develoPment of microSatellite marKerS for the

american SPecieS vachellia aroma (fabaceae,

caeSalPinodeae)

deSarrollo de marcadoreS microSatéliteS Para la eSPecie

americana vachellia aroma (fabaceae, caeSalPinodeae)

Carolina L. Pometti , Cecilia F. Bessega , Mauricio Ewens

& Beatriz O. Saidman

Juan C. Vilardi

Summary

Background and aims:There are currently no microsatellite markers available for any

American species of Vachellia, and particularly, for V. aroma. Then the aims of this

study were to develop SSR markers speciﬁc for V. aroma, for the ﬁrst time, and test

its ampliﬁcation in a close related species.

M&M: For the development of the SSR in V. aroma, total genomic DNA was extracted

and it was sequenced in a one-fourth run on a Roche 454 GS FLX+ platform. The

study area included two Argentinean populations: San José and Robles, Santiago

del Estero province.

Results: We detected 422 sequences containing SSR loci. A set of 39 primer pairs

presented ampliﬁed products in V. aroma and V. caven, but 12 revealed clear,

replicable and polymorphic loci in V. aroma.

Conclusions: The results of this work indicate that a new set of SSR markers

was developed for V. aroma and their transferability to V. caven was assessed.

The analysis of variability, showed that these 12 polymorphic markers are highly

informative, and a powerful tool to investigate population genetics parameters in V.

aroma and related species.

. Laboratorio de Genética,

Departamento de Ecología,

Genética y Evolución, Instituto

IEGEBA (CONICET- UBA), Facultad

de Ciencias Exactas y Naturales,

Universidad de Buenos Aires,

Buenos Aires, Argentina

Estación Experimental

Fernández-UCSE (Convenio

Provincia Santiago del Estero-

Universidad Católica Santiago

del Estero), Santiago del Estero,

Argentina

caritoege@gmail.com ;

cpometti@ege.fcen.uba.ar

KeywordS

Citar este artículo

POMETTI, C. L., C. F. BESSEGA,

M. EWENS, J. C. VILARDI & B. O.

SAIDMAN. 2024. Development

of microsatellite markers for the

American species Vachellia aroma

454 pyrosequencing, polymorphic SSR, Vachellia aroma, Vachellia caven.

reSumen

Introducción y objetivos: Actualmente, no existen marcadores microsatélites

disponiblesparaningunaespecieamericanadelgéneroVachellia, yparticularmente,

para V. aroma. Por lo tanto, los objetivos de este estudio fueron desarrollar

marcadores SSR especíﬁcos para Vachellia aroma por primera vez, y probar su

ampliﬁcación en una especie emparentada.

(

Fabaceae, Caesalpinodeae). Bol.

Soc. Argent. Bot. 59: 27-32.

DOI: https://doi.

org/10.31055/1851.2372.v59.

n1.40763

M&M: Para el desarrollo de los microsatélites en V. aroma, se extrajo ADN genómico

total de un individuo y este fue secuenciado en un cuarto de placa en una plataforma

Roche 454 GS FLX+. El área de estudio comprendió dos poblaciones argentinas:

San José y Robles, provincia de Santiago del Estero.

Resultados: Detectamos 422 secuencias que contenían loci microsatélites. Un set

de 39 pares de cebadores presentó ampliﬁcación de productos en Vachellia aroma

y Vachellia caven, pero doce de ellos mostraron un patrón claro, reproducible y

polimórﬁco en V. aroma.

Conclusiones: Los resultados de este trabajo indican que se ha desarrollado un

nuevo set de marcadores SSR especíﬁcos para V. aroma y su transferencia a V.

caven fue exitosa. El análisis de variabilidad mostró que estos 12 marcadores

polimórﬁcos son altamente informativos, constituyendo una poderosa herramienta

para investigar parámetros de la genética de poblaciones en V. aroma y especies

relacionadas.

PalabraS clave

454 pirosecuenciación, SSR polimórﬁcos, Vachellia aroma, Vachellia caven.

