µL containing: 8 µL of ddH 2 O, 5 µL of 10X Buffer with MgCl 2 , 0.5 µL of dNTP's, 6 µL of primer, 0.5 µL of Taq polymerase (5U/µL) and 3 µL of Moringa genomic DNA. PCR amplifications were programmed in a thermal cycler (Axygen, Maxynene) with the following specifications: a denaturation temperature was used at 94°C for 1 min, followed by 45 amplification cycles, where a different alignment temperature was used for each primer (Table 2 ) for 45 s, followed by a polymerization temperature at 72°C for 2 min, at the end of the 45 cycles, a temperature of 72°C was maintained for 7 min. The amplified products were electrophoretically separated on 1.5% agarose gels, stained with ethidium bromide, using SB 1X buffer and a 100 bp − 3000 bp DNA Ladder molecular weight marker (Axygen biosciences). Table 2 ISSR primers used for PCR amplification of two Moringa oleifera phenotypes. Primer Sequence (5’ – 3’) Annealing temperature (°C) 1 GAGAGAGAGAGAGAGATC 47.7 2 AGAGAGAGAGAGAGAGTC 49.7 3 CTCTCTCTCTCTCTCTAG 40.0 4 CTCCTCCTCCTCCTCCTC 55.0 5 GAGAGAGAGAGAGAGAT 52.0 6 GTGTGTGTGTGTGTGTA 48.0 7 AGCAGCAGCAGCAGCGA 49.0 8 ACACACACACACACACA 47.0 9 GTGTGTGTGTGTGTGTTC 54.0 10 TCTCTCTCTCTCTCTCC 54.1 Analysis of ISSR profiles Analyses were performed using the statistical language R (R Development Core Team, 2011). A binary data matrix of presence (1) and absence (0) of bands was generated to determine the Shannon Index as a measure of genetic diversity. Fisher's exact test was performed to assess the association between markers and short and long capsule traits. In addition, an ANOVA, various genetic measurements and Wright's parameters (Fst, Fis and Fit) were estimated using InfoGen software (2011). Genetic relationships between the two moringa types were determined by cluster analysis using the unweighted pairwise method grouping algorithm with arithmetic mean (UPGMA) and to group the studied individuals a Principal Coordinate analysis (PCoA) was performed using InfoGen software (2011). Results and Discussion Cytogenetic analysis The different mitotic phases identified in the cytological analysis of M. oleifera cells reveal the presence of small chromosomes which, due to their size, present limitations during the identification of the complete karyotype (Liu et al., 2020 ). On the other hand, after hydrolysis with papain, 2N = 28 chromosomes were visualized in a single metaphase cell of the long capsule phenotype with a chromosome size between 0.05 and 0.10 µm (Fig. 1 ) (Table 3 ), having a number of chromosomes similar to that reported in previously studies (Chang et al., 2022 ) and a chromosome size smaller than previously reported in the mitotic metaphase of M. peregrina species with sizes between 1.19 and 2.1 µm which is attributed to genomic differences between species (Nazari et al., 2012 ). Table 3 Mitotic chromosome length of a metaphase cell of the long capsule Moringa oleifera type. Chromosome Length (µm) Chromosome Length (µm) Chromosome Length (µm) 1 0.09 11 0.09 20 0.06 2 0.07 12 0.08 21 0.05 3 0.05 13 0.09 22 0.05 4 0.05 14 0.10 23 0.07 5 0.06 15 0.07 24 0.05 6 0.06 16 0.06 25 0.07 7 0.06 17 0.06 26 0.08 8 0.07 18 0.08 27 0.06 9 0.07 19 0.06 28 0.07 10 0.08 The results of this work in comparison to other moringa species such as M. peregrina sharing the same biological and geographical origin, present differences in chromosome size, with M. oleifera being 7 times smaller than those documented in M. peregrina , which is attributed to the type of treatment and time used in the cytogenetic analysis due to size variations during the cell cycle (Abdel-Hameed, 2015 ; Nazari et al., 2012 ). Likewise, previous reports with M. oleifera and M. Stenopela have documented the presence of 28 small bivalent chromosomes that are difficult to measure and identify, typical of a diploid condition of the species (Mohamed Anwar, 2016 ) Likewise, presence of small chromosomes in both plants are frequent characteristics of the monogenean family Moringaceae which is attributed to their geographical origin or genetic ancestry (Lysak, 2018 ). On the other hand, after performing the cytological technique, it was not possible to observe the chromosomal number in the short capsule phenotype which is attributed to the nature of the sample as well as the short age of the seedling presenting cells in interphase which limits the observation by the established technique (Windham et al., 2020 ). Figure 2 shows micrographs of both types of moringa where the prophase, metaphase, anaphase and telophase were observed during the mitotic phases, where different types of cells were observed in the meristems of both phenotypes of moringa in which cells in mitotic division, meristematic cells with diffuse chromatin and larger cylindrical cells with diffuse chromatin characteristic cells in species of the Moringaceae Family such as M. oleifera and M. Stenopetala (Mohamed Anwar, 2016 ). Genetic Diversity In this study, the ISSR polymorphism pattern in two phenotypes of M. oleifera (short capsule and long capsule) was evaluated by determining the closeness of relationship or distance between the accessions. The analysis detected the presence of 129 polymorphic bands obtained using 10 primers (where primers 7 and 8 were eliminated from the study because they did not amplify during PCR), a number higher than that documented in different works with SSR, where 58 polymorphic bands have been amplified employing 14 primers (Natarajan & Joshi, 2015 ). In addition, a genetic diversity measured as Shannon entropy of 0.81 was presented (Fig. 3 ), indicating a high genetic diversity among moringa types. During performance of the Fisher exact test, probability was corrected by False Discovery Rate, thus, the column (fdr) is the one that should be considered to declare significance (Table 4 ). A total of 67 markers were significant with fdr ≤ 0.05, which could be an indication that the association is due to the population structure between the two types of plants, and not indicative of the markers being associated with the coding region. Table 4 Examples of markers with values from the Fisher exact test. Number Marker p.value Fdr 1 P1_1B6 4.029123e-03 9.281273e-03 2 P1_1B7 6.364236e-04 1.757082e-03 3 P1_1B8 9.545655e-06 4.329636e-05 4 P1_1B9 1.069942e-09 1.235297e-08 5 P1_1B10 6.364236e-04 1.757082e-03 6 P1_1B11 1.921510e-03 4.980239e-03 Table 5 shows the measures of genetic diversity and Wright statistics, which indicate the presence of polymorphic loci with similarities in both types of moringa (short and long capsule), which is reflected in the variation in the population, and is related to the frequency of the most popular allele (less than 0.99 − 0.95). Likewise, in this work the genetic diversity (variability of an inbred population) of both genotypes was observed, in general in previous works have categorized Moringa oleifera as a species of high genetic diversity (Lakshmidevamma et al., 2021 ), in particular in this work a higher genetic diversity was presented in the short capsule population compared to that of the long capsule population with values of 0.33 and 0.31 respectively, similar behaviors in studies of cross-pollinated plants (Tomas et al., 2017 ). On the other hand, the genetic variation of the population (which represents the average number of alleles) was 1.88, a value attributed to cross-pollinated plants which can improve genetic diversity, and provide an advantage by reducing the risk of inbreeding depression (Oliveira et al., 2024 ). Likewise, an average number of alleles close to 1.55 is attributed to free pollination by factors in the environment such as air, and animals such as birds and insects, which could be a form of pollination due to moringa flower morphology and general tree morphology (Lakshmidevamma et al., 2021 ; Singh et al., 2020 ). The Fst value to indicate the