Transference of microsatellite markers from Eucalyptus spp toAcca sellowiana and the successful use of this techniquein genetic characterization, Manuais, Projetos, Pesquisas de Engenharia Agronômica
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Transference of microsatellite markers from Eucalyptus spp toAcca sellowiana and the successful use of this techniquein genetic characterization, Manuais, Projetos, Pesquisas de Engenharia Agronômica

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C:\Typeset\sbg\30-1\2006-002\002.vp

Transference of microsatellite markers from Eucalyptus spp to Acca sellowiana and the successful use of this technique in genetic characterization

Karine Louise dos Santos1, Leocir José Welter1, Adriana Cibele de Mesquita Dantas1,

Miguel Pedro Guerra1, Jean Pierre Henri Joseph Ducroquet2 and Rubens Onofre Nodari1

1Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal, Departamento de Fitotecnia,

Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil. 2Estação Experimental de São Joaquim, Epagri, São Joaquim, SC, Brazil.

Abstract

The pineapple guava (Acca sellowiana), known in portuguese as the goiabeira-serrana or “Feijoa”, is a native fruit tree from southern Brazil and northern Uruguay that has commercial potential due to the quality and unique flavor of its fruits. Knowledge of genetic variability is an important tool in various steps of a breeding program, which can be fa- cilitated by the use of molecular markers. The conservation of repeated sequences among related species permits the transferability of microsatellite markers from Eucalyptus spp. to A. sellowiana for testing. We used primers devel- oped for Eucalyptus to characterize A. sellowiana accessions. Out of 404 primers tested, 180 amplified visible prod- ucts and 38 were polymorphic. A total of 48 alleles were detected with ten Eucalyptus primer pairs against DNA from 119 A. sellowiana accessions. The mean expected heterozygosity among accessions was 0.64 and the mean ob- served heterozygosity 0.55. A high level of genetic diversity was also observed in the dendrogram, where the degree of genetic dissimilarity ranged from 0 to 65% among the 119 genotypes tested. This study demonstrates the possibil- ity of transferring microsatellite markers between species of different genera in addition to evaluating the extent of genetic variability among plant accessions.

Keywords: Feijoa sellowiana, genetic diversity, goiabeira-serrana, pineapple-guava, transferability.

Received: January 17, 2006; Accepted: July 21, 2006.

Introduction

The pineapple guava (Acca sellowiana, synonym

Feijoa sellowiana), known in portuguese as the goiabeira-

serrana or “Feijoa”, is a native of the Brazilian southern pla-

teau with secondary dispersion in Uruguay (Mattos, 1990;

Thorp and Bieleski, 2002). Due to the uniqueness of its fla-

vor, the economic importance of the pineapple guava is

steadily increasing on the world market (Thorp and

Bieleski, 2002) and it is an attractive commercial alterna-

tive for farmers in southern Brazil (Mattos, 1990).

Although the pineapple guava can be found on the

European market or in the countries in which adapted

cultivars are active (e.g. New Zealand, Colombia and the

USA) as yet there are no improved cultivars in Brazil, its

greatest center of diversity. However, there is an A.

sellowiana Active Germplasm Bank (AGB) located at the

São Joaquim Experimental Station (Estação Experimental

de São Joaquim (EPAGRI), São Joaquim-SC, Brazil) in the

town of São Joaquim in the Brazilian state of Santa Cata-

rina. This germplasm bank contains 119 A. sellowiana ac-

cessions from several regions of Brazil and other countries,

and it is possible to use directly an accession as a clone or to

develop a cultivar by means of genetic breeding methods in

order to scale up commercial production

The genetic variability of this species is normally

high at the center of origin, and information on such vari-

ability is essential for A. sellowiana conservation, breeding

and commercial production. In general, specific pheno-

types of discreet variation are used as morphological mark-

ers. However, a limited number of morphological markers

have been identified for this species (Nodari et al., 1997),

which are frequently affected by dominance and epistatic

gene interactions, environmental effects and pleiotropy. To

overcome such problems, molecular markers can be used to

help genetic characterization and breeding (Nodari et al.,

1997; Brondani et al., 1998, 1997).

