Analysis of the genetic diversity of amaranth (Amaranthus spp.) using ISSR markers

Análise da diversidade genética do amaranto (Amaranthus spp.) utilizando marcadores ISSR

Abstract

Amaranth is a short-cycle herbaceous plant with high nutritional value and multiple uses as a vegetable, forage, and ornamental; however, its broad phenotypic diversity in Colombia remains uncharacterized. Therefore, the objective of this study was to evaluate the genetic diversity of 50 amaranth genotypes in the department of Boyacá using ISSR (Inter Simple Sequence Repeat) molecular markers. The research identified 194 amplicons—ranging from 14 for ACA to 28 for GT—with 88% polymorphism, sizes between 200 and 3820 bp, and an average PIC of 0.23, allowing the population to be classified into two groups differentiated by morphological traits and the presence of anthocyanins. General genetic parameters revealed an expected heterozygosity (He) of 0.27, a Shannon index (I) of 0.22, a genetic differentiation coefficient (Gst) of 0.16, and a gene flow (Nm) of 2.67, thus demonstrating significant genetic diversity within the evaluated germplasm. For the two established genetic groups, an He = 0.23 was found, with I values of 0.35 and 0.36, respectively. Finally, the AMOVA (Analysis of Molecular Variance) determined that the highest genetic variation occurs at the intrapopulation level, suggesting higher hierarchical levels than those considered in this study. The results obtained may be highly useful for designing conservation strategies and selecting promising amaranth genotypes in Colombia.

Keywords:
Amaranthus; genetic diversity; germplasm; molecular markers; plant breeding

Resumo

O amaranto é uma planta herbácea de ciclo curto e de alto valor nutricional com múltiplos usos como hortaliça, forragem e ornamental, com ampla diversidade fenotípica na Colômbia, a qual ainda não foi descrita. Por qual motivo o objetivo deste estudo foi avaliar a diversidade genética de 50 genótipos de amaranto no departamento de Boyacá usando marcadores moleculares ISSR (Inter Simple Sequence Repeat). A investigação identificou 194 amplicones – com uma faixa de 14 para ACA até 28 para GT –, um polimorfismo de 88%, tamanhos entre 200 e 3.820 pb e um PIC de 0.23, permitindo a classificação da população em grupos diferenciados por traços morfológicos e presença de antocianinas. Os parâmetros genéticos gerais revelam uma heterocigosidade esperada (He) de 0.27, um índice de Shannon (I) de 0.22, um coeficiente de diferenciação (Gst) de 0.16 e um fluxo gênico (Nm) de 2.67, mostrando assim a existência de diversidade genética significativa dentro do germoplasma avaliado. Para os grupos genéticos conformados obtiveram He = 0.23 e valores de I de 0.35 e 0.36 respectivamente. Finalmente, a análise AMOVA determinou que a maior variação genética ocorre em nível intrapoblacional, o que sugere níveis hierárquicos superiores aos considerados neste estudo. Os resultados obtidos podem ser muito úteis para projetar estratégias de conservação e selecionar genótipos promissores de amaranto na Colômbia.

Palavras-chave:
Amaranthus; diversidade genética; germoplasma; marcadores moleculares; melhoramento de plantas

1. Introduction

The genus Amaranthus L. comprises C4 dicotyledonous herbaceous species with a broad global distribution across Asia, Africa, Australia, and Europe (Waselkov et al., 2018), standing out for its versatility as a grain, vegetable, forage, and ornamental plant (Mukuwapasi et al., 2024). Due to its exceptional nutritional profile and remarkable adaptability to diverse environmental factors, there has been a significant resurgence in scientific interest to explore this underutilized orphan crop (Maurya et al., 2023). Globally recognized as the “miracle grain” or “grain of the future”, amaranth possesses a high nutraceutical value that includes proteins, unsaturated oils, dietary fiber, flavonoids, and vitamins (Segura-Jiménez and Jacobo-Velázquez, 2025). Specifically, it features a balanced concentration of essential amino acids, highlighting lysine—which represents between 0.73% and 0.84% of the seed's total protein content—overcoming the common limitations of other cereals (Segura-Jiménez and Jacobo-Velázquez, 2025). In addition to being a safe option for celiac patients due to its gluten-free nature (Kour et al., 2025), regular consumption of its seeds or oil provides vitamin E and squalene, compounds beneficial for cardiovascular health and hypertension (Sattar et al., 2024). Squalene, in particular, acts as a potent antioxidant against cellular damage and premature aging (Gautam and Khedkar, 2024; Dutta et al., 2026). Its ability to thrive in extreme ecogeographical conditions and its low susceptibility to pests position it as a resilient crop (Oliveira et al., 2024). Therefore, given the growing food demand and global malnutrition, the development of amaranth emerges as a strategic and beneficial alternative for developing countries (Oliveira et al., 2024).