level of genetic variation in a subpopulation compared to total variation was higher in long-capped moringa (more than double) compared to short-capped moringa, indicating a classification with moderate differences and greater interpopulation genetic differentiation (Barrera-Guzmán et al., 2020 ). Fis values (reflecting the degree of inbreeding between the populations studied) was 0.029 and 0 for the short and long capsule population, respectively, populations with Fis values close to 0 present in long capsule moringa indicate that they are in heterozygote balance or similarity, while those with values different from 0 (short capsule moringa) reflect excess or deficiency of heterozygotes. The Fit values determined in this study indicate a heterozygote deficiency in both moringa populations with values of 0.083 (moringa short capsule) and 0.138 (moringa long capsule). Table 5 Genetic diversity measurements determined by ISSR markers in two moringa type populations. Statistical Short capsule type Long capsule type Total Polymorphic loci 0.88 0.88 0.92 Genetic diversity 0.33 0.31 0.35 Nei heterozygosity 0.34 0.31 0.36 Average number of alleles 1.88 1.88 1.96 Fst 0.055 0.138 Fis 0.029 0.000 Fit 0.083 0.138 ANOVA of the polymorphic band ISSR indicated significant differences between (p-value 0.0001) and within moringa populations (p-value 0.0001) (Table 6 ). A 4-fold higher variance was shown between capsule-type populations than within moringa types. This result, as well as Wright's statistics, suggest a subdivision in Moringa oleifera . Table 6 Analysis of variance of polymorphic ISSR bands from two moringa type populations. Source SS df MS p-value Iter.# VC Average Between types 34.65 1 34.65 < 0,0001 400 1.31 13.51 Within types 319.30 38 8.40 < 0,0001 400 8.40 86.49 Total 353.95 39 9.08 9.72 100.00 In the principal coordinate analysis (PCoA) (Fig. 4 ), the first two axes account for 47.5% of the total variation. In addition, a division reflecting the difference in genetic structure between short-capsule, and long-capsule moringa is observed, showing distinct clusters in the projection in Fig. 4 . Individuals of the short capsule moringa phenotype were grouped in a sector of smaller dispersion indicating a greater similarity in genetic structure, while in the long capsule Moringa phenotype, a wide separation is observed between individuals of the long capsule phenotype, indicating a greater difference in genetic structure. This type of behavior is observed in different genetic identification and population variation studies, where one of the important factors is the presence and absence of genes derived from one or multiple common ancestors, and the crossbreeding or isolation of these genes generated by development and evolution in different geographical conditions (Natarajan & Joshi, 2015 ; Nguyen et al., 2023 ). According to the UPGMA clustering (Fig. 5 ), it was observed the formation of two different groups based on the Euclidean distances between the two phenotypes. Cluster analysis confirms the grouping of individuals within the short capsule phenotype, and the separation of individuals from the long capsule phenotype. This indicates that the phenotypic expression of both phenotypes (Moringa short and long capsule) are associated with the genetic structure of each of the phenotypes, ruling out the presence of different capsules due to phenotypic plasticity effects (Hausiku et al., 2020b ). Based on cluster and principal component analyses, it can be deduced that M. oleifera populations have a high level of genetic variability, most likely due to their cross-pollination type. This variability is the basis for establishing a genetic improvement program in this species. Representative profiles of ISSR products in the two moringa phenotypes can be seen in the annex (A1). In Fig. 6 , it can be observed that primer 9 offered the highest number of bands and polymorphism in both phenotypes, followed by primers 5 and 6 (A1). On the other hand, primer 3 presented a lower number of identified bands which were monomorphic bands in the long capsid phenotype, while there was no amplification in the short capsid phenotype (Fig. 7 ). Finally, the long capsid phenotype showed greater amplification of bands than the short capsid phenotype with most of the primers (A1), indicating greater variability in the long capsid phenotype population, with the exception of primer 10 where greater amplification was observed in the short capsule in contrast to the long capsule as only 2 to 3 bands were observed (Fig. 8 ), a number of bands similar to the averages documented in other studies with a frequency between 2 and 6 bands with Moringa oleifera species from Tamil Nadu (Natarajan & Joshi, 2015 ). The observed polymorphisms indicate that ISSR primers are good and reliable for the assessment of genetic diversity in M. oleifera with potential applications in breeding programs (Lakshmidevamma et al., 2021 ; Natarajan & Joshi, 2015 ). Conclusions The number of chromosomes observed was 2N = 28 chromosomes in the long capsule phenotype of Moringa oleifera with a size between 0.05 to 0.10 µm, in prophase, metaphase, anaphase and telophase, in addition, ISSR markers with the capacity to be used for genetic diversity studies in Moringa oleifera with a high degree of polymorphism were identified. Furthermore, the short and long capsule phenotypes belong to two different genotypes with genetic variability between and within genotypes, which will allow the implementation of a genetic improvement program in this specie. Declarations Declaration of competing interest: M. Gómez-Martínez, J. D. García-Ortiz, S. Gómez-Martínez, A. C. Flores-Gallegos, M. H. Reyes-Valdés, J. A. 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South African Journal of Botany , 131 , 335–341. https://doi.org/10.1016/j.sajb.2020.03.002 . Hausiku, M. K., Kwembeya, E. G., Chimwamurombe, P. M., & Mbangu, A. (2020b). Assessment of species boundaries of the Moringa ovalifolia in Namibia using nuclear its DNA sequence data. South African Journal of Botany , 131 , 335–341. https://doi.org/10.1016/j.sajb.2020.03.002 . Islam, Z., Islam, S. M. R., Hossen, F., Mahtab-Ul-Islam, K., Hasan, M. R., & Karim, R. (2021). Moringa oleifera is a Prominent Source of Nutrients with Potential Health Benefits. International Journal of Food Science , 2021 (July 2015). https://doi.org/10.1155/2021/6627265 . Lakshmidevamma, T. N., Ugalat, J., Apoorva, K. A., Suresh, S. P. G., Doddamani, M., Kadam, S., Nayana, R. S., Jagadeesha, R. C., & Fakrudin, B. (2021). Genetic Diversity of Moringa (Moringa Oleifera Lam.) BT - The Moringa Genome. In N. M. Boopathi, M. Raveendran, & C. Kole (Eds.), The Moringa Genome (pp. 57–65). Springer International Publishing. https://doi.org/10.1007/978-3-030-80956-0_7 . Li, X., Liu, X. T., Wei, J. T., Li, Y., Tigabu, M., & Zhao, X. Y. (2020). Genetic Improvement of Pinus koraiensis in China: Current Situation and Future Prospects. Forests , 11 (2). https://doi.org/10.3390/f11020148 . Liu, X., Sun, S., Wu, Y., Zhou, Y., Gu, S., Yu, H., Yi, C., Gu, M., Jiang, J., Liu, B., Zhang, T., & Gong, Z. (2020). Dual-color oligo-FISH can reveal chromosomal variations and evolution in Oryza species. Plant Journal , 101 (1), 112–121. https://doi.org/10.1111/tpj.14522 . Lysak, M. A. (2018). Brassicales: an update on chromosomal evolution and ancient polyploidy. Plant Systematics and Evolution , 304 (6), 757–762. https://doi.org/10.1007/s00606-018-1507-2 . Mohamed Anwar, G. (2016). Studies of some cytological features on two Moringa Species (M. Oleifera And M. Stenopetala) Cultivated In Egypt. Minia Journal of Agricultural Research and Development , 34 (4), 601–611. https://www.researchgate.net/publication/313701739%0ASTUDIES . Natarajan, S., & Joshi, J. (2015). Characterisation of Moringa (Moringa oleifera