Marcadores moleculares de Eucaliptus para genética de Acca sellowiana, Manuais, Projetos, Pesquisas de Engenharia Agronômica

Baixe Marcadores moleculares de Eucaliptus para genética de Acca sellowiana e outras Manuais, Projetos, Pesquisas em PDF para Engenharia Agronômica, somente na Docsity! 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. 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. References Billote N, Risterucci AM, Barcelos E, Noyer JL, Amblard P and Baurens FC (2001) Development, characterization and across-taxa utility of oil palm (Elaeis guineensis Jacq) microsatellites markers. Genome 44:413-425. Brondani RPV, Brondani C, Tarchini R and Grattapaglia D (1998) Development, characterization and mapping of micro- satellite markers in Eucalyptus grandis and E. urophylla. Theor Appl Genet 97:816-827. Cipriane G, Lot G, Huang WG, Marrazzo MT, Peterlunger E and Testolin R (1999) AC;GT and AG;CT microsatellite repeats in peach (Prunus persica (L) Batsch): Isolation, character- ization and cross-species amplification in prunus. Theor Appl Genet 99:65-72. Choumane W, Winter P, Weigand F and Kahl G (2000) Conserva- tion and variability of sequence-tagged microsatellite sites (STMSs) from chickpea (Cicer aerietinum L.) within the genera Cicer. Theor Appl Genet 101:269-278. Coart E, Vekemans X, Smulders MJM, Wagner I, Huylenbroeck JV, Bockstaele EV and Roldán-Ruiz I (2003) Genetic varia- tion in the endangered will apple (Malus sylvestris) in Bel- gium as revealed by amplified fragment length polymorphism and microsatellite markers. Mol Ecol 12:845-857. Creste S, Tulmann-Neto A and Figueira A (2001) Detection of single sequence repeat polymorphism in denaturing poly- acrylamide sequencing gels by silver staining. Plant Mol Biol Reptr 19:1-8. Decroocq V, Fave MG, Hagen L, Bordenave L and Decroocq S (2003) Development and transferability of apricot and grape EST microsatellite markers across taxa. Theor Appl Genet 106:912-922. Dirlewanger E, Cosson P, Tavaud M, Aranzana MJ, Poizat C, Zanetto A, Arus P and Laigret F (2002) Development of microsatellite markers in peach (Prunus persica (L) Batsch) and their use in genetic diversity analysis in peach and sweet cherry (Prunus avium L). Theor Appl Genet 105:127-138. Doyle JJ and Doyle JL (1987) Isolation of plant DNA from fresh tissue. Focus 12:13-15. Hodgetts RB, Aleksiuk MA, Brown A, Clarke C, Macdonald E, Nadeem S and Khasa D (2001) Development of micro- satellite markers for white spruce (Picea glauca) and related species. Theor Appl Genet 102:1252-1258. Hokanson SC, Szewc-Mcfadden AK, Lamboy WF and Mcferson JR (1998) Microsatellite (SSR) markers reveal genetic iden- tities, genetic diversity and relationships in a Malus x domestica Borkh. Core subset collection. Theor Appl Genet 97:671-683. Kijas JMH, Fowler JCS and Thomas MR (1995) An evaluation of sequence tagged microsatellite site markers for genetic anal- ysis within Citrus and related species. Genome 38:349-355. Liewlaksaneeyanawin C, Ritland CE, El-Kassaby YA and Ritland K (2004) Single-copy, species-transferable microsatellite markers developed from loblolly pine ESTs. Theor Appl Genet 109:361-369. Lorieux M, Ndjiondjop M-N and Ghesquière A (2000) A first interspecific Oryza sativa & Oryza glaberrima microsatel- lite-based genetic linkage map. Theor Appl Genet 100:593- 601. Marques CM, Brondani RPV, Grattapaglia D and Sederoff R (2002) Conservation and synteny of SSR loci and QTLs for vegetative propagation in four Eucalyptus species Theor Appl Genet 105:474-478. Mattos JR (1990) Goiabeira Serrana Fruteiras Nativas do Brasil. 