Amaranth germplasm has a wide distribution. In the United States, it is cultivated as a health food, while in Peru, Bolivia, India, Mexico, and Colombia, it is a species that lost importance after the introduction of cereals such as wheat and rice (Abrar et al., 2024). The United States conserves approximately 3300 accessions of A. hypochondriacus from 40 different countries (Trucco et al., 2011). Additionally, there are 3081 accessions (Das, 2016) of A. hypochondriacus in the National Bureau of Plant Genetic Resources (NBPGR). Peru has a collection of 740 accessions of A. caudatus (Kalinowski et al., 1992). However, the genetic diversity of these collections has not been fully characterized, thus missing the important role it plays in the development of superior cultivars for crop improvement, which gives plant breeders the opportunity to develop improved cultivars with variable characteristics, including traits preferred by farmers, consumers, and plant breeders alike (Begna and Begna 2021; Anuradha et al., 2023).

Historically, plant diversity was assessed through natural variants at the cytological, morphological, and biochemical levels (Jamalluddin et al., 2022); however, advances in sequencing and bioinformatics have established molecular markers as the preferred method due to their superior accuracy, speed, and reliability (Anuradha et al., 2023). In the case of amaranth, the development of high-yielding cultivars with enhanced nutritional quality is currently hindered by the insufficient genetic characterization of germplasm and a lack of knowledge regarding the inheritance patterns of key traits (Chauhan et al., 2025). Consequently, molecular genotyping markers have become essential not only for germplasm characterization and diversity assessment but also for identifying redundant accessions in gene banks, developing core molecular collections, quantitative trait loci (QTL) mapping, and implementing marker-assisted selection (Singh et al., 2025).

To characterize intra- and interspecific genetic diversity in amaranth, different DNA markers have been used, such as Restriction Fragment Length Polymorphism (Park et al., 2014), Random Amplified Polymorphic DNA (RAPDs) (Sammour et al., 2020), Amplified Fragment Length Polymorphisms (AFLPs) (Wassom and Tranel, 2005), Inter-Simple Sequence Repeats (ISSRs) (Gelotar et al., 2019), Simple Repeat Sequences (SSRs) (Vats et al., 2023), and Single Nucleotide Polymorphism (SNPs) (Lin et al., 2022). These markers have allowed the identification of potential genotypes to meet different breeding objectives and thus achieve more sustainable agriculture. In the last decade, ISSR markers have been successfully used in diversity studies. These markers exhibit high reproducibility, allowing the revelation of numerous informative bands in a single amplification, thus becoming one of the most widely used markers in intraspecific diversity analysis in Amaranthus.

The cultivation of Amaranthus sp. in Colombia remains underdeveloped, with less than 3000 ha planted annually, hindered by low technological adoption and the use of low-yielding local cultivars characterized by late maturation and excessive plant height (García Parra et al., 2019; Kumar et al., 2024). Given the recent inclusion of amaranth in the basic food basket, it is imperative to characterize the genetic variations of regional materials to guide productive development and ensure the supply of certified planting material that meets the requirements of both production systems and consumers (Gelpud et al., 2023). Although institutions in the department of Boyacá, such as the Governor's Office and the CIDE research group, possess germplasm collections with prior morpho-agronomic characterizations, transitioning toward genetic characterization using molecular markers is essential to overcome the limitations of morphological descriptors and establish the breeding foundations demanded by the current market; therefore, this research aimed to evaluate the genetic diversity of amaranth genotypes using ISSR markers.

2. Material and Methods

2.1. Plant material

The genetic diversity of amaranth was characterized using 50 genotypes that had shown high morphological variability in previous studies. These genotypes are part of the germplasm collection held by the Government of the Department of Boyacá and were donated by producers. The genotypes were established under greenhouse conditions in a completely randomized block design at an average temperature of 16 °C, a relative humidity of 87%, and a photoperiod of 12:12 h. Young leaves were collected from each genotype and stored at −80 °C until use.

