Keywords:

Genetic diversity

Genotype

Microsatellite markers

Sesamum indicum L.

Molecular study of the “Escoba Blanca” variety of Sesamum indicum L. used in Paraguay

Estudio molecular de la variedad “Escoba Blanca” de Sesamum indicum L. utilizado en Paraguay

Estudo molecular da variedade “Escoba Blanca” do Sesamum indicum L. usado no Paraguai

Roberto Martínez-López

Walter Esfrain Pereira

Andrea Alejandra Arrua

1,3

Danilo Fernández Ríos

Liz Mariela Centurión

Rev. Fac. Agron. (LUZ). 2024, 41(1): e244104

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron.v41.n1.04

Crop production

Associate editor: Dra. Lilia Urdaneta

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Centro Multidisciplinario de Investigaciones Tecnológicas,

Universidad Nacional de Asunción, Paraguay.

Federal University of Paraiba, Brazil.

Facultad de Ciencias Exactas y Naturales, Universidad

Nacional de Asunción, Paraguay.

Abstract

The high quality of sesame seeds originating in the country has led

Paraguay to be among the main exporters. Of the varieties available in

Paraguayan territory, the most widespread is ‘‘Escoba Blanca’’, which,

possibly due to the multiplication process, could promote changes in its allele

frequency, diversity, and genetic purity. This work was carried out, aiming to

determine the genetic dierentiation between 50 populations/seedbeds/banks

from seven Paraguayan companies collecting ‘‘Escoba Blanca’’ sesame,

using microsatellite markers. These seven banks/companies/cooperatives

collect and represent samples from all the producers/seedbeds located in

dierent departments of the Eastern and Western Region (Chaco) of the

country, with whom they work, market, and collect sesame. Plant tissue was

obtained to extract DNA, from seedlings planted especially for the purpose,

using all the included samples/accessions. Six microsatellite markers were

used: GBssrsa184, GBssrsa123, GBssrsa182, GBssrsa108, GBssrsa08, and

GBssrsa72. The following were calculated: number and frequency of alleles,

distance/groupings, dierentiation between populations, and their genetic

structure. The mean number of alleles per locus ranged from 1.33 to 3.00.

In the markers, GBssrsa184 and GBssrsa108, three populations presented

a higher frequency of alleles. The populations examined exhibited a wide

degree of genetic dierentiation between them, with the identication of four

groups, with greater and less purity respectively.

Received: 27-10-2023

Accepted: 18-12-2023

Published: 09-02-2024

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Resumen

La alta calidad en los granos de sésamo originados en el país ha

conducido al Paraguay a situarse entre los principales exportadores.

De variedades disponibles en territorio paraguayo, la más difundida

es ‘Escoba Blanca’, que, posiblemente debido al proceso de

multiplicación, pudo impulsar modicaciones en su frecuencia alélica,

diversidad y pureza genética. Este trabajo fue realizado, objetivando

determinar la diferenciación genética existente entre 50 poblaciones/

semilleros/bancos provenientes de siete empresas paraguayas

acopiadoras de sésamo ‘Escoba Blanca’, utilizando marcadores

de microsatélites. Estos/as siete bancos/empresas/cooperativas,

colectan y representan muestras de todos los productores/semilleros

ubicados en diferentes departamentos de la Región Oriental y

Occidental (Chaco) del país, con quienes trabajan, comercializan y

acopian sésamo. Se obtuvo tejido vegetal para extraer ADN, desde

plantines sembrados especialmente para el n, usando todas las

muestras/accesiones incluidas. Fueron utilizados seis marcadores

microsatélites: GBssrsa184, GBssrsa123, GBssrsa182, GBssrsa108,

GBssrsa08 y GBssrsa72. Fueron calculados: número y frecuencia de

alelos, distancia/agrupamientos, diferenciación entre poblaciones y

su estructura genética. El número medio de alelos por locus varió

de 1,33 a 3,00. En los marcadores GBssrsa184 y GBssrsa108 tres

poblaciones presentaron mayor frecuencia de alelos. Las poblaciones

examinadas exhibieron amplio grado de diferenciación genética entre

ellas, con identicación de cuatro grupos, con mayor y menos pureza

respectivamente.

Palabras clave: diversidad genética, genotipo, marcadores

microsatelites, Sesamum indicum L.