Genetics and Molecular Biology, 30, 1, 73-79 (2007)

Copyright by the Brazilian Society of Genetics. Printed in Brazil

www.sbg.org.br

Send correspondence to Karine Louise dos Santos. Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Caixa Postal 476, 88040-900 Florianópolis, SC, Brazil. E-mail: karinesantos@cca.ufsc.br.

Research Article

Among the classes of molecular markers available to

identify variation at DNA level, the microsatellites, or sim-

ple sequence repeats (SSRs), are considered ideal markers

for genetic studies because they combine several suitable

features: (i) co-dominance; (ii) multiallelism; (iii) high

polymorphism, allowing precise discrimination even of

closely related individuals; (iv) abundance and uniform

dispersion in plant genomes; and (v) the possibility of effi-

cient analysis by a rapid and simple polymerase chain reac-

tion (PCR) assay (Morgante and Olivieri, 1993; Rafalski

and Tingey, 1993; Sharma et al., 1995; Brondani et al.,

1998). In addition, for the amplification of microsatellite

loci, a knowledge of their DNA sequence is required, and

this is an expensive and time consuming process (Zucchi et

al., 2003). However, the approach of using enriched librar-

ies with repetitive sequences has been very successful in

developing SSRs at a reasonable cost (Zane et al., 2002).

The ability to use the same microsatellite primers in

different plant species, called transferability, depends on

the extent of sequence conservation in the primer sites

flanking the microsatellite loci and the stability of those se-

quences during evolution (Choumane et al., 2000;

Decroocq et al., 2003; Zucchi et al., 2003). It has been

shown that closely related species are more likely to share

microsatellite priming sites than more distantly related

ones, but it is possible to transfer functional microsatellite

primers even from more distantly related species (Lorieux

et al., 2000).

Because there are no microsatellites available for A.

sellowiana, the Eucalyptus spp. primers of microsatellite

loci (Brondani et al., 1998) can be used as an alternative to

find similar regions on the A. sellowiana genome, since

they belong to the same family (Zucchi et al., 2003).

Thus, the objectives of the work described in this pa-

per were to evaluate the transferability of microsatellite

markers from Eucalyptus to A. sellowiana (both members

of the Myrtaceae) and to characterize the genetic variability

present in the Active Germplasm Bank (AGB) of this spe-

cies.

Material and Methods

Genetic material

The 119 accessions tested shown in Table 1 were ob-

tained from the pineapple guava Active Germplasm Bank

(AGB) located at the São Joaquim Experimental Station

(Estação Experimental de São Joaquim - EPAGRI, São

Joaquim-SC, Brazil). Most of the accessions came from the

Brazilian state of Santa Catarina, although a few accessions

came from other countries (Table 1).Samples of DNA were

obtained following the protocol developed by Doyle and

Doyle (1987). The extracted DNA was quantified in aga-

rose gel (Sambrook et al., 1989) and diluted to 3 ng μL-1 for further use in the amplification reactions. Leaf DNA from

Eucalyptus grandis was used as a control.

Microsatellite markers and DNA isolation

For amplification in A. sellowiana we used 404 pri-

mer pairs developed by Brondani et al. (1998) for the Euca-

lyptus complex E. grandis x E. Urophylla and they were

obtained from the Genetics and Biotechnology unit of the

Brazilian agricultural company Embrapa (Empresa Brasi-

leira de Pesquisa Agropecuária-Recursos Genéticos e Bio-

74 Santos et al.

Table 1 - Accession number and origin of the 119 accessions from the Active Germoplasm Bank of Goiabeira-serrana located at the São Joaquim/Epagri

Experimental Station in the Brazilian state of Santa Catarina. All the Brazilian cities are located in the state of Santa Catarina.