2.2. DNA extraction

DNA was extracted from 200 mg of young leaf tissue from each genotype, using the modified protocol of Dellaporta et al. (1983). DNA integrity was verified by electrophoresis on 0.8% agarose gels stained with Gelred dye (Biotum, USA). The concentration was determined using a spectrophotometer (Biotek EPOCH|2 device) and the absorbance ratios were expressed in ng/µL at A260/A280. The samples were diluted in HPLC water to a total volume of 100 µL to 10 ng/µL and stored at −20 °C.

2.3. ISSR amplification

Amplification was performed using eight ISSR markers selected for their high level of polymorphism and use in genetic diversity studies of plant germplasm (Morillo et al., 2024). The PCR amplification reaction consisted of 20 ng of DNA, 2 µmol of the primers, 1 U of Taq DNA polymerase, 0.2 mM of dNTPs, 1.5 mM of MgCl2, and 1X buffer in a 25 µL reaction chamber. The amplification program was: 5 min at 95 °C (initial denaturation), followed by 37 cycles at 95 °C for 30 s, annealing at 50-58 °C (depending on the primer, Table 1) for 45 s, 72 °C for 2 min, and a final extension at 72 °C for 7 min. All reactions were carried out with the PTC 1000 programmable thermal controller thermocycler (M.J. Research, Inc). PCR product was then separated by electrophoresis in 2% agarose gel, with running TBE 0.5X at 100 V for approximately 3 h in a Maxicell Primo EC-340 Electrophoresis Gel System chamber and stained with Gelred dye and then visualized under transilluminator.

Table 1
ISSR markers used to determinate the genetic diversity in the Amaranthus sp. genotypes.

Primer	Sequence (5′to 3′)
ACA	BDB(ACA)5
CCA	DDB(CCA)5
CGA	DHB(CGA)5
GT	VHV(GT)5G
AG	HBH(AG)7A
CT	DYD(CT)7C
TG	HVH(TG)7T
CA	DBDA(CA)7

• Single-letter abbreviations for multiple base positions: H: (A, C, T); B: (C, G, T); V: (A, C, G); D: (A, G, T); Y (C, T).

2.4. Statistical analysis

The data analysis was performed only on the bands that showed clear amplification. A binary matrix of presence (1) and absence (0) was constructed. Cluster analysis was performed using the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA). The SIMQUAL program was used to calculate the Jaccard coefficients using NTSYS-pc 2.1 (Rohlf, 2020).

The dendrograms were constructed using the algorithm with the SAHN module. The cut-off values of dendrograms were then determined based on calculation method described by Jamshidi and Jamshidi (2011). Genetic similarity (GS) was estimated for all cultivars pairs using the following Equation 1 (Nei and Li, 1979):

$G{s}_{ij}=\frac{2N_{ij}}{2N_{ij}}+N_i+N_j$ (1)

where $G{s}_{ij}$ represents the similarity estimated between the genotypes i and j, based on the ISSR data, $N_{ij}$ is the total number of bands common to i and j, and $N_i$ and $N_j$ correspond to the number of bands found in genotypes i and j, respectively. Cophenetic correlation coefficient (CCC) between similarity matrix and dendrogram cophenetic values was estimated to validate the dendrogram in relation to the original similarity estimates and the binary data matrix analyzed using COPH and MXCOMP programs in NTSYSpc.

The Nei’s genetic distance (H), Shannon information index (I), coefficient of genetic differentiation (Gst), number and percentage of polymorphic loci, and gene flow (Nm) (McDermott and McDonald, 1993), heterozygosity were estimated with the statistical package POPGENE version 3.2. Polymorphic information content (PIC) was calculated according to the Equation 2 proposed by Botstein et al. (1980):

$PIC=1-{\displaystyle \sum }_{j=1}^n{P}_{ij}^2$ (2)

where P_ij is the frequency of allele j at marker i.

According to the authors, indices below 0.25 are slightly informative; between 0.25 and 0.50, informative; an above 0.50 highly informative, where 0.50 is the maximum value reached in dominant markers such as ISSR (Botstein et al., 1980).