Lam.) Genotypes for Growth, Pod and Seed Characters and Seed Oil using Morphological and Molecular markers. Vegetos- An International Journal of Plant Research , 28 , 64. https://doi.org/10.5958/2229-4473.2015.00039.7 . Nazari, Z., Mirzaie-Nodoushan, H., Bakhshi-Khaniki, G., & Asadicorom, F. (2012). Karyotypic characteristics of Moringa peregrina (Forssk.) Fiori in Iran. Iranian Journal of Medicinal and Aromatic Plants Research , 27 (4), 635–646. https://doi.org/10.22092/ijmapr.1390.4513 . Nguyen, B. L. Q., Chanthanousone, H., Ho, H. N., Ho, N. T. H., Le, M. H. D., Rasphone, S., Nguyen, C. Q., & Truong, H. T. H. (2023). Waterlogging tolerance, phenolic and flavonoid contents, and genetic diversity among Moringa oleifera self-pollinated lines. South African Journal of Botany , 157 , 287–296. https://doi.org/10.1016/j.sajb.2023.04.012 . Oliveira, A. M., Ramos, S. L. F., Ferreira, M. J., Lopes, R., Meneses, C. H. S. G., Valente, M. S. F., da Silva, R. F., Batista, J. da, Muniz, S., A. W., & Lopes, M. T. G. (2024). Mating System Analysis and Genetic Diversity of Parkia multijuga Benth. One Native Tree Species of the Amazon. Forests , 15 (1). https://doi.org/10.3390/f15010172 . Orozco-Sifuentes, M. M., Castillo-Godina, R. G., Campos-Muzquiz, L. G., Palomo-Ligas, L., Nery-Flores, S. D., García-Ortiz, J. D., Flores-Gallegos, A. C., & Rodríguez-Herrera, R. (2023). DNA barcoding of crop plants. Genomics Transcriptomics Proteomics and Metabolomics of Crop Plants , 199–228. https://doi.org/10.1016/B978-0-323-95989-6.00010-3 . Ortiz-Curiel, S., Carrillo-Castañeda, G., Corona-Torres, T., Cortés-Cruz, M., & Muratalla-Lua, A. (2018). Karyotype Determination In Native Heliconias Of Mexico. Revista Fitotecnia Mexicana , 41 (3), 221–226. https://doi.org/10.35196/rfm.2018.3.221-226 . Peter, A. F., Wagiran, A., Rahmat, Z., Yusop, M. R., & Ridzuan, R. (2023). Exploring Genetic Variation and Therapeutic Properties of Moringa oleifera: Progress and Future Potential for Crop Improvements. Pharmacognosy Reviews , 17 (34), 426–438. https://doi.org/10.5530/phrev.2023.17.18 . Rice, A., & Mayrose, I. (2021). Model adequacy tests for probabilistic models of chromosome-number evolution. New Phytologist , 229 (6), 3602–3613. https://doi.org/10.1111/nph.17106 . RS, D. R., Nair, B. R., & Siril, E. A. (2022). Fingerprinting and genetic variability in drumstick (Moringa oleifera Lam.) elite trees using RAPD markers- An underutilized and wild edible future crop. South African Journal of Botany , 145 , 370–377. https://doi.org/10.1016/j.sajb.2021.11.009 . Sánchez-García, Y., Imery, J., & Raymúndez, M. (2018). Citogenética convencional y molecular aplicadas a propuestas de fitomejoramiento en Aloe vera (L.) Burm.f. Revista Cientifica Unet , 30 (1), 167–178. Saraswathi, T., Meena, P., Pugalendhi, L., & Manikanda Boopathi, N. (2023). Assessment of genetic divergence and molecular characterization of moringa (Moringa oleifera Lam.) genotypes. South African Journal of Botany , 157 , 502–507. https://doi.org/10.1016/j.sajb.2023.04.020 . Shahzad, U., Khan, M. A., Jaskani, M. J., Khan, I. A., & Korban, S. S. (2013). Genetic diversity and population structure of Moringa oleifera. Conservation Genetics , 14 (6), 1161–1172. https://doi.org/10.1007/s10592-013-0503-x . Silva, N., Mendes-Bonato, A. B., Sales, J. G. C., & Pagliarini, M. S. (2011). Meiotic behavior and pollen viability in Moringa oleifera (Moringaceae) cultivated in southern Brazil. Genetics and Molecular Research: GMR , 10 (3), 1728–1732. https://doi.org/10.4238/vol10-3gmr1490 . Singh, M., Singh, S., & Verma, D. (2020). Morphological and Pharmacognostical Evaluation of Moringa oleifera Lam. (Moringaceae): A Plant with High Medicinal Value in Tropical and Subtropical Parts of the World. Pharmacognosy Reviews , 14 (28), 138–145. https://doi.org/10.5530/phrev.2020.14.17 . Tamboli, A. S., Yadav, P. B., Gothe, A. A., Yadav, S. R., & Govindwar, S. P. (2018). Molecular phylogeny and genetic diversity of genus Capparis (Capparaceae) based on plastid DNA sequences and ISSR markers. Plant Systematics and Evolution , 304 (2), 205–217. https://doi.org/10.1007/s00606-017-1466-z . Tomas, P. A., Ziets, R., & Cerino, M. C. (2017). Análisis de diversidad genética en Ziziphus mistol griseb. Mediante marcadores moleculares ISSR. Fave Sección Ciencias Agrarias , 16 (1), 153–162. http://www.scielo.org.ar/scielo.php?pid=S1666- 77192017000100009&script=sci_arttext&tlng=en. Windham, M. D., Pryer, K. M., Poindexter, D. B., Li, F. W., Rothfels, C. J., & Beck, J. B. (2020). A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited. Applications in Plant Sciences , 8 (4), e11342. https://doi.org/10.1002/aps3.11342 . Additional Declarations No competing interests reported. Supplementary Files ANNEXES.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4202934","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287148090,"identity":"94d53bed-f7ca-41bd-a893-8118748795e3","order_by":0,"name":"Martha Gómez-Martínez","email":"","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":false,"prefix":"","firstName":"Martha","middleName":"","lastName":"Gómez-Martínez","suffix":""},{"id":287148091,"identity":"e9c903f6-2415-44d4-a7f8-990e7e638ca9","order_by":1,"name":"Jesús David García-Ortiz","email":"","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"David","lastName":"García-Ortiz","suffix":""},{"id":287148092,"identity":"9d9c011c-6011-42e8-b704-1383065e09d9","order_by":2,"name":"Susana Gómez-Martínez","email":"","orcid":"","institution":"Universidad Autónoma Agraria Antonio Narro","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Gómez-Martínez","suffix":""},{"id":287148093,"identity":"720d9dac-651a-4de7-889c-ee95ddadf1da","order_by":3,"name":"Adriana C. Flores-Gallegos","email":"","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"C.","lastName":"Flores-Gallegos","suffix":""},{"id":287148094,"identity":"348259a5-4f3f-4bed-a3fa-20caa5b272ac","order_by":4,"name":"M. Humberto Reyes-Valdés","email":"","orcid":"","institution":"Universidad Autónoma Agraria Antonio Narro","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"Humberto","lastName":"Reyes-Valdés","suffix":""},{"id":287148095,"identity":"c1085590-aed5-4e28-92a8-8bcf45cbce9a","order_by":5,"name":"Jesús A. Morlett-Chávez","email":"","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"A.","lastName":"Morlett-Chávez","suffix":""},{"id":287148096,"identity":"dda6bd8e-05c4-410b-8fc1-cd2eeaa1e2b5","order_by":6,"name":"Raúl Rodríguez-Herrera","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYHACxgMgooEZzLEB4gQQgxmvHmQtaaRogbAPE9Zi3n7G4ADjHhvZ7e3s1yQ+/Dmf2N+efPADQ4V1YoPYGQNsWmTO5BgcYHiWZjznME+Z5Aye24kzzjxLlmA4k57YIJ2WgE2LBANIy4HDiTOYedKkeSRuJzbcyDFjYGw7DNSSfACrFv43SFr+GJxLnH8j/xsD4z+QlsQGrFok4LawH5NmSDiQuOFGDhswNPDYIvGs4EDCgTRjoC3Mlj0Hko03nnlmLJFwLN24DZdf+JM3PvhwwEZ2Bv/xhzd+/LGTnXc8+eGHDzXWsv3SOVhDDAwghvEYoIqw4VQPB+wPCKsZBaNgFIyCEQkArEpmF+SP8g8AAAAASUVORK5CYII=","orcid":"","institution":"Autonomous University of Coahuila","correspondingAuthor":true,"prefix":"","firstName":"Raúl","middleName":"","lastName":"Rodríguez-Herrera","suffix":""}],"badges":[],"createdAt":"2024-04-01 23:44:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4202934/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4202934/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54322562,"identity":"e02c012d-379a-433d-ac4b-839691465db9","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471568,"visible":true,"origin":"","legend":"\u003cp\u003eMetaphasic chromosomes in the long capsule \u003cem\u003eMoringa oleifera\u003c/em\u003e type\u003c/p\u003e","description":"","filename":"Fig.1..png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/5bb39677aee19f2d61769bfc.png"},{"id":54322560,"identity":"e96b7404-34d2-4127-a747-33ac74c18c90","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1540092,"visible":true,"origin":"","legend":"\u003cp\u003eCells