Recibido: 24 Mar 2023

Aceptado: 4 Mar 2024

Publicado impreso: 31 Mar 2024

Editora: Viviana Solis Neffa

ISSN versión impresa 0373-580X

ISSN versión on-line 1851-2372

Bol. Soc. Argent. Bot. 59 (1) 2024

introduction

species of Vachellia, and particularly, for V. aroma.

Then, this motivated us to the aims of this study

Vachellia aroma, synonym Acacia aroma (Gillies that were to develop, for the ﬁrst time, SSR markers

ex Hook. & Arn.) Seigler & Ebinger (Seigler & speciﬁc for Vachellia aroma, an American species,

Ebinger, 2006), (Fabaceae, Caesalpinoideae) is a and test its ampliﬁcation in a close related species.

tree that inhabits Central and South America, and the

Caribbean. In South America, the applications for

this tree are several. The wood has many uses, like materialS and methodS

charcoal and fuel, and more important, construction of

hard structures (del Valle Perea et al., 2007). A study

One silica gel-dried leaf sample of V. aroma

of the properties of its wood, yielded to a hard, dense belonging to one individual from Robles, Santiago

and durable one with the possibility of even more del Estero, Argentina (S 63°58’ 59.76”; W 28° 3’

potential uses (Pometti et al., 2009). Furthermore, 12.78”) was used in a simple sequence repeat (SSR)

its twigs and leaves are used in traditional medicine scan at the whole genome level. Total genomic

as infusion to treat gastritis, liver and stomach DNA was extracted with Mini Plant DNAeasy Kit

disorders, and as a digestive aid (Carrizo et al., 2005); (Quiagen) according to manufacturer manual. Then,

also, the leaves have antiseptic properties, useful in it was sequenced in a one-fourth run on a Roche

several aﬀections of skin, throat, eyes and canker 454 GS FLX+ platform (454 Life Sciences, a Roche

(

Martínez Crovetto, 1981; Cialdella, 1984; Alonso Company, Branford, Connecticut, USA) by the

Desmarchelier, 2005; del Valle Perea et al., 2007). INDEAR, Rosario, Argentina, service.

As regards its cultivation, it adapts to silvopastoral In order to identify microsatellite sequences

production systems. Their fruits and leaves serve as in the contigs obtained, we used the software

forage for goats, sheep and cattle (Demaio et al., 2002; MSATCOMMANDER v. 0.8.2 (Faircloth, 2008).

del Valle Perea et al., 2007), but the size of the thorns We used the option Design primer, in which the

turn the access to the higher branches very diﬃcult. In software searches for microsatellite repeats and

this line, several years ago, we started an improvement identiﬁes possible primer annealing sites in one step.

program of controlled crossings aiming to reduce Primer3 (Rozen & Skaletsky, 2000) is implemented in

the size of the thorns in the Estación Experimental MSATCOMMANDER for primer design according

Fernández, Santiago del Estero, Argentina.

to the following criteria: amplification products

The microsatellites or Simple Sequence Repeat within the size range of 100-500 bp, optimal melting

(

SSR) are short sequences, from two to six nucleotides, temperature (range 57-62 ºC), optimal GC content of

tandem repeated that can vary in number and sequence 50%, possession of at least 1-bp GC clamp, low levels

across the diﬀerent individuals. The main advantage of self- or pair-complementary, and maximum end

that these markers oﬀer for the genome analysis stability (D G) of 8.0 (Faircloth, 2008).

is their abundance and dispersion in the DNA.

Ampliﬁcation through PCR was performed on

Its value consists in their multialellic nature and individual loci in 50µl reactions containing 10/30

codominat inheritance, allowing the detection of ng DNA, 0.6 mM each primer, 0.2 mM dNTPs, 0.3

several variants in the population. The SSR can be U Taq DNA polymerase (Invitrogen, Buenos Aires,

found in all the eukaryote organisms, they are highly Argentina), and 1.5 mM MgCl . A T21 IVEMA

polymorphic, and, although they are genome-speciﬁc, thermal cycler (IVEMA, Buenos Aires, Argentina)

they could be transferred across species, according was used for ampliﬁcations, where the cycling

to the phylogenetic distance between them and the proﬁle was initial denaturation at 94 ºC for 5 min;

loci conservation (Ferreira, 2003). Therefore, these followed by 40 cycles at 94 ºC for 45s denaturation,

markers have become a very valuable tool for the primer-speciﬁc annealing temperature (53-58 ºC;

genetic mapping of species, diversity analyses, studies see Table 1) for 45 s, extension at 72 ºC for 45 s;

of gene ﬂow and diﬀerentiation between populations, and a ﬁnal extension at 72 ºC for 10 min. PCR

and genetic improvement and breeding (Contreras et products were run in a 6% (w/v) polyacrylamide gel

al., 2020).