The genetic structure analysis was done based on Bayesian model (Hubisz et al., 2009) with STRUCTURE program version 2.3.4. The runs for K values ranging from 1 to 10 were executed with a burn-in length of 100,000 tailed by 1,000,000 Monte Carlo Markov Chain (MCMC) interactions using admixture model. The number of subpopulations was determined using the Delta K (ΔK) ad hoc method proposed by Evanno et al. (2005) and implemented in the online tool Structure Harvester (Earl and vonHoldt, 2012) to estimate the most likely K in each set of Amaranthus spp. The difference between and within the groups was evaluated by molecular variance analysis AMOVA, using GenAlex 6.5 program.

3. Results

The eight ISSR markers evaluated in 50 amaranth genotypes produced a total of 194 bands, with an average percentage of polymorphic loci of 88% (Table 2). The number of bands per marker ranged from 14 for ACA to 28 for CT, for a total of 194 amplified loci. The Polymorphic Information Content ranged from 0.16 (ACA) to 0.21 (TG), with a mean of 0.23 for the entire population (Table 2).

Table 2
Genetic parameters measured in the 50 genotypes of Amaranthus sp. and the genetic groups formed in the UPGMA cluster analysis and the one based on the Bayesian model, using ISSR markers.

Total Population									POP I		POP II
Primer	Nº Bands	Nº Polymorphic Loci	% Polymorphic Loci	He	I	Gst	PIC	Nm	He	I	He	I
ACA	18	14	77.78	0.22	0.21	0.26	0.16	1.44	0.13	0.22	0.20	0.31
AG	26	20	76.92	0.25	0.37	0.30	0.18	1.16	0.16	0.24	0.18	0.28
CA	22	17	77.27	0.25	0.38	0.03	0.17	17.75	0.27	0.40	0.23	0.35
CCA	28	25	89.29	0.30	0.45	0.24	0.24	1.57	0.25	0.37	0.21	0.32
CGA	22	22	100	0.30	0.45	0.04	0.25	12.49	0.29	0.46	0.27	0.43
CT	28	27	96.43	0.32	0.47	0.06	0.27	7.85	0.29	0.42	0.30	0.46
GT	28	24	85.71	0.27	0.41	0.28	0.20	1.27	0.20	0.31	0.17	0.28
TG	22	21	95.45	0.27	0.43	0.06	0.21	7.72	0.22	0.33	0.29	0.46
Average	194	170	87.63	0.27	0.22	0.16	0.23	2.67	0.23	0.35	0.23	0.36

• He: Expected heterozygosity; I: Shannon Index; Gst: Coefficient of genetic differentiation; PIC: Polymorphic Information Content; Nm: Gene flow.

The dendrogram obtained from the Nei and Li (1979) distance of approximately 0.60 and the UPGMA method grouped the amaranth genotypes into two large groups (Figure 1), results that were corroborated by the Bayesian analysis of the Structure program (Figures 22b).

Figure 1
Dendrogram of passion fruit cultivars based on the Nei-Li similarity coefficient and estimated with eight ISSR markers with UPGMA, SAHN and TREE classification methods of NTSYS-pc, version 1.8 (Exeter Software, NY, USA).

Figure 2
(a) Delta K values obtained from Harvester Structure, estimated as the mean of the probability of K divided by the standard deviation of the probability of K. Evanno’s method with an optimal model of K=2; (b) Population genetic structure of 50 Amaranthus genotypes analyzed, calculate using the Structure software. Each vertical bar represents an individual sample, and the color of the bar indicates the probability that an individual will be assigned to one of the identified groups.

The Bayesian clustering method with a generalized linear mixed model indicated that the 50 amaranth genotypes evaluated were grouped into two groups (K=2) (Figure 2a). Figures 22b show the population structure estimated based on Delta K (ΔK) when it reaches its maximum value following the ad hoc method and the subpopulation clusters (K) that are represented by different colors, respectively. The formation of two genetic groups represented by the color green for the different genotypes (approximately 56%, AM-34, AM-35, AM-29, AM-37, AM-48, AM-36, AM-38, AM-39, AM-45, AM-49, AM-30, AM-31, AM-32, AM-46, AM-50, AM-33, AM-47, AM-40, AM-44, AM-41, AM-42, AM-43.), while the second genetic group represented by the color red for the rest of the genotypes evaluated (44%, AM-26, AM-28, AM-15, AM-18, AM-13, AM-14, AM-17, AM-8, AM-12, AM-1, AM-7, AM-9, AM-11, AM-19, AM-2, AM-3, AM-16, AM-20, AM-22, AM-6, AM-23, AM-21, AM-4, AM-5, AM-10, AM-24, AM-25, AM-27). The groupings corresponded mainly to phenotypic characteristics such as stem, leaf, and inflorescence color, as well as the presence or absence of anthocyanins in different parts of the plant (Figure 3). The cophenetic correlation coefficient was 95%, thus showing a good correspondence between the genetic distances and the groups formed.