Country, city of isolation and accession number

Brazil Other counties

Bom Jardim: 370, 371, 372, 373,

373B, 374, 376, 527

Lages: 228, 229, 246, 247, 249,

250, 259, 276, 276B, 276-20A, 27

8-1, 278-2, 294, 301, 306B, 321, 3

26B, 331, 332, 337, 401

Urupema: 233, 234, 238, 239-2,

240, 242, 244, 390, 392

Israel: 459, Israel*

Caçador: 66, 511, 512, 522 Palmas: 159-27, 159-30 Vacaria: 902, 903 New Zealand: 451, 454, 456, 457,

457A, 457B

Campos Novos: 85 Papanduvas: 755 Vargem Bonita: 804, 805, 805-2 Unknown origin: 438

Curitibanos: 80, 735A, 735B, 735 Ponte Alta152-24, 732, 732B, 740 Uruguai: 441

Frei Rogério: 79 São Joaquim:

103, 110, 117, 118, 119, 120, 124,

260, 277, 300, 358, 359, 360, 366,

369, 387

Videira: 50, 50-2, 53, 53B7,

59-30, 91, 98A, 98B, 101, 101PR,

132, 135, 152-12, 333, 393, 509,

526, 528

USA: 452-Califórnia, 453-USA

Fraiburgo: 148, 501, 502B, 504,

519, 521

Tangará: 141

Iomerê:150B

Lebon Régis: 138, 707, 711, 712, 716

*Unspecified source.

tecnologia, Brasilia, DF, Brazil). Polymerase chain

reaction (PCR) amplification of the microsatellite markers

was performed in 96-well plates containing a 13 μL reac- tion volume composed of buffer (10 mM Tris-HCl pH 8.3,

50 mM KCl, 1.5 mM MgCl2), 5% (w/v) dimethyl sulfoxide,

9 ng of template DNA, 0.3 μM of each primer, 0.02 mM of each dNTP (Invitrogen) and one unit of Taq DNA polymer-

ase (Invitrogen). Amplifications were performed using an

MJ Research PT-100 thermal controller adjusted to the fol-

lowing conditions: 96 °C for 2 min, then 30 cycles of 94 °C

for 1 min, 56 °C for 1 min and 72 °C for 1 min followed by a

final elongation step at 72 °C for 7 min.

The screening of the pairs of primers was done in two

steps. The first step employed the DNA of two A.

sellowiana plants and one control E. grandis plant, the am-

plification products being visualized on 1.5% (w/v) agarose

gel. In the second step, the pairs of primers showing posi-

tive amplification were confronted with an additional

group of eight A. sellowiana genotypes to detect polymor-

phism and 10 selected primers were then utilized to analyze

the genetic variability in the 119 A. sellowiana accessions,

the amplification products being separated on 6% (w/v) de-

naturing polyacrylamide gel. A 100 bp DNA ladder was

used as a molecular weight reference to estimate the sizes

of the amplification products. The gels were stained with

silver nitrate, as described by Creste et al. (2001).

Data analysis

The genetic diversity characterization potential of the

primers was based on allele frequency estimates of the

mean observed heterozygosity (Ho), mean expected

heterozygosity (He) (Nei, 1978) and the number of alleles

per locus for the AGB accessions. These estimates were ob-

tained using the BIOSYS-1 program (Swofford and Se-

lander, 1989). In addition, a dendrogram was plotted from

an unbiased genetic similarity matrix (Nei, 1978) grouped

by the unweighted pair group method with arithmetic mean

(UPGMA) of Sneath and Sokal (1973).

Results

Of the 404 primer pairs tested we found that 180

(44.5%) amplified visible products in A. sellowiana. Fur-

thermore, 38 (9.4%) primer pairs allowed the detection of

clear bands and easy fragment recognition for eight A.

sellowiana genotypes, generating an average of 1.5 alleles

per locus. Satisfactory amplification products were ob-

tained using an annealing temperature of 56 °C.

When we screened the 119 accessions with ten se-

lected polymorphic primer pairs we detected 49 alleles,

varying from 120 bp to 320 bp. The quality of the amplifi-

cation products is shown in Figure 1.

