Microsatellite Markers in Hazelnut: Isolation, Characterization, and Cross-Species Amplifi Cation
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J. AMER. SOC. HORT. SCI. 130(4):543–549. 2005. Microsatellite Markers in Hazelnut: Isolation, Characterization, and Cross-species Amplifi cation Nahla V. Bassil1 USDA–ARS, NCGR, 33447 Peoria Road, Corvallis, OR 97333 R. Botta Università degli Studi di Torino, Dipartimento di Colture Arboree, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy S.A. Mehlenbacher Oregon State University, Department of Horticulture, 4017 Agricultural and Life Sciences Building, Corvallis, OR 97331 ADDITIONAL INDEX WORDS. Corylus avellana, simple sequence repeats, SSRs, DNA fi ngerprinting, genetic diversity ABSTRACT. Three microsatellite-enriched libraries of the european hazelnut (Corylus avellana L.) were constructed: library A for CA repeats, library B for GA repeats, and library C for GAA repeats. Twenty-fi ve primer pairs amplifi ed easy-to-score single loci and were used to investigate polymorphism among 20 C. avellana genotypes and to evaluate cross-species amplifi cation in seven Corylus L. species. Microsatellite alleles were estimated by fl uorescent capillary electrophoresis fragment sizing. The number of alleles per locus ranged from 2 to 12 (average = 7.16) in C. avellana and from 5 to 22 overall (average = 13.32). With the exception of CAC-B110, di-nucleotide SSRs were characterized by a relatively large number of alleles per locus (≥5), high average observed and expected heterozygosity (Ho and He > 0.6), and a high mean polymorphic information content (PIC ≥ 0.6) in C. avellana. In contrast, tri-nucleotide microsatellites were more homozygous (Ho = 0.4 on average) and less informative than di-nucleotide simple sequence repeats (SSRs) as indicated by a lower mean number of alleles per locus (4.5), He (0.59), and PIC (0.54). Cross-species amplifi cation in Corylus was demonstrated. These microsatellite markers were highly heterozygous and polymorphic and differentiated among genotypes of C. avellana irrespective of geographical origin. They will aid in fi ngerprinting genotypes of the european hazelnut and other Corylus species, genome mapping, and genetic diversity assessments. Hazelnuts are members of the Betulaceae family of the or- colchica Alb. are considered distinct species by some authorities der Fagales. They are deciduous wind-pollinated monoecious but are more commonly placed in one polymorphic species C. trees or shrubs that grow in temperate regions of the northern avellana. The paperbark hazel (C. papyraceae Hickel) is a tree hemisphere. Hazelnut is the fourth most economically impor- that was briefl y described by Hickel (1928). tant tree nut crop, following almond, walnut, and cashew in Molecular markers offer a direct estimation of genetic diversity world production. Cultivars are genotypes of Corylus avellana through a determination of the differences in the genetic material. selected for large kernels of high quality. In the United States, Published results on DNA markers in hazelnut have been limited hazelnut production is mostly confi ned to the Willamette Valley to random amplifi ed polymorphic DNA (RAPD) markers linked in Oregon. Corylus species are diploid and have a chromosome to eastern fi lbert blight resistance (Davis and Mehlenbacher, number of 2n = 2x = 22 (Thompson et al., 1996). They exhibit 1997; Mehlenbacher et al., 2004) and to self-incompatibility sporophytic self-incompatibility and are thus cross-pollinated. (Bassil and Azarenko, 2001; Pomper et al., 1998). Microsatellite Great morphological variation exists among and within Corylus or SSR markers have become the marker of choice for “DNA species. More than 25 species have been described. Of these, fi ve fi ngerprinting” and for genetic mapping in various organisms. shrub species (C. avellana, C. americana Marshall, C. cornuta The relatively random distribution of microsatellites in the ge- Marshall, C. heterophylla Fischer, and C. sieboldiana Blume) nome, as well as the high level of polymorphism, co-dominant and four tree species (C. colurna L., C. jacquemontii Decne., C. inheritance, and high reproducibility and transportability across chinensis Franch., and C. ferox Wall.) are most commonly rec- laboratories, make them useful for assessing genetic diversity, ognized (Thompson et al., 1996). Corylus californica Marshall structure, and differentiation. is recognized as a distinct species by some authorities and as a The U.S. Dept. of Agriculture (USDA), Agricultural Research subspecies or botanical variety of C. cornuta by others (Thompson Service (ARS), National Clonal Germplasm Repository (NCGR) et al., 1996). Corylus maxima Mill., C. pontica Koch., and C. in Corvallis, Ore., preserves more than 740 hazelnut genotypes, including representatives of all commonly recognized species. Microsatellite markers will provide genetic fi ngerprints for the Received for publication 22 Nov. 2004. Accepted for publication 29 Jan. 2005. We acknowledge Isabela Mackey for her dedication in performing PCR, frag- accessions, allow an assessment of genetic variability in the col- ment separation and tabulating the data. A special thank you is extended to Kim lection, and address suspected cases of duplication. Preliminary Hummer for editorial suggestions and to all the technicians who keep the hazelnut results of microsatellite marker development from three micro- trees healthy including Dave Smith, Joe Snead, and Ray Gekosky. We thank the satellite-enriched libraries were recently reported (Bassil et al., Oregon Hazelnut Commission and the USDA–ARS CRIS 5358-150-033-00D for their fi nancial support. 