containing 5 M urea in 1% TBE buﬀer (89 mM Tris,

There are currently, to our knowledge, no 89 mM boric acid, 2 mM EDTA, pH 8) with a 10-bp

microsatellite markers available for any American DNA Ladder (Invitrogen) size marker. Gels were

C. L. Pometti et al. - New SSR markers for Vachellia aroma

Table 1. Characteristics of the polymorphic microsatellite markers developed in Vachellia aroma.

Abbreviations= F: forward; R: reverse; Ta: annealing temperature.

Locus

Primer sequence (5´-3´)

F: TGAGAGTTAGCGGTACAGGC

R: GCCAACTCTAAGAAGCAGCG

F: ATCCCATTCGATCCCTGAGC

R: GTGTTGTAAGTCGCGACCTG

F: TCCCTTTACTTCCCTTGGCC

R: AGAGAGCAAGACAGTCCAGC

F: CGACCCACACTTGAGAAAGC

R: CGCCATGATTCTTCTCCTGC

F: TCCTCCTTCAGCACTCCTTC

R: CTCAGAAGTGCAGGGAGGAG

F: CATGTCCGTTGTCCCTTCTC

R: CTCTCTTTGCAGCACCTTGG

F: CAGAAAGCCGAGCAAGAGTG

R: TCAGGGTCAAGAAACGTTGTG

F: TTTCTTCCCATCCAAGCGG

R: CCTTTACACCATCACCGCAC

F: CATCTAGAGGCTTTGCTTGGTG

R: TGACATACTTCTGCTGGGCG

F: GGGAGTGCGCATTAAGATCAG

R: GATTCATGCATGTCCCACAAG

F: GTGGCAGACTCACCTCCTAC

R: GACGAGGAGTTCAATGTTTGTC

F: TCGGTTGCATAATTGGAGGG

R: TCGAAGGCCAACTGTAATATCC

Ta (ºC)

Motif

Product size (bp)

Accession

TU03

(AATC)₄

300- 306

OR231272

TU04

TU08

(AATG)₄

(AACTG)₄

(ATC)₄

382- 390

385- 390

213-222

400- 410

284- 294

326- 334

357- 362

324- 328

317- 325

354- 358

170- 180

OR231273

OR231274

OR231275

OR231276

OR231277

OR231278

OR231279

OR231280

OR231281

OR231282

OR231283

TU13

TU39

(AACTG)₄

(AAAAT)₄

(ACTG)₄

(AAAAG)₄

(ACGC)₅

(AAAG)₅

(AAAT)₅

TU61

TU80

TU114

TU123

TU131

TU134

TU154

(AAAGG)₄

stained with silver nitrate. For all the polymorphic Argentinean populations (10 from San José (SJ): S

loci that generated clear and reproducible patterns, 63°49’54.90”; W 27°52’52.92” and 10 from Robles

we calculated observed, expected, and unbiased (RO): S 63°58’59.76”; W 28° 3’12.78”, Santiago

expected heterozygosity, the eﬀective number of del Estero province). The primers were also assayed

alleles, Nei’s genetic distance (Nei, 1978), Wright in two individuals of Vachellia caven (Molina)

(

1978) ﬁxation indices (F or inbreeding coeﬃcient Seigler & Ebinger, a related species, to test cross

and F_S_Tthat estimates the genetic diﬀerentiation ampliﬁcation.

between populations) and Chi-Square Hardy-

Weinberg equilibrium test using GenAlEx 6.5

(

Peakall & Smouse, 2012) and the polymorphism reSultS and diScuSSion

information content (PIC) using MolMarker

Jahnke et al., 2022). The PIC index describes

(

The 454 sequencing of V. aroma was assembled

diversity within populations and characterizes the into 9449 contigs where we detected 422 sequences

degree of polymorphism at each locus (Botstein et containing SSR loci; 239 (56.7%) di-, 113 (26.8%)

al., 1980). In order to calculate all these indices, tri-, 15 (3.5%) tetra-, 27 (6.4%) penta-, 12 (2.8%)

we used 20 individuals in total, belonging to two hexanucletide and 16 (3.8%) with complex motifs.