The number of detected alleles per locus ranged from

2 to 9, averaging 4.9 alleles per locus, with the EMBRA 26

marker being the most polymorphic one (Table 2). The ten

Transference of microsatellite markers from Eucalyptus to Acca sellowiana 75

Table 2 - Sequence of the 10 used pairs of primers developed for Eucalyptus genera, allele size range, number of alleles per locus (A), observed

heterozygosity (Ho) and expected heterozygosity (He) of amplified microsatellite loci in Acca sellowiana.

Primer* Sequence (F- forward/ R- Reverse) Allele size range (bp) A Ho He

EMBRA 26 F-CAT GAG TTA CTG CAA GAA AAG

R- ACA GCC AAA AAC CAA ATC

155-320 9 0.600 0.868

EMBRA 69 F-TGT GTT CTC GGT TTC AAA ACT

R- TGT GAA GTG ATG CGA AGC

200-290 5 0.434 0.758

EMBRA 85 F- CAC CTC TCC AAA CTA CAC AA

R- CTC CTC TCT CTT CAC CAT TC

140-300 7 0.685 0.831

EMBRA 99 F- AAT ACA ATT GAG GGG TCT C

R- ACC AAA AAC AAA TGT CGT

230-250 3 0.120 0.499

EMBRA 108 F- CGG TTA CTT GCT TCA TTC G

R- GTA CGG ATG GGT GGA CAC

150-160 2 0.505 0.442

EMBRA 123 F- AGA ACC CTC TAT AAA ACC CC

R- GGG CTA GAC ATG ATG GAG

180-300 5 0.813 0.656

EMBRA 148 F- TGG ATG CTG TTC TCA TCC T

R- GGG TTT CTT TGT GAA ACG A

200-320 6 0.702 0.695

EMBRA 166 F- CTG GTC AAC GTC CGA AAG

R- ATG CTG CAG AGG GCA TAA

130-300 5 0.781 0.724

EMBRA 265 F- TAT CAC TGG CAG GAC GCA

R- ACC GAC GCC GAT AAG AGA

200-250 5 0.602 0.652

EMBRA 267 F- GAC CTC CGC TTC AAC GAT

R- GTG CGA GAC CCT CAA TTC TA

120-140 2 0.274 0.275

Average - - 4.9 0.551 0.640

*The primers were those reported by Brondani et al. (1998) and were obtained from the Genetics and Biotechnology unit of the Brazilian agricultural

company Embrapa (Empresa Brasileira de Pesquisa Agropecuária-Recursos Genéticos e Biotecnologia, Brasilia, DF, Brazil).

76 Santos et al.

Figure 1 - Profile of the PCR products of 21 Acca sellowiana accessions

from the Active Germoplasm Bank. The products were amplified by the

Eucalyptus primer EMBRAPA 123 and resolved on silver stained poly-

acrylamide gel. Left line: 1 kb Plus DNA Ladder (Invitrogen).

Figure 2 - Unweighted pair group method with arithmetic mean

(UPGMA) dendrogram reflecting the genetic similarities, based on 10

microsatellite loci, among 119 accessions from the Acca sellowiana Ac-

tive Germplasm Bank.

Table 3 - Origin of the plant accessions presenting alleles with a low fre-

quency (f < 0.05). All Brazilian cities are located in the state of Santa

Catarina.

Local Primer* Local Primer*

Brazil Brazil (cont)