in different mitotic phases from two types of \u003cem\u003eM. oleífera\u003c/em\u003e (a = Short capsule, b = Long capsule, 1= Prophase, 2= Metaphase, 3= Anaphase and 4= Telophase)\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/f568ac2d65dba17c4a555dd4.png"},{"id":54322563,"identity":"af0948c5-fa20-4fb3-9757-b12dbe909d4c","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75241,"visible":true,"origin":"","legend":"\u003cp\u003eShannon entropy distribution through markers\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/a1c7b353e4b510f07ac0ac3c.png"},{"id":54322561,"identity":"c77274bd-0e31-4d92-9754-0b5f22c3b6a6","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":370759,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCoA) obtained from Jaccard similarity using ISSR markers in two \u003cem\u003eMoringa oleifera\u003c/em\u003e phenotypes\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/4a280e731e2ac3c94b17313a.png"},{"id":54322559,"identity":"2a131e01-f4d8-40d1-866c-3c2fd4a90947","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54223,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram showing genetic diversity in two phenotypes of \u003cem\u003eMoringa oleifera\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/487e9537eb3e48100d0ae90f.png"},{"id":54322568,"identity":"7f10efbb-d3c5-4065-af9f-aeb3b321f787","added_by":"auto","created_at":"2024-04-08 19:50:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":952560,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of ISSR bands generated by the primer 9 in both phenotypes of \u003cem\u003eMoringa oleifera\u003c/em\u003e. A high polymorphism is observed. ISSR-9 = Primer; M = DNA Ladder; Short capsule and long capsule = \u003cem\u003eMoringa oleifera\u003c/em\u003e phenotypes\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/71a5e0d7fc4d4cc724bc533e.png"},{"id":54322567,"identity":"a63b5e67-49e0-477a-bbf0-4e3669e14e20","added_by":"auto","created_at":"2024-04-08 19:50:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":744603,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of ISSR bands generated by primer 3 in the long capsule phenotype and absence of bands in the short capsule phenotype of \u003cem\u003eMoringa oleifera\u003c/em\u003e. ISSR-3 = Primer; M = DNA Ladder; Short capsule and Long capsule = \u003cem\u003eMoringa oleifera\u003c/em\u003e phenotypes\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/cb5db4929fbd41ca3a3ec58f.png"},{"id":54322564,"identity":"a2eb62f2-2a07-4015-b911-3f3613b84ac7","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":176469,"visible":true,"origin":"","legend":"\u003cp\u003eISSR band profile generated by primer 10 in the long capsule and short capsule phenotypes of \u003cem\u003eMoringa oleifera\u003c/em\u003e. ISSR-9 = Primer; M = DNA Ladder; Short capsule and Long capsule = \u003cem\u003eMoringa oleifera\u003c/em\u003ephenotypes\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/a5abf403eabe6c82baff9a06.png"},{"id":54752391,"identity":"e5af51ab-a35a-4f08-a8c5-666c404ffef8","added_by":"auto","created_at":"2024-04-16 08:58:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4047099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/61d9c544-6ab1-4e44-8169-1df4015e7e86.pdf"},{"id":54322565,"identity":"666e7c75-d96e-4223-b966-149a9807dc21","added_by":"auto","created_at":"2024-04-08 19:50:23","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":891614,"visible":true,"origin":"","legend":"","description":"","filename":"ANNEXES.docx","url":"https://assets-eu.researchsquare.com/files/rs-4202934/v1/0785cc93801db71a80d2817c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic and cytogenetic analysis of Moringa oleifera short and long capsule phenotypes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eMoringa\u003c/em\u003e is a monophyletic group comprising 13 species (AbdAlla et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), whose research has focused on their nutritional, antioxidant, pharmacological and pharmacokinetic properties. At present, chromosomal analysis is of utmost importance in plant breeding programs, as well as in phylogenetic and evolutionary plant studies, allowing to obtain multiple data such as the length of chromosomes, the presence of primary and secondary constrictions, the position of the centromere (S\u0026aacute;nchez-Garc\u0026iacute;a et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) the number of chromosomes, which is a central feature in eukaryotic genomes (Rice \u0026amp; Mayrose, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003eMoringa\u003c/em\u003e genus has been characterized mostly by its morphological characteristics (Hausiku et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e) with few cytological studies (both in mitosis and meiosis); and presenting limitations in karyotype analysis due to the presence of too small chromosomes (Boopathi \u0026amp; Raveendran, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mohamed Anwar, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) which hinders a complete analysis. One species of scientific importance for its biological properties in the pharmaceutical and food area is \u003cem\u003eM. oleifera\u003c/em\u003e (Hassan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Islam et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), whose short capsule (15 to 25 cm) and long capsule (30 to 80 cm) variants are of scientific importance (Castillo-L\u0026oacute;pez et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and whose cytological information is limited describing the presence of 14 bivalents in diakinesis as meiotic behavior (Boopathi \u0026amp; Raveendran, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, the use of molecular genetic markers is of great importance to detect genetic variability of genotypes of interest at the DNA level, and to develop effective selection strategies for breeding programs (Alavilli et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; RS et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saraswathi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), since detection of genetic polymorphisms can contribute to improve the accuracy and efficiency of conventional breeding programs (Darkwa et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The most commonly used molecular marker techniques are based on DNA amplification employing the PCR technique, which generates copies of a DNA region, providing genetic information for genetic variability analysis (Orozco-Sifuentes et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. oleifera\u003c/em\u003e has been studied previously using AFLP (amplified fragment length polymorphism) markers showing variations among the studied populations (Peter et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), likewise, studies by employing RAPD molecular markers recorded low genetic diversity in \u003cem\u003eMoringa\u003c/em\u003e accessions from the Brazilian Germplasm Bank and a high level of polymorphism (81.5%) in Nigerian populations (Bernard et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, reports on the use of ISSR (Inter Simple Sequence Repeat) markers, are very limited (Hassanein \u0026amp; Al-Soqeer, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Peter et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), being this information for the detection of genetic variability in multiple plant species by the its high polymorphism rate, co-dominance, high reproducibility, wide variability and economic feasibility (Cardoso et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tamboli et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ISSR markers are based on the amplification of intermediate zones between microsatellites, presenting the favorable characteristics of markers such as microsatellite (SSR), AFLP and RAPD (Aydin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to the limited information on chromosome number and molecular characterization of moringa diversity, the objectives of this research were to determine the cytological behavior, chromosome number and estimate genetic diversity by ISSR molecular markers of two phenotypes of \u003cem\u003eM. oleifera\u003c/em\u003e (short capsule and long capsule).