Figure 3
Phenotypic variability present in some of the Amaranthus genotypes evaluated with ISSR markers.

Regarding the estimation of genetic parameters for the entire population, the average expected heterozygosity (He) was 0.27 (Table 2). The Shannon diversity index ranged from 0.21 to 0.47, with an average of 0.22. The estimated genetic differentiation coefficient for the entire population was 0.16. Gene flow, represented by Nm = 2.67, was moderate-high for the 50 amaranth genotypes evaluated. The molecular analysis of variance (AMOVA) (Table 3) showed that the diversity between groups and within the evaluated genotypes was 22% and 78%, respectively. The groups formed by the cluster analysis showed similar He values ​​(He = 0.23) and average Shannon indices of 0.35 and 0.36, respectively (Table 2).

Table 3
Analysis of molecular variance (AMOVA) among and within groups using eight ISSR markers.

Source	df	Sum of squares	Variance of Components	%	p-value
Among Pops	1	220.78	7.84	22%	0.001
Within Pops	48	1320.31	27.51	78%	0.01
Total	49	1541.10	35.36	100%

• Significance test after 1000 permutations. df: Degree of freedom.

4. Discussion

The eight ISSR markers used for the assessment of the genetic diversity of 50 Amaranthus genotypes produced a total of 194 loci, which is considered appropriate for a genetic study, results similar to those found in other studies of genetic diversity in Amaranthus (Gelotar et al., 2019; Rathod et al., 2021; Hamidzadeh Moghadam et al., 2023; Siamey et al., 2025). The percentage of polymorphic loci was greater than 80%, which agrees with results obtained in characterization studies of amaranth germplasm in India using ISSR markers, where Gelotar et al. (2019) reported a polymorphism percentage of 87% with a PIC of 0.85. Rathod et al. (2021), in the morphological, biochemical, and molecular evaluation of seven Amaranthus varieties in India, found that the 14 ISSRs produced 84 amplicons, of which four (4) were monomorphic, while the remaining 80 were polymorphic. Most of the ISSR primers used showed 100% polymorphism for the seven varieties evaluated, except for the primers that were monomorphic. Hamidzadeh Moghadam et al. (2023) in the evaluation of Amaranthus retroflexus and Chenopodium album showed that the level of polymorphism revealed by the ISSRs was very high and reached values ​​of 98.46% for A. retroflexus and 74.81% for C. album. Therefore, these differentiating loci are suitable for evaluating the genetic diversity of these populations.

Table 2 shows that the ISSR primers used in this study accurately measure the variation present in the evaluated genotypes with a sufficient degree of polymorphism and are powerful enough to discriminate differences; therefore, they are suitable for studies of plant genetic diversity. Furthermore, the high levels of polymorphism detected may be due to the percentage of cross-pollination in the species, the seed selection processes carried out by producers in their fields without pollination control, and the coevolutionary processes to which the species is subjected in its natural environment.

Regarding Botstein et al. (1980), the average PIC value for the evaluated ISSR markers was 0.23, indicating that they are informative (Table 2). These results contrast with those obtained in other genetic diversity studies using ISSRs, where the estimated PIC exceeds 70%, demonstrating high efficiency for genetic diversity analyses in Amaranthus in different producing regions worldwide (Yan et al., 2019; Kwiecińska-Poppe et al., 2020; Alotaibi and Abd-Elgawad, 2022; Flihi et al., 2022; Ghanbari et al., 2022; Haq et al., 2022). Serrote et al. (2020) stated that markers with a PIC value between 0.5 and 1.0 are very informative for genetic studies, as they express significant variability between alleles, allowing for better discrimination between species or populations of individual plants.