Bom Jardim Ponte Alta

527 EMBRA 26 740 EMBRA 26

372 EMBRA 123 732 EMBRA 26

152 EMBRA 108

Caçador

511 EMBRA 26 São Joaquim

522 EMBRA 69 103-110 EMBRA 26

511 EMBRA 123 119-120 EMBRA 85

277 EMBRA 108

Campos Novos

85 EMBRA 26 Vargem Bonita

805 EMBRA 69

Curitibanos 805 EMBRA 123

735 EMBRA 26

Videira

Fraiburgo 101 EMBRA 26

521 EMBRA 26 101-333 EMBRA 69

501 EMBRA 85 50-91-526 EMBRA 85

501-502 EMBRA 123 50 EMBRA 108

Lages Other countries

246-247 EMBRA 26 Israel,

229 EMBRA 26 Unspecified

source

EMBRA 26

276 EMBRA 69 Unspecified

source

EMBRA 108

250-276 EMBRA 85

326 EMBRA 108 New Zealand

246-249 EMBRA 123 456-457 EMBRA 108

332 EMBRA 265

Unknown origin

438 EMBRA 85

*The primers were those reported by Brondani et al. (1998) and were

obtained from the Genetics and Biotechnology unit of the Brazilian agri-

cultural company Embrapa (Empresa Brasileira de Pesquisa de Agrope-

cuária-Recursos Genéticos e Biotecnologia, Brasilia, DF, Brazil).

Local: Country, city of origin and accession number.

pairs of primers used were able to detect low frequency al-

leles (≤ 0.05), which were distributed in accessions of dif- ferent origins (Table 3).

The mean expected heterozygosity among loci was

He = 0.640, while the mean observed heterozygosity was

Ho = 0.551 (Table 2). In the dendrogram (Figure 2), the 119

accessions were distributed in different groups and the de-

gree of genetic similarity ranged from 35% to 100%. Two

sub-groups contained two accessions with 100% similarity,

one sub-group consisting of accessions 228 and 331 from

Lages and the other sub-group consisting of the New Zea-

land accessions 456 and 457. On the other hand, three ac-

cessions formed two sub-groups, one of which contained

accession 80 from Curitibanos (37.5% similarity with the

other 117 accessions) and the other accession 247 from

Lages plus accession 501 from Fraiburgo (35% similarity

with the other 117 accessions), these two sub groups being

very different from the other sub-groups. Besides this main

feature of grouping, the other sub-groups did not reveal any

special structure, except one, which included 7 out of 9 ac-

cessions from outside Brazil. Interestingly, the two geno-

types from Israel were located outside the group that inclu-

ded accessions from the USA, New Zealand and Brazil.

Discussion

Considering the time-consuming and expensive pro-

cess of microsatellites isolation (Powell et al., 1996), we

took advantage of the availability of Eucalyptus primer se-

quences and used them in Acca sellowiana. Our study dem-

onstrated the transferability of microsatellite markers from

Eucalyptus to Acca across different genera belonging to the

same family (Myrtaceae). This demonstration of transfer-

ability means that future genetic studies can be carried out,

this marker type being extremely useful due to its ease of

use and high amount of information generated. Because of

these features, microsatellites are considered as useful mo-

lecular markers in plant breeding, and are widely used for

cultivar fingerprinting, paternity testing and genome map-

ping.

The transferability across related species and genera

makes these markers very powerful for comparative ge-

netic studies (Szewc-Mcfadden et al., 1996; Smulders et

al., 1997 Roa et al., 2000). A high cross-species conserva-

tion of microsatellite loci within genera has been reported

in tree species such as Citrus (Kijas et al., 1995), Prunus

(Cipriani et al., 1999; Dirlewanger et al., 2002; Wünsh and

Hormaza, 2002), Elaieis (Billote et al., 2001), Picea

(Hodgetts et al., 2001), Pinus (Shepherd et al., 2002;

Liewlaksaneeyanawin et al., 2004), Olea (Olive) (Sefc et

al., 2000), Malus (Coart et al., 2003) and Eucalyptus (Mar-

ques et al., 2002).

However, a cautious approach is required when com-

paring similar PCR products obtained across different spe-

cies, since various factors can cause size homoplasy. Over

long periods of evolution, the interspecific allelic differ-

ences at one locus are often more complex than simple

changes in repeat number. Products amplified in different

species might include mutation, rearrangements and dupli-

cations in the flanking region and/or changes in the number

of repeats (Peakall et al., 1998).