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial vegetal\u003c/h2\u003e \u003cp\u003eTo obtain meristems (cytogenetic analysis) and leaf tissue (DNA extraction), seeds of two phenotypes of \u003cem\u003eM. oleifera\u003c/em\u003e were sown: short capsule (15 to 25 cm) and long capsule (30 to 80 cm) (Castillo-L\u0026oacute;pez et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moringa seeds were purchased from the Autonomous University of Nuevo Le\u0026oacute;n, San Nicol\u0026aacute;s, Nuevo Le\u0026oacute;n, M\u0026eacute;xico.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCytogenetic analysis\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eChromosome number determination\u003c/h2\u003e \u003cp\u003eFor this section, 10 moringa seeds of each phenotype (short and long capsule) previously disinfected with 1% NaClO and treated with N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide fungicide (Captan 50 WP, ADAMA, M\u0026eacute;xico) were used. Seeds were placed in Petri dishes with a moistened filter paper base and subjected to germination conditions in a germinator (Achieva Basic Style, Seedburo, USA) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C (Garcia-Ortiz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Then, for cytological study, root tips of 1 to 2 cm were collected and pretreated with 0.04% 8-hydroxyquinoline at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 2 h. Treated roots were fixed for 24 h with Farmer's 3:1 (ethanol:glacial acetic acid) solution (Ortiz-Curiel et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Once the roots were fixed, they were washed three times with distilled water for 30 min in each rinse. Subsequently, roots were subjected to aceto-carmine staining (carmine dissolved in 45% acetic acid), then the flasks were placed in a burner flame (7 times) with 7 s of exposure and 7 s without exposure. The meristems were placed for five days in carmine dye, and finally a drop of FeCl3 ammonia was added for staining (Almejo Vazquez et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDegradation with papain and microscopic observation\u003c/h2\u003e \u003cp\u003eFor this section, the technique reported by (Ortiz-Curiel et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was applied with some modifications. To promote alteration in the cell wall and pectin, \u003cem\u003eMoringa\u003c/em\u003e roots were immersed in papaya extract (obtained from the filtrate of the mixture of 100 g of papaya and 20 mL of H\u003csub\u003e2\u003c/sub\u003eO) at room temperature for 30 min, subsequently, samples were washed with distilled water for 30 min, finally, roots were stained using the acetocarmine technique (Almejo Vazquez et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMeristems were sampled and placed on a slide which had a drop of carmine dye, then the sample was heated and gently pressed on a filter paper to remove excess dye. Cells were observed and photographed at 100X using a visible light microscope (Axiostar plus, Karl Zeiss, Germany) coupled to a camera (Canon, PowerShot G5, Japan). Chromosome measurements and analysis were performed with AxioVision Rel 4.8 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversity with ISSR molecular markers\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eVegetal material\u003c/h2\u003e \u003cp\u003eTo obtain leaf tissue, 25 seeds of each type of moringa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were sown in biodegradable germination boxes under greenhouse conditions. Once the seedlings emerged, they were transplanted into one-liter polyurethane pots. Subsequently, young leaves were collected separately for analysis. Samples were transported on ice and in the absence of light. Briefly, samples were disinfected with 70% EtOH for 2 min and 1% NaClO for 1 min, rinsed with distilled water, weighed 200 mg of each sample and placed to dry. Finally, they were stored at -80\u0026deg;C in a freezer (Panasonic, MDF-V56VC-PA, USA) until genomic DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of short and long capsule \u003cem\u003eMoringa oleifera\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShort capsule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLong capsule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShort capsule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLong capsule\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 23\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL135-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 46\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 95\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 33\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 137-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 59\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 100-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 44\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 132-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 62\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 102-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 2\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 136-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 63\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 103-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 4\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 133-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 64\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 109-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 5\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 128-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 65\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 120-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 6\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 129-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 67\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 121-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 7\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 138-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 70\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 11\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 8\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 139-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 71\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 13\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 21\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 70\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 72\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 15\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 22\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 71\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 73\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 24\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 26\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 90\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS 18\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL 28\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS 32\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL 92\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDNA isolation\u003c/h2\u003e \u003cp\u003eIn this case, the CTAB extraction method (Doyle \u0026amp; Doyle, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Shahzad et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) with slight modifications was used, where samples were frozen at -80\u0026deg;C and ground employing a mortar with previously sterilized pistil, subsequently placed in 1.5 mL vials and 800 \u0026micro;L CTAB buffer solution with 20 \u0026micro;L of 20% bovine serum albumin were added, then sample was heated at 55\u0026deg;C for 20 min in a water bath (Premiere, HH-4). Then, samples were centrifuged at 13400 rfc for 5 min at 4\u0026deg;C in a refrigerated centrifuge (Select Bioproducts, Spin-17R). After, the supernatant was recovered and transferred to a vial with a chloroform:isoamyl alcohol solution (24:1 v/v), subsequently, it was mixed by gentle inversions for 2 min, finally, samples were centrifuged at 13400 rfc for 10 min. The aqueous phase was transferred to 50 \u0026micro;L of a solution of 7.5 M ammonium acetate and 800 \u0026micro;L of cold 96% EtOH, the solution was mixed by gentle inversions and left to stand for 12 h at -20\u0026deg;C. Samples were centrifuged at 13400 rfc for 5 min and the sediment was recovered. The DNA sediments were washed with 70% EtOH, after that, samples were allowed to dry at room temperature for 2 h, then resuspended in 80 \u0026micro;L of milli-Q water and 12 \u0026micro;L of RNAase enzyme and placed in a dry bath (Labnet, AccuBlock Digital, USA) at 37\u0026deg;C for 30 min. Upon completion of drying, samples were stored at -20\u0026deg;C for quantification and use in PCR reactions. DNA concentration was determined from the ratio of wavelength absorption at 260 and 280 nm obtained from Spectrophotometer (Biotek, Epoch). Finally, DNA integrity was observed by electrophoresis in 1% agarose gels using a transilluminator (Spectroline) with UV light and Launch VisionsWorks L5 software (version 6.8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePCR Amplification\u003c/h2\u003e \u003cp\u003eTo determine polymorphism within and between both moringa types, 10 ISSR primers were tested (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). DNA amplification was performed in a total volume per reaction of 23 \u0026micro;L containing: 8 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO, 5 \u0026micro;L of 10X Buffer with MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 \u0026micro;L of dNTP's, 6 \u0026micro;L of primer, 0.5 \u0026micro;L of Taq polymerase (5U/\u0026micro;L) and 3 \u0026micro;L of \u003cem\u003eMoringa\u003c/em\u003e genomic DNA. PCR amplifications were programmed in a thermal cycler (Axygen, Maxynene) with the following specifications: a denaturation temperature was used at 94\u0026deg;C for 1 min, followed by 45 amplification cycles, where a different alignment temperature was used for each primer (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) for 45 s, followed by a polymerization temperature at 72\u0026deg;C for 2 min, at the end of the 45 cycles, a temperature of 72\u0026deg;C was maintained for 7 min. The amplified products were electrophoretically separated on 1.5% agarose gels, stained with ethidium bromide, using SB 1X buffer and a 100 bp \u0026minus;\u0026thinsp;3000 bp DNA Ladder molecular weight marker (Axygen biosciences).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eISSR primers used for PCR amplification of two \u003cem\u003eMoringa oleifera\u003c/em\u003e phenotypes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence (5\u0026rsquo; \u0026ndash; 3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnnealing temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGATC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAGTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCTCTCTCTCTCTCTAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCCTCCTCCTCCTCCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGTGTGTGTGTGTGTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGCAGCAGCAGCAGCGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACACACACACACACACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGTGTGTGTGTGTGTTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCTCTCTCTCTCTCTCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of ISSR profiles\u003c/h2\u003e \u003cp\u003eAnalyses were performed using the statistical language R (R Development Core Team, 2011). A binary data matrix of presence (1) and absence (0) of bands was generated to determine the Shannon Index as a measure of genetic diversity. Fisher's exact test was performed to assess the association between markers and short and long capsule traits. In addition, an ANOVA, various genetic measurements and Wright's parameters (Fst, Fis and Fit) were estimated using InfoGen software (2011). Genetic relationships between the two moringa types were determined by cluster analysis using the unweighted pairwise method grouping algorithm with arithmetic mean (UPGMA) and to group the studied individuals a Principal Coordinate analysis (PCoA) was performed using InfoGen software (2011).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCytogenetic analysis\u003c/h2\u003e \u003cp\u003eThe different mitotic phases identified in the cytological analysis of \u003cem\u003eM. oleifera\u003c/em\u003e cells reveal the presence of small chromosomes which, due to their size, present limitations during the identification of the complete karyotype (Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the other hand, after hydrolysis with papain, 2N\u0026thinsp;=\u0026thinsp;28 chromosomes were visualized in a single metaphase cell of the long capsule phenotype with a chromosome size between 0.05 and 0.10 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), having a number of chromosomes similar to that reported in previously studies (Chang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and a chromosome size smaller than previously reported in the mitotic metaphase of \u003cem\u003eM. peregrina\u003c/em\u003e species with sizes between 1.19 and 2.1 \u0026micro;m which is attributed to genomic differences between species (Nazari et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMitotic chromosome length of a metaphase cell of the long capsule \u003cem\u003eMoringa oleifera\u003c/em\u003e type.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLength (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLength (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results of this work in comparison to other moringa species such as \u003cem\u003eM. peregrina\u003c/em\u003e sharing the same biological and geographical origin, present differences in chromosome size, with \u003cem\u003eM. oleifera\u003c/em\u003e being 7 times smaller than those documented in \u003cem\u003eM. peregrina\u003c/em\u003e, which is attributed to the type of treatment and time used in the cytogenetic analysis due to size variations during the cell cycle (Abdel-Hameed, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nazari et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Likewise, previous reports with \u003cem\u003eM. oleifera\u003c/em\u003e and \u003cem\u003eM. Stenopela\u003c/em\u003e have documented the presence of 28 small bivalent chromosomes that are difficult to measure and identify, typical of a diploid condition of the species (Mohamed Anwar, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Likewise, presence of small chromosomes in both plants are frequent characteristics of the monogenean family \u003cem\u003eMoringaceae\u003c/em\u003e which is attributed to their geographical origin or genetic ancestry (Lysak, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, after performing the cytological technique, it was not possible to observe the chromosomal number in the short capsule phenotype