Cluster analysis was performed to deduce the genetic structure, estimate lineage, and identify the possible presence of duplicates. The ISSRs evaluated using UPGMA cluster analysis and the Bayesian model produced two groups (Δk = 2) for the 50 Amaranthus genotypes (Figures 22b), where a small degree of admixture among individuals can be observed, likely due to gene flow, the species' reproductive system, seed selection processes in production lots, and other factors (Brackenridge et al., 2024). The groupings were defined more by phenotypic characteristics of the germplasm associated with the shape and color of the stem, leaves, inflorescence, and seeds than by their geographic origin, as well as the type of reproduction, percentage of cross-pollination, and absence of targeted selection processes (Delgado and Martín, 2023). Admixture is also attributed to the exchange of plant material between different producing regions in Colombia and to natural hybridization processes (Sokolova et al., 2024).

Similar results were obtained by Hamidzadeh Moghadam et al. (2023), where the UPGMA clustering algorithm grouped the 16 A. retroflexus populations into four clusters with a similarity index of 0.46. In this case, there was no correlation with geographic origin, as the Spanish and Iranian populations showed a loose distribution among the clusters. In the evaluation of black amaranth germplasm in Ecuador, the clusterings, as in this study, are not defined by the origin of the genotype but rather by the morphological characteristics of the germplasm (Delgado and Martín, 2023). Hence, the importance of interrelating different types of markers to obtain a more precise evaluation of the germplasm.

Regarding the expected heterozygosity values ​​(He), these ranged between 0.13 (ACA) and 0.29 (CGA and CT) with an average value for the entire population of He = 0.27 and for each of the genetic groups formed He = 0.23, lower values ​​than those obtained by Siamey et al. (2025) in the molecular characterization of 21 amaranth genotypes (He = 0.61) as well as in other investigations (Hamidzadeh Moghadam et al., 2023; Delgado and Martín (2023) in the evaluation of the genetic diversity of 139 black amaranth accessions collected in three representative provinces of Ecuador and using nine (9) microsatellite markers (SSR) found observed (Ho = 0.014) and expected (He = 0.13) heterozygosity values, lower than those found in this study, these differences may be due to the type of marker used as well as the genetic characteristics of the germplasm evaluate.

On the other hand, Mallory et al. (2008), evaluating 13 amaranth accessions from different South American countries (one from Argentina, two from Bolivia, two from Brazil, four from Peru, and four from Ecuador), found 27 different alleles, with an average of 5.4 alleles per locus, and a high He value (0.71), which may be due to the diverse origin of the accessions. In our study, however, we obtained small heterozygosity values, indicating that the vast majority of the analyzed samples are likely highly homozygous. This may also be explained by the predominantly self-pollinating nature of the species, which has already been reported (Peralta et al., 2013), and, in general, cultivated species of the genus Amaranth (Suresh et al., 2014; Basantes et al., 2022). Nguyen et al. (2019) also obtained low heterozygosity values ​​(0.14) in A. tricolor accessions.

Additionally, the genetic diversity values ​​detected among the evaluated genotypes may imply that the markers capture the variation or that their usefulness in distinguishing between individuals was not limited by their genetic diversity. Serrote et al. (2020) state that the heterozygosity of a marker is based on the number and frequency of alleles in the population. The range of values ​​for heterozygosity is between 0 and 1, with zero indicating no heterozygosity and 1 indicating alleles with equal frequency. Allele diversity plays an essential role in the survival of species, as it allows them to adapt to adverse environmental conditions (Begna and Begna, 2021).

The Shannon diversity index found for the 50 genotypes evaluated with eight ISSR markers (Table 2) was 0.22 for the total population and 0.35 and 0.36 for the genetic groups, respectively. These values ​​are higher than those reported by Hamidzadeh Moghadam et al. (2023) in the evaluation of ISSRs in A. retroflexus and C. album (I = 0.094 and I = 0.11, respectively), but lower than that found by Delgado and Martín (2023) in black amaranth germplasm in Ecuador (I = 0.30). Low values ​​for this index indicate low diversity and high dominance of a few species. An index of 0 means there is only one species, while a low value (<2) reflects a simple population, poor in species, or with a very uneven distribution of individuals. Given the selection and domestication processes to which the species is subjected in its natural environment and within production systems, the values ​​of this parameter tend to vary greatly depending on the germplasm, its origin and the corresponding evaluation site (Sánchez del Pino et al., 2026).