Microsatellite transferability has also been confirmed

to occur between species from different genera. In the work

described in this paper we have demonstrated that primer

pairs developed for Eucalyptus were able to amplify in the

A. sellowiana genome. Zucchi et al. (2003) used a sample

from the same set of Eucalyptus complex primer pairs to

test their transferability to other Myrtaceae species such as

Eugenia dysenteria and found that of the 356 microsatellite

primer pairs tested it was possible to transfer 10, represent-

ing a transferability of 2.8%. Interestingly, none of the

microsatellite primer pairs transferred to Eugenia

dysenteria by Zucchi et al. (2003) coincided with the prim-

ers transferred to A. sellowiana in our study. Although it is

premature to make inferences about relatedness before ob-

taining further evidence, by considering these results to-

gether it can be hypothesized that there is more similarity

between Acca and Eucalyptus than between Eugenia and

Eucalyptus. According to Palop et al. (2000), micro-

satellites loci are more likely to be amplified in closely re-

lated species.

The number of primer pairs transferred from Euca-

lyptus to A. sellowiana (44.5%) can be considered very

high in comparison to other studies (Padian et al., 2000). In

addition, at least 26% of the primer pairs transferred were

able to detect polymorphism among the 119 A. sellowiana

genotypes screened, which can be considered to be a high

level in light of the fact that in the study by Zucchi et al.

(2003) cited above with 10 pairs of Eucalyptus primers

transferred to E. dysenteria the level of polymorphism was

only 2%.

It is relevant to mention the existence of a large

amount of genetic variability among the A. sellowiana ac-

cessions. Besides supporting such a conclusion, the pres-

ence of alleles with a low frequency in accessions of

different origins suggests that the genetic variability is dis-

persed across locations. Genotypes with a low level of simi-

larity in comparison with others were also found in this

study. This high amount of genetic variability is no sur-

prise, since the A. sellowiana germplasm bank contains a

collection of 119 representatives from 14 locations distrib-

uted across southern Brazil. Most of the accessions have

agronomic importance, since they express one or more ag-

ronomic traits that can be further integrated into breeding

programs.

The high value of expected heterozygosity in compar-

ison with observed heterozygosity among the accessions in

the A. sellowiana germplasm bank indicates that hetero-

zygote deficit is present in the germplasm bank accessions.

However, this deficit is relatively low and the heterozy-

Transference of microsatellite markers from Eucalyptus to Acca sellowiana 77

gosity is high, which suggests the existence of high genetic

diversity in the natural populations from which the A.

sellowiana accessions were collected.

The dendrogram which we obtained based on 10 loci

showed two sub-groups consisting of two accessions each,

with 100% of similarity. At the other extreme, three acces-

sions showed a low degree of similarity (from 35.0 to

37.5%) with the other 117 plant accessions. These results

agree with the high values of heterozygosity and the 48 al-

leles detected in the Active Germplasm Bank. It is impor-

tant to mention that the accessions from New Zealand and

the United States were included in a sub-group, indicating

the narrowing of the genetic base for these accessions and a

substantial degree of relationship among them.

This study has demonstrated the transferability of Eu-

calyptus spp. microsatellite markers to Acca sellowiana,

which belong to the same family (Myrtaceae) but are dis-

tinct genera. Because the Eucalyptus microsatellite loci

were able to detect the existence of a large amount of ge-

netic variability among the A. sellowiana accessions they

can be used for genetic characterization of both accessions

and natural populations, knowledge of which helps to

accelerate not only the establishment of appropriate conser-

vation strategies but also marker assisted selection, the se-

lection of parents for controlled crosses and the monitoring

of the segregation of genomic regions of agronomic interest

in segregating progenies. In addition, the Eucalyptus prim-

ers can be used for genetic studies in other Myrtaceae spe-

cies for which there are no species-specific microsatellites

available.

Acknowledgments

The authors thank Embrapa Recursos Genéticos e

Biotecnologia for the primer sequences and the Brazilian

agencies CNPq and PRODETAB for a research grant. The

Brazilian agency CAPES provided scholarships to KLS

and ACMD.

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Associate Editor: Márcio de Castro Silva Filho

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