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).