Resumo

A alta qualidade das sementes de gergelim originárias do país fez

com que o Paraguai estivesse entre os principais exportadores. Das

variedades disponíveis no território paraguaio, a mais difundida é a

‘Escoba Blanca’, que, possivelmente pelo processo de multiplicação,

poderia promover alterações na sua frequência alélica, diversidade

e pureza genética. Este trabalho foi realizado com o objetivo de

determinar a diferenciação genética entre 50 populações/canteiros/

bancos de sete empresas paraguaias que coletam gergelim ‘Escoba

Blanca’, utilizando marcadores microssatélites. Estes sete bancos/

empresas/cooperativas coletam e representam amostras de todos os

produtores/sementeiros localizados em diferentes departamentos

da Região Leste e Oeste (Chaco) do país, com os quais trabalham,

comercializam e coletam gergelim. Foi obtido tecido vegetal para

extração de DNA, de mudas plantadas especialmente para esse m,

utilizando todas as amostras/acessos incluídos. Foram utilizados seis

marcadores microssatélites: GBssrsa184, GBssrsa123, GBssrsa182,

GBssrsa108, GBssrsa08 e GBssrsa72. Foram calculados: número

e frequência de alelos, distância/agrupamentos, diferenciação entre

populações e sua estrutura genética. O número médio de alelos

por locus variou de 1,33 a 3,00. Nos marcadores GBssrsa184 e

GBssrsa108, três populações apresentaram maior frequência de alelos.

As populações examinadas exibiram amplo grau de diferenciação

genética entre si, com identicação de quatro grupos, com maior e

menor pureza respectivamente.

Palavras-chave: diversidade genética, genótipo, marcadores

microssatélites, Sesamum indicum L.

Introduction

In Paraguay, the cultivation of sesame (Sesamun indicum L.) has

become one of the main income items for small and medium-scale

family farming in the last decade (González and Causarano, 2014;

Rabery et al., 2020). The high quality of sesame seeds originating in

the country has led Paraguay to become one of the world’s leading

exporters, gaining space in international markets (Melgarejo et al.,

2020). At the same time, this signicant boost in the production and

marketing system has tripled the area planted per harvest, making it

an item with a high socioeconomic impact.

Among the varieties available in Paraguay, the most widespread

is the ‘‘Escoba Blanca’’ (Rabery et al., 2020). Although this situation

was positive, elds with “Foundation” category seeds were not

maintained to guarantee varietal purity, in addition to the widespread

practice among farmers of using their seeds. This is complemented

by the dubious phylogenetic origin of this variety, which drives the

concern to molecularly characterize the material. In that context,

consideration of microsatellites, also known as simple sequence

repeats (SSRs), is important. Due to their robustness, these SSRs have

become an ideal molecular marker in population genetics research

(Zhang et al., 2023).

So far, ve stable and morphologically uniform genotypes have

been identied, which means that they belong to the ‘‘Escoba Blanca’’

variety, but the level of genetic diversity between the selected and non-

selected materials is still unknown, so performing a genetic analysis

with microsatellite markers would provide more precise information

than an analysis with morphological descriptors (Nyongesa et al.,

2013). Thus, it was visualized to use microsatellite markers to develop

the molecular study from samples from the most important national

seedbeds in the country, in order to know distances, dierentiations,

and genetic structure between them, contributing to determining the

purity and/or ow of this variety, widely disseminated.

Materials and methods

Sample collection

Existing samples were collected in the seven most important

seedbeds in Paraguay, places where the reproductive material of

the ‘‘Escoba Blanca’’ variety is periodically collected; directly from

its production centers, located in areas of San Pedro, Concepción,

Caaguazú, Cordillera, Central, Caazapá, Canindeyú, Alto Paraná and

Misiones (Eastern Region), as well as from Boquerón and Presidente

Hayes (Western Region or Chaco). These seedbeds correspond to

companies, private cooperatives, and a university academic center,

here denominated by discretion and ethics: A, B, C, N, S, F, and L. A

total of 50 accessions/samples were obtained from the seven seedbeds.

Each sample consisted of 200 g of seeds, randomly taken from the key

batches, collected, and available in the seven seed banks, according to

geographical origin. For sampling, metal drills were used, obtaining

the reproductive material from the collection bag center (20 kg each).

The samples were carefully stored and transported to a national

biotechnology laboratory, where grain moisture (5 to 10 %) was

quantied and kept in conservative chambers.

Obtaining plant tissue

Ten seeds from each population were sown in commercial

substrates, composed of combinations of peat, nutrients, bark,

vermiculites, and homogeneous black soil. They were placed in

small plastic pots, taking care of the planting depth, watering, and

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monitoring them permanently. After germination and emergence of

the seedling, the leaf tissue of three seedlings per population was

extracted, at the time that each one presented between four and six

leaves/plant, becoming a biological sample. Then, DNA extraction

was carried out with everyday material, a typical practice, which

Alda et al. (2019) highlight as a simple procedure, but key for

biotechnological services.

