Transference of microsatellite markers from Eucalyptus spp toAcca sellowiana and the successful use of this techniquein genetic characterization

Transference of microsatellite markers from Eucalyptus spp toAcca sellowiana and...

(Parte 1 de 3)

Transference of microsatellite markers fromEucalyptusspp to Acca sellowianaand 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 facilitated 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 developed for Eucalyptus to characterize A. sellowiana accessions. Out of 404 primers tested, 180 amplified visible products 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 observed heterozygosity 0.5. 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 possibility 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 goiabeiraserrana or “Feijoa”, is a native of the Brazilian southern plateau with secondary dispersion in Uruguay (Mattos, 1990; Thorp and Bieleski, 2002). Due to the uniqueness of its flavor, the economic importance of the pineapple guava is steadily increasing on the world market (Thorp and Bieleski, 2002) and it is an attractive commercial alternative 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 Catarina. This germplasm bank contains 119 A. sellowiana accessions 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 variability is essential for A. sellowiana conservation, breeding and commercial production. In general, specific phenotypes of discreet variation are used as morphological markers. 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 w.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 simple sequence repeats (SSRs), are considered ideal markers for genetic studies because they combine several suitable features: (i) co-dominance; (i) multiallelism; (ii) high polymorphism, allowing precise discrimination even of closely related individuals; (iv) abundance and uniform dispersion in plant genomes; and (v) the possibility of efficient analysis by a rapid and simple polymerase chain reaction (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 libraries 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 sequences 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 paper 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 species.

Material and Methods

Genetic material

The 119 accessions tested shown in Table 1 were obtained 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 primer pairs developed by Brondani et al. (1998) for the Eucalyptus complex E. grandis x E. Urophylla and they were obtained from the Genetics and Biotechnology unit of the Brazilian agricultural company Embrapa (Empresa Brasileira 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

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

USA: 452-Califórnia, 453-USA

*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 reaction volume composed of buffer (10 mM Tris-HCl pH 8.3,

50mMKCl,1.5mMMgCl2),5%(w/v)dimethylsulfoxide, 9 ng of template DNA, 0.3 μM of each primer, 0.02 mM of eachdNTP(Invitrogen)andoneunitofTaqDNApolymerase (Invitrogen). Amplifications were performed using an MJ Research PT-100 thermal controller adjusted to the following conditions: 96 °C for 2 min, then 30 cycles of 94 °C for1min,56°Cfor1minand72°Cfor1minfollowedbya 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 amplification products being visualized on 1.5% (w/v) agarose gel. In the second step, the pairs of primers showing positive amplification were confronted with an additional group of eight A. sellowiana genotypes to detect polymorphism 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) denaturing 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 obtained using the BIOSYS-1 program (Swofford and Selander, 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 (4.5%) amplified visible products in A. sellowiana. Furthermore, 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 obtained using an annealing temperature of 56 °C.

When we screened the 119 accessions with ten selected polymorphic primer pairs we detected 49 alleles, varying from 120 bp to 320 bp. The quality of the amplification 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

EMBRA26 F-CATGAGTTACTGCAAGAAAAG R-ACAGCCAAAAACCAAATC 155-320 9 0.600 0.868

EMBRA69 F-TGTGTTCTCGGTTTCAAAACT R-TGTGAAGTGATGCGAAGC 200-290 5 0.434 0.758

EMBRA85 F-CACCTCTCCAAACTACACAA R-CTCCTCTCTCTTCACCATTC 140-300 7 0.685 0.831

EMBRA99 F-AATACAATTGAGGGGTCTC R-ACCAAAAACAAATGTCGT 230-250 3 0.120 0.499

EMBRA108 F-CGGTTACTTGCTTCATTCG R-GTACGGATGGGTGGACAC 150-160 2 0.505 0.442

EMBRA123 F-AGAACCCTCTATAAAACCCC R-GGGCTAGACATGATGGAG 180-300 5 0.813 0.656

EMBRA148 F-TGGATGCTGTTCTCATCCT R-GGGTTTCTTTGTGAAACGA 200-320 6 0.702 0.695

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