Bol. Soc. Argent. Bot. 59 (1) 2024

The most frequent number of tandem repeats

Across the sample size, we found between two

within dinucleotide motifs was 8, in the case to four alleles (A), with an average of 2.417 and

of trinucleotides and tetranucleotides the most 2.5 alleles in San José and Robles, respectively.

frequent conﬁguration included 5 repeats, and In all cases, Ne is lower than A, the mean number

ﬁnally, 4 times was most frequent for penta- and of eﬀective alleles (Ne) was 1.787 and 1.911 in

hexanucleotides.

San José and Robles respectively, ranging from

A set of 39 primer pairs was selected according 1.308 to 2.916 for the two populations. Mean

to ascending order on primer pair penalty value to heterozygosity was 0.336/0.425, 0.412/0.463

test the ampliﬁcation success. All these primers and 0.433/0.488, in San José/Robles (Observed

presented ampliﬁed products in V. aroma and V. Heterozygosity [H ], Expected Heterozygosity

caven with the same intensity, but only 12 revealed [He] and Expected and Unbiased Heterozygosity

clear and polymorphic loci in V. aroma (Table [uHe], respectively) (Table 2). All these estimates

) (the polymorphism was not tested in V. caven are similar to those calculated for the SSRs of

because we only tested the cross ampliﬁcation). Acacia nilotica ssp. indica (synonym Vachellia

Table 2. Descriptive statistics for polymorphic microsatellites in Vachellia aroma. Abbreviations= N: Sample size;

A: Number of diﬀerent alleles; Ne: Number of eﬀective alleles, 1 / (Sum pi^2); Ho: Observed heterozygosity, No.

of Hets / N; He: Expected heterozygosity, 1 - Sum pi^2; uHe: Unbiased expected heterozygosity, (2N / (2N-1)) *

He; F : Fixation index; P H-W test: P value for the Hardy-Weinberg equilibrium test (the asterisks show the level

of signiﬁcance for Hardy-Weinberg equilibrium test: *P< 0.05, **P<0.01); PIC: Polymorphic information content;

SE: Standard error; 95% Conﬁdence interval of F between square brackets.

Population Locus

UHe

Fis

P H-W test PIC

San José

TU13

TU61

1,847 0,545 0,459 0,481

2,180 0,091 0,541 0,567

1,862 0,182 0,463 0,485

2,916 0,455 0,657 0,688

1,322 0,273 0,244 0,255

1,308 0,273 0,236 0,247

1,342 0,100 0,255 0,268

1,541 0,455 0,351 0,368

-0,189

0,832

0,607

0,308

-0,119

-0,158

0,608

-0,294

0,175

-0,429

0,214

0,200

0,300

0,011*

0,044*

0,427

0,965

0,601

0,055

0,329

0,445

0,175

0,477

0,527

0,387

0,539

0,356

0,598

0,228

0,208

0,222

0,290

0,409

0,332

0,356

0,305

0,445

0,260

0,556

0,379

0,351

0,247

0,462

0,365

0,332

0,305

0,485

0,365

0,372

0,383

0,083

TU123

TU131

TU80

TU39

TU154

TU03

TU04

1,942 0,400 0,485

0,511

TU08

1,724 0,600 0,420 0,442

1,862 0,364 0,463 0,485

1,600 0,300 0,375 0,395

TU134

TU114

Mean 10,000 2,417 1,787 0,336 0,412 0,433 0,146 [-0,078/0,371]