DNA Extraction with CTAB + DTT

DNA was extracted using the cetyltrimethylammonium bromide

(CTAB) plus dithiothreitol (DTT) method, which is also used in other

living beings (Angulo et al., 2020; Suazo et al., 2020), here, modied

and described below:

The material to be extracted was placed in a porcelain mortar and

ground with the addition of liquid nitrogen until the total homogeneity

of the product was achieved; it was then collected and placed in 2 mL

tubes to which 750 μL of CTAB+DTT were added to each sample

inside the fume hood. The samples were then placed in a thermal

block and incubated at 65 °C for 30 min. After 15 minutes, they

were taken out and reversed six to eight times, and then completed

with the remaining 15 minutes. The samples were then given 750 μL

of chloroform at -20 °C. They were again mixed by inversion and

centrifuged at 1136 g for 8 min and at 4 °C. Labeled 1.5 mL eppendorf

tubes were prepared and 450 μL of isopropanol or propanol-2 at -20

°C were added to each, and the supernatant was then added to the

corresponding tube. It was mixed by inversion about six to eight

times and incubated for 5 min at room temperature while resting, and

then centrifuged at 441 g for 5 min. The isopropanol or propanol-2

was removed from a glass, taking care not to throw the pellet away.

One milliliter of 75 % ethanol was added to wash it, passed for 30

seconds (s) through a vortex, and was subsequently centrifuged at

1153 g for 5 min. The supernatant was removed and the samples were

dried in the hood. To dissolve the pellet well, ultrapure water was

added. The samples were placed in the oven at 37 °C until the next

day, and then the DNA content was quantied with a DS-11-DeNovix

microvolume spectrophotometer. DNA integrity was determined by

electrophoresis at 60 volts for 60 min in a 0.8 % agarose gel with

tris-acetate-ethylenediaminetetraacetic (TAE) buer that was stained

with Promega’s Diamond Nucleic Acid Dye intercalant and visualized

using Promega’s Gel Doc photodocumentation system. Samples of

adequate quality and integrity were conveniently stored at -20 °C.

Molecular and statistical analysis

After a literature review of molecular studies with sesame, six

SSR markers were chosen: GBssrsa184, GBssrsa123, GBssrsa182,

GBssrsa108, GBssrsa08, and GBssrsa72 (Cho et al., 2011). To

perform the Polymerase Chain Reaction (PCR), each PCR reaction

was carried out in a volume of 10 μL containing ultrapure water, 1.5

millimolar (mM) of MgCl

, 1x 1.5 mM of MgCl

of 10x PCR buer,

0.2 mM of deoxyribonucleotides (dNTPs), 250 nanomolar (nM) of

each direct and reverse primer, 0.5 units of Taq DNA polymerase

and 10 nanograms (ng) of DNA. The temperature prole used for

PCR amplication comprised a denaturation step at 94 °C for 1 min,

followed by the hybridization temperature of the primers at 45.2 - 53

°C for 1 min, and elongation at 72 °C for 1 min. After 34 cycles, the

reaction was nished with 10 min at 72 °C for the nal extension.

Subsequently, the PCR amplication products were run with a

commercially available automatic sequence analyzer, typically used

in paternity and genetic analysis in general, in a private laboratory

of the national environment, considered a reference, which follows

international protocols, validated in the academic and scientic

community. Several alleles and allele frequencies were then tested

using the Microsatellites Toolkit (Excel® MS Toolkit). Likewise,

Nei’s genetic distances were calculated considering the Neighbor-

Joining clustering method, granting condence intervals with 1,000

repeats (bootstrap) through Populations® version 1.2.28. Treeview®

was used to visualize the genetic dendrogram. Correspondence factor

analysis was performed in the process of genetic dierentiation

between populations, using Genetix®. Finally, the genetic structure

between the sesame analysis groups and the correct allocation to

the populations under study were analyzed (Structure® 2.1). These

procedures were followed by other authors (Martínez-López, 2017;

Martínez-López et al., 2019a, 2019b).

Results and discussion

Table 1 shows the allele frequencies for each of the six loci used,

the average number of alleles, and the standard deviation of each

group.

The highest average number of alleles was in the L population

(3.00). This value would be due to the high number of samples

obtained from it, considering that it is a seedbed that: a) harvests and

distributes its germplasm in at least 12 communities distant from

each other, b) It is one of the oldest to operate with this agricultural

item in the Paraguayan Chaco. Concerning this parameter of genetic

diversity, Surapaneni et al. (2014) in sesame cultivars (India);

indicated an average of 2.5 alleles per locus. On the other hand,

Pandey et al. (2015), veried an average of 3.37; while Dossa et al.