which is attributed to the nature of the sample as well as the short age of the seedling presenting cells in interphase which limits the observation by the established technique (Windham et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows micrographs of both types of moringa where the prophase, metaphase, anaphase and telophase were observed during the mitotic phases, where different types of cells were observed in the meristems of both phenotypes of moringa in which cells in mitotic division, meristematic cells with diffuse chromatin and larger cylindrical cells with diffuse chromatin characteristic cells in species of the \u003cem\u003eMoringaceae\u003c/em\u003e Family such as \u003cem\u003eM. oleifera\u003c/em\u003e and \u003cem\u003eM. Stenopetala\u003c/em\u003e (Mohamed Anwar, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGenetic Diversity\u003c/h2\u003e \u003cp\u003eIn this study, the ISSR polymorphism pattern in two phenotypes of \u003cem\u003eM. oleifera\u003c/em\u003e (short capsule and long capsule) was evaluated by determining the closeness of relationship or distance between the accessions. The analysis detected the presence of 129 polymorphic bands obtained using 10 primers (where primers 7 and 8 were eliminated from the study because they did not amplify during PCR), a number higher than that documented in different works with SSR, where 58 polymorphic bands have been amplified employing 14 primers (Natarajan \u0026amp; Joshi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, a genetic diversity measured as Shannon entropy of 0.81 was presented (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating a high genetic diversity among moringa types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring performance of the Fisher exact test, probability was corrected by False Discovery Rate, thus, the column (fdr) is the one that should be considered to declare significance (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A total of 67 markers were significant with fdr\u0026thinsp;\u0026le;\u0026thinsp;0.05, which could be an indication that the association is due to the population structure between the two types of plants, and not indicative of the markers being associated with the coding region.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExamples of markers with values from the Fisher exact test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFdr\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.029123e-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.281273e-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.364236e-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.757082e-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.545655e-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.329636e-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.069942e-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.235297e-08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.364236e-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.757082e-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1_1B11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.921510e-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.980239e-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the measures of genetic diversity and Wright statistics, which indicate the presence of polymorphic loci with similarities in both types of moringa (short and long capsule), which is reflected in the variation in the population, and is related to the frequency of the most popular allele (less than 0.99\u0026thinsp;\u0026minus;\u0026thinsp;0.95). Likewise, in this work the genetic diversity (variability of an inbred population) of both genotypes was observed, in general in previous works have categorized \u003cem\u003eMoringa oleifera\u003c/em\u003e as a species of high genetic diversity (Lakshmidevamma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), in particular in this work a higher genetic diversity was presented in the short capsule population compared to that of the long capsule population with values of 0.33 and 0.31 respectively, similar behaviors in studies of cross-pollinated plants (Tomas et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, the genetic variation of the population (which represents the average number of alleles) was 1.88, a value attributed to cross-pollinated plants which can improve genetic diversity, and provide an advantage by reducing the risk of inbreeding depression (Oliveira et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Likewise, an average number of alleles close to 1.55 is attributed to free pollination by factors in the environment such as air, and animals such as birds and insects, which could be a form of pollination due to moringa flower morphology and general tree morphology (Lakshmidevamma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The Fst value to indicate the level of genetic variation in a subpopulation compared to total variation was higher in long-capped moringa (more than double) compared to short-capped moringa, indicating a classification with moderate differences and greater interpopulation genetic differentiation (Barrera-Guzm\u0026aacute;n et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Fis values (reflecting the degree of inbreeding between the populations studied) was 0.029 and 0 for the short and long capsule population, respectively, populations with Fis values close to 0 present in long capsule moringa indicate that they are in heterozygote balance or similarity, while those with values different from 0 (short capsule moringa) reflect excess or deficiency of heterozygotes. The Fit values determined in this study indicate a heterozygote deficiency in both moringa populations with values of 0.083 (moringa short capsule) and 0.138 (moringa long capsule).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic diversity measurements determined by ISSR markers in two moringa type populations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStatistical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShort capsule type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLong capsule type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymorphic loci\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenetic diversity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNei heterozygosity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage number of alleles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFst\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFit\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eANOVA of the polymorphic band ISSR indicated significant differences between (p-value 0.0001) and within moringa populations (p-value 0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). A 4-fold higher variance was shown between capsule-type populations than within moringa types. This result, as well as Wright's statistics, suggest a subdivision in \u003cem\u003eMoringa oleifera\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of polymorphic ISSR bands from two moringa type populations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIter.#\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBetween types\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWithin types\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e319.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e86.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e353.