According to Yeh (2000), estimates of genetic divergence between 0.151 and 0.250 represent a high level of differentiation. Therefore, the results obtained with a Gst (0.16) were high, even compared to other studies of amaranth genetic diversity, such as those conducted by Hamidzadeh Moghadam et al. (2023) (Gst = 0.85; Gst = 0.82) and Siamey et al. (2025) (Gst = 0.60). Furthermore, this value of 0.16 indicates that 16% of the genetic variation exists between populations, while 84% corresponds to individual variations within each individual. Gst values ​​close to 0 show little differentiation and high gene flow between populations, while values ​​close to 1 indicate high differentiation and genetic isolation.

The average gene flow value Nm = 2.67 corroborates the results obtained for the previous genetic parameters and is also higher than that reported by Hamidzadeh Moghadam et al. (2023) (Nm = 0.10), where genetic exchange between populations was low. Factors such as pollen and seed dispersal determine the gene flow of populations, playing an essential role in their structure. Furthermore, the results obtained seem to indicate that in recent decades, within local production systems, there has been some mobility and exchange of amaranth germplasm. In this sense, it is worth mentioning that the flow of plant material from different Andean crops has been governed by an informal seed exchange system that connects farmers from different Andean zones, and that this has largely favored the generation of small collections which have served to conserve much of the native agrobiodiversity that we can find today in the different Andean crops (Salazar et al., 2019; Delgado and Martín, 2023).

Finally, the analysis of the genetic diversity of the 50 amaranth genotypes was completed with the analysis of molecular variance (AMOVA) (Table 3). In all cases and hierarchical levels, the estimates obtained for the different components of molecular variance were highly significant (P < 0.0001). When analyzing the results for the two genetic groups formed in the cluster analyses, it was observed that the largest percentage of variance was due to differences between the genotypes that made up each group (78%), but the percentage of variance due to differences between groups was lower (22%).

Similar results were obtained by Delgado and Martín (2023) in the evaluation of black amaranth germplasm in Ecuador, where 17% of the total variance was due to differences between provinces, and the remaining 82.1% to differences within accessions (72.4% and 9.7%, respectively), with the percentage of variation being higher between accessions than within provinces. Furthermore, Hamidzadeh Moghadam et al. (2023) found a strongly significant genetic differentiation between and within populations (P < 0.001), where 81% of the total variation was due to differences between populations, while the remaining 19.0% was attributed to intrapopulation differences.

Genetic variability represents vital information about the historical effects of bottlenecks and diversification since their establishment, and understanding a population's history allows for informed decisions regarding germplasm conservation and genetic improvement options (Gonçalves-Dias et al., 2023). Therefore, knowing the level of genetic variation within and between populations is essential for developing strategic agronomic management practices and for the effective selection of individuals that could potentially become a production alternative for amaranth farmers throughout the Andean region.

The results of this study suggest the existence of a limited gene pool among the evaluated amaranth genotypes, consistent with previous research indicating that domestication processes have led to a reduction in genetic diversity due to the selection of only a few specific genotypes (Delgado and Martín, 2023). Despite its nutritional significance and various potential applications in industry and medicine, amaranth remains an underutilized crop in Colombia, confined to small areas where much of its original variability has been lost. Nevertheless, its recent inclusion in the Colombian basic food basket, along side other Andean crops such as quinoa, underscores the importance of pioneering research like this, which establishes the ground work for germplasm selection processes aimed at potential agroindustrial uses and food security (Stetter et al., 2025).

Genetic diversity analyses performed using ISSR markers revealed a high level of divergence among the studied amaranth genotypes. The interaction of variables such as genetic variation, heterozygosity, gene flow, and population structure suggests that the current genetic configuration and its spatial distribution stem from processes of introduction and germplasm exchange between various producing regions (Hamidzadeh Moghadam et al., 2023). Furthermore, biological and evolutionary factors—including self-fertilization, genetic drift events, colonization by small groups of individuals, and differential selective pressures acting even within limited geographic areas—have significantly shaped the genetic diversity of these populations (Siamey et al., 2025).

Acknowledgments

The authors are grateful to the VIE (Vicerrectoría de Investigación y Extensión) from UPTC, Universidad Pedagógica y Tecnológica de Colombia (UPTC), for the financial and technical support in the development of the research, and to the private amaranth producers for the contribution of their plant material.

Data Availability Statement

Research data is only available upon request.

References

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  11 May 2026
• Date of issue
  2026

History

• Received
  30 Dec 2025
• Accepted
  25 Mar 2026