(2016); conrmed 4.15 alleles per locus. On the other hand, Cho et

al. (2011), showed that the number of alleles/locus depends on the

diversity of the material, which in turn would be inuenced by the

geographical distribution and age of operation in the eld.

In the present study, these concordances could not be veried.

On the other hand, for the allele frequencies (table 1) in the

GBssrsa184 marker, populations A, C, and L exhibited greater

extension in their distribution, occurring between the base pairs (bp)

169:177. In contrast, L showed an absence of fragments at the height of

169 bp. In GBssrsa08, reduced polymorphism was recorded, with the

F population having a slightly larger extension of allele frequencies

and relatively low variability with fragments of 138 and 148 bp. The

other populations showed low variability. This dynamic was observed

for the GBssrsa72 locus, where the only sample that revealed a slight

amplitude in gene frequencies was B. The GBssrsa108 locus revealed

that the A, C, and L populations were more polymorphic, while

the frequencies for GBssrsa182 indicated that the sample with the

highest polymorphism was “L”. According to Abdellatef et al. (2008),

employing RAPD (Random amplied polymorphic DNA) markers

revealed high levels of genetic variability in sesame, even with the

use of a limited set of primers. Previously, Laurentin and Karlovsky

(2006) reported a high degree of variability in the sesame population

studied with AFLP (Amplied fragment length polymorphism).

Molecular techniques as a basis of calculations for genetic

distances are a very useful tool to delve into possible origins and

formation of populations (Martínez-López et al., 2019a). Along

these lines, gure 1 shows the dendrogram obtained based on the Nei

distance.

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Table 1. Allele frequencies per microsatellites and population, and average number of alleles of white sesame collected in Paraguay.

Locus/Marker

Population/Seedbeds

A B C N S F L

n=6 n=4 n=5 n=5 n=15 n=3 n=12

GBssrsa184

161 4.17

169 16.67 20.00

171 50.00 75.00 40.00 100.00 86.67 33.33 66.67

175 16.67 20.00 66.67 20.83

177 16.67 25.00 20.00 13.33 8.33

GBssrsa08

138 100.00 100.00 100.00 100.00 100.00 83.33 100.00

148 16.67

GBssrsa72

272 100.00 75.00 100.00 100.00 96.67 100.00 100.00

284 25.00

288 3.33

GBssrsa123

262 12.50 6.67

264 25.00 12.50 60.00 10.00 66.67 25.00

266 16.67 12.50 20.00 8.33

267 20.00

268 58.33 62.50 20.00 80.00 76.67 33.33 66.67

282 6.67

GBssrsa182

204 16.67 20.00 8.33

206 50.00 50.00 60.00 16.67 66.67 25.00

260 33.33 25.00 20.00 100.00 83.33 33.33 58.33

262 25.00

264 8.33

GBssrsa108

173 4.17

189 8.33 12.50 20.00 20.00 3.33

190 6.67

191 58.33 75.00 30.00 80.00 76.67 66.67 75.00

193 16.67 10.00 4.17

199 8.33 10.00

203 8.33 12.50 13.33 8.33

213 30.00 33.33 8.33

Average number of alleles

2.83 2.50 2.83 1.33 2.50 1.83 3.00

Standard Deviation

1.60 1.05 1.60 0.52 1.22 0.41 1.67

bp: Base pairs,

n: number of samples

Three main lines/clusters have been found (gure 2), where the L

population is the rst, and most distant from the rest. This population

(L) represents a productive cooperative in the center of the western

region, which has been spreading and marketing this agricultural item

for about three decades, perhaps being the pioneer in the area. The

second group was made up of A, C, and F. Among these, C and F

were genetically very close to each other. In terms of commercial

and geographical dynamics, these three populations have had a lot of

germplasm exchange in the last 15 years, both as collectors of “cross-

producer", except for F, which represents an agrarian academic center

and not an objective economic return. The cross-producer is the one

who receives germplasm from a company, for its cultivation, but after

the harvest, chooses to sell to another collector, looking for a better

price, which would be the fundamental circuit for the mixture or

deterioration of varietal purity.

The third group consisted of B, N, and S, where samples of N

and S were genetically close to each other. However, the B samples

were close to the N and S populations. All the proximities recorded

between some of the accessions of the populations (C and F; N and S),

would be due to the frequent exchange of germplasms given annually

for the development of the crop in the country, taking into account

that the company S and the cooperatives F and N, started the eld

in the 90s (Chaco). It is worth noting that the bootstrap indices of

distance analysis give consistency to the results (Nei and Takezaki,

1996).