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the principal coordinate analysis (PCoA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the first two axes account for 47.5% of the total variation. In addition, a division reflecting the difference in genetic structure between short-capsule, and long-capsule moringa is observed, showing distinct clusters in the projection in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Individuals of the short capsule moringa phenotype were grouped in a sector of smaller dispersion indicating a greater similarity in genetic structure, while in the long capsule \u003cem\u003eMoringa\u003c/em\u003e phenotype, a wide separation is observed between individuals of the long capsule phenotype, indicating a greater difference in genetic structure. This type of behavior is observed in different genetic identification and population variation studies, where one of the important factors is the presence and absence of genes derived from one or multiple common ancestors, and the crossbreeding or isolation of these genes generated by development and evolution in different geographical conditions (Natarajan \u0026amp; Joshi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nguyen et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the UPGMA clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), it was observed the formation of two different groups based on the Euclidean distances between the two phenotypes. Cluster analysis confirms the grouping of individuals within the short capsule phenotype, and the separation of individuals from the long capsule phenotype. This indicates that the phenotypic expression of both phenotypes (Moringa short and long capsule) are associated with the genetic structure of each of the phenotypes, ruling out the presence of different capsules due to phenotypic plasticity effects (Hausiku et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on cluster and principal component analyses, it can be deduced that \u003cem\u003eM. oleifera\u003c/em\u003e populations have a high level of genetic variability, most likely due to their cross-pollination type. This variability is the basis for establishing a genetic improvement program in this species. Representative profiles of ISSR products in the two moringa phenotypes can be seen in the annex (A1). In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, it can be observed that primer 9 offered the highest number of bands and polymorphism in both phenotypes, followed by primers 5 and 6 (A1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, primer 3 presented a lower number of identified bands which were monomorphic bands in the long capsid phenotype, while there was no amplification in the short capsid phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, the long capsid phenotype showed greater amplification of bands than the short capsid phenotype with most of the primers (A1), indicating greater variability in the long capsid phenotype population, with the exception of primer 10 where greater amplification was observed in the short capsule in contrast to the long capsule as only 2 to 3 bands were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), a number of bands similar to the averages documented in other studies with a frequency between 2 and 6 bands with \u003cem\u003eMoringa oleifera\u003c/em\u003e species from Tamil Nadu (Natarajan \u0026amp; Joshi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The observed polymorphisms indicate that ISSR primers are good and reliable for the assessment of genetic diversity in \u003cem\u003eM. oleifera\u003c/em\u003e with potential applications in breeding programs (Lakshmidevamma et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Natarajan \u0026amp; Joshi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe number of chromosomes observed was 2N\u0026thinsp;=\u0026thinsp;28 chromosomes in the long capsule phenotype of \u003cem\u003eMoringa oleifera\u003c/em\u003e with a size between 0.05 to 0.10 \u0026micro;m, in prophase, metaphase, anaphase and telophase, in addition, ISSR markers with the capacity to be used for genetic diversity studies in \u003cem\u003eMoringa oleifera\u003c/em\u003e with a high degree of polymorphism were identified. Furthermore, the short and long capsule phenotypes belong to two different genotypes with genetic variability between and within genotypes, which will allow the implementation of a genetic improvement program in this specie.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest:\u003c/h2\u003e \u003cp\u003eM. G\u0026oacute;mez-Mart\u0026iacute;nez, J. D. Garc\u0026iacute;a-Ortiz, S. G\u0026oacute;mez-Mart\u0026iacute;nez, A. C. Flores-Gallegos, M. H. Reyes-Vald\u0026eacute;s, J. A. Morlett-Ch\u0026aacute;vez, Ra\u0026uacute;l Rodr\u0026iacute;guez-Herrera declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. G. M: Conceptualization, Writing \u0026ndash; original draft, and Investigation. J. D. G. O: Formal Analysis and Writing \u0026ndash; review \u0026amp; editing. S. G. M: Supervision and Validation. M. H. R. V: Supervision and Validation. A. C. F. G: Supervision and Validation. J. A. M. C: Supervision and Validation. R. R. H: Project administration, Writing \u0026ndash; review \u0026amp; editing, Supervision, and Validation.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eIn memory of Martha G\u0026oacute;mez-Mart\u0026iacute;nez.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdAlla, H. A. M., Wanga, V. O., Mkala, E. M., Amenu, S. G., Amar, M. H., Chen, L., \u0026amp; Wang, Q. F. (2023). Comparative genomics analysis of endangered wild Egyptian Moringa peregrina (Forssk.) 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A step-by-step protocol for meiotic chromosome counts in flowering plants: A powerful and economical technique revisited. \u003cem\u003eApplications in Plant Sciences\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(4), e11342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/aps3.11342\u003c/span\u003e\u003cspan address=\"10.1002/aps3.11342\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chromosomal number, ISSR, Genetic diversity, diploid","lastPublishedDoi":"10.21203/rs.3.rs-4202934/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4202934/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytogenetic and molecular studies hold significant importance in plant breeding programs. In the case of moringa, such studies are scarce. Therefore, the objective of this research was to determine the chromosomal number and genetic diversity parameters in two phenotypes of \u003cem\u003eMoringa oleifera\u003c/em\u003e (short and long capsule) using chromosomal analysis and ISSR markers, respectively. Cytogenetic analyses were conducted using the \"Somatic chromosomes in root apices\" technique, with acetocarmine and papain for staining. It was possible to identify 2N\u0026thinsp;=\u0026thinsp;28 chromosomes in a single cell of the long capsule phenotype, ranging in size from 0.05 to 0.10 \u0026micro;m. Cells in both moringa phenotypes were observed in prophase, metaphase, anaphase, and telophase. High genetic variability was found in both phenotypes of moringa, as indicated by a Shannon index of 0.81. Additionally, the principal component analysis and UPGMA groups revealed genetic isolation between the studied moringa phenotypes. The significant polymorphism obtained with most primers suggests that the tested ISSR markers are highly useful for studying genetic diversity in moringa.\u003c/p\u003e","manuscriptTitle":"Genetic and cytogenetic analysis of Moringa oleifera short and long capsule phenotypes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 19:50:18","doi":"10.21203/rs.3.rs-4202934/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ed33207-f5c0-4107-9dff-2619135b92f7","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-16T08:50:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 19:50:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4202934","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4202934","identity":"rs-4202934","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}