2º edição. Gráfica Ceue, Porto Alegre, 120 pp. Morgante M and Olivieri AM (1993) Hypervariable micro- satellites in plants. Plant J 3:175-182. Nei, M (1978) Estimation of averageheterozigosity and genetic distance from a small number of individuals. Genetics 89:583-590. Nodari RO, Ducroquet JP, Guerra MP and Meler K (1997) Ge- netic variability of Feijoa sellowiana germplasm. Acta Hort 452:41-46. Pandian A, Ford R and Taylor PWJ (2000) Transferability of Se- quence Tagged Microsatellite Site (STMS) primers across four major pulses. Plant Mol Biol Reptr 18:395-395. Palop M, Palacios C and Gonzáles-Candelas F (2000) Develop- ment and across-species transferability of microsatellite 78 Santos et al. markers in the genera Limonium (Plumbaginaceae). Conserv Genet 1:177-179. Peakall R, Gilmore S, Keys W, Morgante M and Rafalski A (1998) Cross species amplification of Soybean (Glycine max) simple-sequence-repeats (SSRs) within the genera and other legume genera: Implications for the transferability of SSRs in plants. Mol Biol Evol 15:1275-1287. Powell W, Machray GC and Provan J (1996) Polymorphism re- vealed by simple sequence repeats. Trends Plant Sci 1:215- 222. Rafalski JA and Tingey SV (1993) Genetic diagnostics in plant breeding: RAPD’s, microsatellites and machines. Theor Appl Genet 8:275-280. Roa AC, Chavarriaga-Aguirre P, Duque MC, Maya MM, Bonier- bale MW, Iglesias C and Thome J (2000) Cross species amplification of cassava (Manhihot esculenta) (Eurphorbia- ceae) microsatellites: Allelic polymorphism and degree of relationship. Amer J Bot 87:1647-1655. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Clon- ing: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press, New York. Sefc KM, Lopes MS, Mendonça D, Rodrigues Dos Santos M, Laimer Da Câmara Machado M and Da Câmara Machado A (2000) Identification of microsatellites loci in olive (Olea europaea) and their characterization in Italian and Iberian ol- ive trees. Mol Ecol 9:1171-1173. Sharma PC, Winter P, Bünger T, Hüttel B, Weingand F, Weising K and Kahl G (1995) Abundance and polymorphism of di, tri and tetranucleotide tandem repeats in chickpea (Cicer arietinum L.). Theor Appl Genet 90:90-96. Shepherd M, Cross M, Maguire TL, Dieters MJ, Williams CG and Henry RJ (2002) Transpecific microsatellites for hard pines. Theor Appl Genet 104:819-827. Smulders MJM, Bredemeijer G, Rus-Kortekaas W, Arens P and Vosman B (1997) Use of short microsatellites to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species. Theor Appl Genet 94:264-272. Sneath PHA and Sokal RR (1973). Numeral Taxonomy. The Prin- ciples and Practice of Numerical Classification. W.H. Free- man, San Francisco, 573 pp. Swofford DL and Selander RBA (1989) A computer program for the analysis of allelic variation in population genetics and biochemical systematics. Release 1.7. Natural History Sur- vey, Illinois, 43 pp. Szewc-McFadden AK, Kresovich S, Bliek SM, Mitchell SE and McFerson JR (1996) Identification of polymorphic, con- served simple sequence repeats (SSRs) in cultivated Bras- sica species. Theor Appl Genet 93:534-538. Thorp G and Bieleski R (2002) Feijoas: Origins, Cultivation and Uses. HortResearch, Ed. David Bateman, 87 pp. Wünsch A and Hormaza JI (2002) Molecular characterization of sweet cherry (Prunus avium L) genotypes using peach (Pru- nus persica (L) Batsch) SSR sequences. Heredity 89:56-63. Zane L, Bargelloni L and Patanello T (2002) Strategies for micro- satellite isolation: A review. Mol Ecol 11:1-16. Zucchi MI, Brondani RPV, Pinheiro JB, Chaves LJ, Coelho ASG and Vencovsky R (2003) Genetic structure and gene flow in Eugenia dysenterica DC in the Brazilian Cerrado utilizing SSR markers. Genet Mol Biol 26:449-457. Associate Editor: Márcio de Castro Silva Filho Transference of microsatellite markers from Eucalyptus to Acca sellowiana 79