0,000 0,149 0,129 0,047 0,037 0,039

0,114

-0,311

0,753

0,341

0,623

-0,359

0,167

0,524

-0,333

-0,062

-0,250

-0,414

0,798*

Robles

TU13

TU61

TU123

TU131

TU80

TU39

TU154

TU03

TU04

TU08

TU134

TU114

2,564 0,800 0,610 0,642

1,681 0,100 0,405 0,426

1,835 0,300 0,455 0,479

1,361 0,100 0,265 0,279

2,062 0,700 0,515 0,542

1,923 0,400 0,480 0,505

1,724 0,200 0,420 0,442

1,600 0,500 0,375 0,395

2,299 0,600 0,565 0,595

1,923 0,600 0,480 0,505

1,980 0,700 0,495 0,521

1,980 0,100 0,495 0,521

0,844

0,004**

0,281

0,018*

0,407

0,598

0,098

0,292

0,963

0,429

0,190

0,012*

Mean 10,000 2,500 1,911 0,425 0,463 0,488 0,123 [-0,142/0,387]

SE 0,000 0,195 0,091 0,075 0,026 0,027 0,135

C. L. Pometti et al. - New SSR markers for Vachellia aroma

nilotica) (Number of alleles between 2-3, Ho concluSionS

ranging from 0- 0.493, He ranging from 0.139-

. 493), an African species that also belongs to

The results of this work indicate that a set of SSR

the genus Vachellia (Wardill et al., 2004). The markers was developed, for the ﬁrst time, for V.

values of heterozygosity, particularly He, were aroma, an American species of the genus Vachellia

larger than that estimated from 401 AFLP loci and their transferability to V. caven was assessed.

in six Argentinean populations of V. aroma (He= The analysis of variability, shows that these 12

.2) (Pometti et al., 2018). The Hardy-Weinberg polymorphic markers are highly informative, and

equilibrium was tested locus by locus, and a powerful tool to investigate population genetics

showed that two out of twelve loci were not at the parameters in V. aroma and related species.

equilibrium for San José and three out of twelve

In conclusion, the present SSR markers could be

were not for Robles (Table 2). In those cases, used for estimating genetic variability and structure,

where the loci are not in equilibrium, a possible include in developing breeding programs, and in

cause could be the excess of homozygotes for each population studies where informative markers are

locus. This could be concluded because of the F_i_sneeded, with the potential transferability across the

values (Table 2). However, the ﬁxation index F_i_sgenus Vachellia.

(

or inbreeding coeﬃcient) was also estimated

in the two populations and the mean value was

.146 and 0.123 for SJ and RO, respectively, authorS’ contributionS

indicating that although the estimates are positive

suggesting some amount of inbreeding, both

Conceptualization: CLP; Formal analysis:

populations are in Hardy-Weinberg equilibrium CLP, CFB; Funding acquisition: CLP, CFB;

because its 95% conﬁdence interval contains Investigation: CLP, CFB, ME; Methodology: CLP;

the 0, P>0.05 in both cases (Table 2). Estimates Project administration: CLP, CFB; Supervision:

of Nei’s genetic distance (0.091) and Wright’s CLP; Visualization: CLP, CFB, ME, JCV, BOS;

F_S_T(0.051) between populations showed an Writing-original draft: CLP, CFB, JCV, BOS

appreciable diﬀerentiation between them with

the SSR markers, and were also consistent with

previous estimates for the same populations acKnowledgmentS

with AFLP markers (Pometti et al., 2018). In

V. aroma, the PIC ranged from 0.208 to 0.598

in San José and from 0.247 to 0.556 in Robles the following funding: Consejo Nacional de

Table 2). PIC values lower than 0.25 indicate Investigaciones Cientíﬁcas y Técnicas (CONICET)

low polymorphism, values between 0.25 and PIP 11220200100477CO to CLP; ANPCyT PICT-

.5 indicate average polymorphism and values 2021-00307 to CLP and PICT 2020-1402 to

The present work was conducted thanks to

(

higher than 0.5 indicate high polymorphism. CFB; Universidad de Buenos Aires, UBACYT

Two loci were highly polymorphic for SJ (TU61 20020190200106BA to CFB.

and TU131) and three for RO (TU13, TU80

and TU04), the remaining ones were average Competing interests

polymorphic for both populations. Although,

The authors have declared that no competing

these results could be biased by the use of interests exist.

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