Next, the correspondence factor analysis of the seven Paraguayan

white sesame populations.

The populations exhibited a high degree of dierentiation between

them, presenting an extensive distribution along the three axes of the

graph that explain most of the genetic variation, except for the N and

S populations, which did not present considerable dierentiation

between them. Likewise, a low degree of genetic dierentiation

between S, B, and N accessions was observed, showing congruence

with results derived from the distance and dierentiation analysis,

discussed above (gure 1).

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Figure 1. Dendrogram of genetic distances (Nei) among the seven

Paraguayan populations (seedlings) of sesame var.

“Escoba blanca”.

Figure 2: Genetic dierentiation between white sesame

populations, using correspondence factor analysis

for the axes of greatest inertia.

al. (2021) and Teklu et al. (2022), who report that single-nucleotide

polymorphisms (SNPs) are also widely used in plant species

analyses. Although SSRs are considered in sesame, Pandey et al.

(2015), argue that their application is limited in studies with this item,

despite their relevance and usefulness. On the other hand, Dossa et al.

(2016), reported limited studies considering large samples of sesame

accessions from Africa and Asia. Also, Dar et al. (2017), indicated

few studies on genetic diversity in sesame.

The result of the analysis of the genetic structure among all the

accessions that constituted the germplasms analyzed is shown below

(gure 3).

Figure 3. Graphical representation of the results of the genetic

structure analysis of the seven Paraguayan populations

of sesame var. “Escoba blanca”.

Genetic structure sharing between C, F populations and some

L constituent samples was observed to be very consistent. This

strong similarity supports the hypothesis that there is some kind of

genotypic relationship of common origin between them; which leads

to believe that there is still varietal purity of white sesame in these

seed banks. The practical importance of genetic structure results

is to identify samples or seedbeds that can be used for breeding

programs, emphasizing the need for distant materials. Pham (2011)

suggested that 40 % of the total, intra-varietal genetic variation can

be found between accessions or dierent seed banks. On the other

hand, Laurentin and Karlovsky (2006) postulated that the inuencing

factor in the current genetic structure of sesame is the human activity

associated with the management of seed banks and their commercial

distribution.

This work showed that the S, N, and B populations are genetically

well related, as well as C and F; giving consistency to the hypothesis

that these groups (S-N-B and C-F) share origin, a fact that can be

attributed to the dynamic ow of germplasm exchange between them,

or the unique origin in the distribution of white sesame seeds in the

early years of cultivation in Paraguay. In the structure analysis (gure

3B), “K7” revealed four clusters (G1, G2, G3, and G4), where G1 had

the highest number of individuals (84 %). These results conveniently

contribute to the determination of selection criteria useful for making

strategic decisions regarding the future of the crop, which is essential

for eective breeding programs in the genetic management of this

variety (Gediya et al., 2019).

In a study by Abdellatef et al. (2008) on Sudanese sesame, two

main clusters were found, one of which was made up of the Elgadaref-1

and Elobaied-1 genotypes, while the other grouped the Abusitta and

Ali Mahdi genotypes. Ali et al. (2007), showed that accessions from

Japan, India, Myanmar, and Pakistan were closely related genetically,

based on their origin. According to Arriel et al. (2006), the low

diversity evidenced in sesame in Brazil can be attributed to the fact

that sesame is an introduced crop. However, Pham (2011) found high

genetic variation in sesame accessions evaluated, indicating that these

were correlated with their geographical origin. However, the analysis

also showed that some accessions were not grouped with others from

the same geographical region, referring to the exchange of varieties

between farmers from dierent regions as a possible cause. On

the other hand, Surapaneni et al. (2014), did not nd any relevant

dierentiation between sesame genotypes, according to their origin.

Molecular marker techniques, such as AFLP, RAPD, ISSR (inter

simple sequence repeats), and SSR, have been widely used in studies

of genetic diversity in sesame (Pham, 2011), agreeing with Teklu et

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Conclusions

Sesame populations exhibit an interesting degree of genetic

dierentiation among them, with the identication of four groups of

seedlings. The mean number of alleles per locus varies at low levels,

showing reduced genetic variability in the white sesame used in

Paraguay. Likewise, the GBssrsa184 and GBssrsa108 markers better

determine the highest allele frequencies, identifying three populations

with this characteristic in molecular diversity. Finally, it should be

mentioned that the molecular study carried out is contributing and

enriching, leaving to the discretion of the decision-makers of the

sector, the type of use and program that they intend to establish in the

future, from more or less genetically pure lineages, all useful in work

of national impact.

Acknowledgment

To the Institute of Agricultural Biotechnology of Paraguay

(INBIO).

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