2003). In this study, we characterize 25 loci from the libraries. 1To whom reprint requests should be addressed. E-mail address: cornb@ars- Our goals were to 1) develop and characterize polymorphic mi- grin.gov crosatellite markers from each of the three libraries, 2) use them J. AMER. SOC. HORT. SCI. 130(4):543–549. 2005. 543 BBookook 11.indb.indb 554343 66/13/05/13/05 33:29:44:29:44 PMPM to characterize 20 genotypes of C. avellana, and 3) evaluate their tral Services Laboratory (CSL) of the Center for Gene Research cross-species amplifi cation. and Biotechnology at Oregon State Univ. (OSU) using an ABI 377 (Applied Biosystems, Foster City, Calif.). Sequences were Materials and Methods compared using ClustalW and identical sequences were elimi- nated. Primers were designed by GIS or at OSU using Primer PLANT MATERIAL AND DNA PREPARATION. For development 3 software (Rozen and Skaletsky, 2000). The primer design of the microsatellite-enriched libraries, dark-germinated seeds parameters included an optimum annealing temperature of 60 of a mixture of Corylus avellana genotypes were used. For SSR °C, a GC content of 50%, and an amplicon size of 100–350 base analysis, DNA was extracted from young leaf tissue (in the spring, pairs (bp). Primers were purchased from Operon Technologies April–June) of 20 genotypes of C. avellana, two genotypes of C. (Qiagen, Valencia, Calif.). Primer pairs are described (Table 2) colurna, and a single representative of six other Corylus species along with their motifs and the annealing temperatures (Ta) used (Table 1). The Puregene kit (Gentra Systems, Minneapolis) was during PCR amplifi cation. used according to the manufacturerʼs instructions. PCR CONDITIONS. PCR was performed in a total reaction vol- Corylus avellana cultivars were chosen to represent the vari- ume of 10 μL containing: 1× PCR buffer, 2 mM MgCl2, 0.2 mM ous countries that grow hazelnut, including Turkey, Italy, Spain, of each dNTP, 0.6 μM of each primer, 0.05 U of Biolase enzyme and the United States (Table 1). OSU 252.146 and OSU 414.062 (Bioline USA, Randolph, Mass.), and 2.5 ng of DNA template. are the parents of a hazelnut mapping population segregating for For each primer pair, a gradient PCR ranging from 45 to 65 °C resistance to eastern fi lbert blight (Mehlenbacher et al., 2003). was carried out to determine the optimum annealing temperature MICROSATELLITE-ENRICHED LIBRARIES AND PRIMER DESIGN. using an Eppendorf Gradient thermocycler (Brinkmann Instru- Genetic Identifi cation Services (GIS, Chatsworth, Calif.) con- ments, Westbury, N.Y.) or an MJ Research Tetrad thermocycler structed hazelnut genomic libraries enriched for di-nucleotide (MJ Research, Watertown, Mass.). The PCR conditions consisted repeats CA (library A) and GA (library B) and for tri-nucleotide of an initial denaturation cycle of 94 °C for 3 min, 35 cycles of repeat GAA (library C). Inserts were sequenced by GIS or Cen- denaturation at 94 °C for 40 s, annealing temperature for 40 s, and extension at 72 °C for 30 s. A fi nal extension at 72 Table 1. List of 20 Corylus avellana genotypes, eight genotypes of Corylus species, °C for 30 min was used to maximize nontemplated dA their accession numbers, and geographical origins. addition to the 5´ ends. Accession Name Species Origin Loci were given an acronym to indicate the species from which they were isolated (CA for Corylus avellana), None OSU 252.146 C. avellana Oregon followed by C for Corvallis, and A, B, or C to indicate None OSU 414.062 C. avellana Oregon the library from which the sequence was isolated. When PI 557035 Tonda Gentile C. avellana Italy two markers were isolated from the same sequences, a delle Langhe fi nal letter (a or b) was added. PI 557233 Römische Nuss C. avellana Italy SSR MARKER GENOTYPING. SSR primers were initially PI 271105 Imperial de C. avellana Turkey screened for length polymorphism and for cross-spe- Trebizonde PI 557039 Cosford C. avellana United Kingdom cies amplifi cation on 3% agarose gels using a random PI 557057 Giresun 54.021 C. avellana Turkey set of 11 hazelnut genotypes (six C. avellana cultivars PI 557125 B-4 Pelargonia C. avellana Macedonia and a single representative of each of fi ve species of PI 557117 San Giovanni C. avellana Italy Corylus). If polymorphism was observed in this set PI 557219 Bulgaria XI-8 C. avellana Bulgaria of 11 genotypes, forward primers were fl uorescently PI 634202 Ganja C. avellana Georgia labeled with 6FAM or HEX purchased from Operon PI 557025 Tonda Gentile C.