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Cross-Transferability of SSR Markers in Osmanthus

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Cross-Transferability of SSR Markers in Osmanthus Publication-2 Genet Resour Crop Evol DOI 10.1007/s10722-017-0514-4 RESEARCH ARTICLE Cross-transferability of SSR markers in Osmanthus Lisa W. Alexander . Chandra S. Thammina . Matthew Kramer Received: 30 September 2016 / Accepted: 28 March 2017 Ó Springer Science+Business Media Dordrecht (outside the USA) 2017 Abstract Developing a molecular tool kit for hybrid Genotyping with 42 microsatellite markers yielded a breeding of Osmanthus species and related genera is an total of 367 loci. Number of alleles per locus ranged important step in creating a systematic breeding from 2 to 17 with a mean of 8.7 ± 4.8. Mean observed program for this species. To date, molecular resources and expected heterozygosities were 0.560 ± 0.225 have been aimed solely at Osmanthus fragrans with and 0.688 ± 0.230, respectively. Percent of polymor- little work to develop markers for other species and phic loci ranged from 40% in Osmanthus delavayi to cultivars. The objectives of this study were to (1) 100% in O. fragrans. Osmanthus fragrans had the determine cross-transferability of O. fragrans and highest mean number of alleles per locus (4.2) while O. Chionanthus retusus derived SSRs in diverse Osman- delavayi had the lowest (1.1). A reduced suite of eight- thus taxa, (2) quantify the influence of locus-specific markers can distinguish between accessions with non- factors on cross-transferability, and (3) determine the exclusion probabilities of identity from 3.91E-04 to genetic relationships between accessions. We tested 70 2.90E-07. The SSR markers described herein will be SSR markers derived from O. fragrans and C. retusus immediately useful to characterize germplasm, iden- in 24 accessions of Osmanthus. Sixty-seven markers tify hybrids, and aid in understanding the level of showed transfer to at least one other Osmanthus species genetic diversity and relationships within the culti- with an overall transfer rate of 84% of loci across taxa. vated germplasm. Electronic supplementary material The online version of Keywords Oleaceae Á Microsatellites Á Marker- this article (doi:10.1007/s10722-017-0514-4) contains supple- assisted breeding Á Ornamental horticulture mentary material, which is available to authorized users. L. W. Alexander (&) C. S. Thammina Floral and Nursery Plants Research Unit, US National Department of Plant Biology and Pathology, Rutgers Arboretum, US Department of Agriculture, Agricultural University, 59 Dudley Road, New Brunswick, NJ 08901, Research Service, Otis L. Floyd Nursery Research Center, USA 472 Cadillac Lane, Mcminnville, TN 37110, USA e-mail: [email protected] M. Kramer US Department of Agriculture, Agricultural Research C. S. Thammina Service, Statistics Group, 10300 Baltimore Avenue, Bldg. Floral and Nursery Plants Research Unit, US National 010A, Beltsville, MD 20705, USA Arboretum, US Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Bldg. 010A, Beltsville, MD 20705, USA 123 Genet Resour Crop Evol Introduction assessing relationships between wild germplasm or cultivated material, genetic map construction, clonal The genus Osmanthus Lour. (Family: Oleaceae Tribe: identification, controlled cross certification, species Oleinae) consists of about 30 species of evergreen and hybrid identification, and paternity determination trees and shrubs distributed primarily throughout (Grocer and Sharma 2016). temperate and tropical China. Osmanthus fragrans To date, Osmanthus molecular resource develop- (Thunb.) Lour., commonly known as sweet osmanthus ment has been aimed solely at O. fragrans with little or fragrant tea-olive, is the most popular Osmanthus work to develop markers for other species and species with at least 166 named cultivars (Xiang and cultivars. Because SSR markers are often transferable Liu 2008). Osmanthus fragrans is a common land- within families, markers developed for other species scape commodity throughout its native range where it and genera within Oleaceae are a good starting point is prized for its fragrant flowers and leaf extracts used for developing an Osmanthus molecular toolkit (van in traditional medicine (Shang et al. 2003). Four Heusden and Arens 2010). The objectives of this study groups of O. fragrans (Albus, Asiaticus, Aurianticus, were to (1) determine cross-transferability of O. fra- and Luteus, a.k.a Thunbergii) are defined by morpho- grans and Chionanthus retusus derived SSRs in logical and phenological traits including flower color, diverse Osmanthus taxa, (2) quantify the influence of peduncle length, and flowering time (Xiang and Liu locus-specific factors on cross-transferability, and (3) 2008). Other popular Osmanthus species include determine the genetic relationships between acces- Osmanthus heterophyllus (Don.) Green (holly tea sions. The development of these SSR markers will aid olive, false holly) and Osmanthus 9 fortunei Carr., an in assessing relationships and diversity within the O. fragrans 9 O. heterophyllus hybrid, Osmanthus genus Osmanthus, prioritizing germplasm for breed- armatus Diels, and Osmanthus delavayi Franch. These ing, and providing a tool for cultivar and hybrid species have cultivars with diverse plant architecture, identification. leaf forms, and flowering times and are commonly found in landscape plantings throughout USDA Har- diness zones 7 and 8 in the United States. Nursery growers desire Osmanthus cultivars with Materials and methods vibrant flower color, flower fragrance, increased growth rate, and tolerance to colder climates through- Twenty-four Osmanthus accessions representing six out both the US and China (Dong 2010). Germplasm species were included in this study (Table 1). Plants evaluation and breeding are underway to find species from Nurseries Caroliniana (North Augusta, South and cultivars suitable for the US market and incorpo- Carolina, USA) were maintained in 3-gallon pots rate favorable traits into Osmanthus through hybrid under full sun and micro-irrigated using spray stakes. breeding. Developing a molecular tool kit for hybrid Growing media consisted of pine bark amended with -3 breeding of Osmanthus species and related genera is 6.6 kg m 19 N-2.1P-7.4 K Osmocote Pro fertilizer an important step in improving this species. Initial (Scotts-Sierra Horticultural Products Co., Maryville, -3 molecular studies using chloroplast and nuclear inter- Ohio, USA), 0.6 kg m Micromax (Scotts-Sierra -3 genic regions to resolve Osmanthus phylogeny Horticultural Products Co.), 0.6 kg m iron sulfate, -3 showed that Osmanthus is a polyphyletic genus with and 0.2 kg m Epsom salts. Accessions from the US close genetic relationships to Chionanthus L. and National Arboretum were received as semi-hardwood other species (Arias et al. 2009; Yuan et al. 2010). cuttings, dipped in liquid K-IBA (1000 ppm), and Molecular work targeted at O. fragrans includes the rooted in pine bark under mist. Growing media -3 development of SSRs from O. fragrans using repeat- consisted of pine bark amended with 3.3 kg m enriched libraries (Zhang et al. 2011; Duan et al. 19 N-2.1P-7.4 K Osmocote Pro fertilizer. 2013), sequencing of the O. fragrans transcriptome (Mu et al. 2014), and shot-gun sequencing of the Primer selection O. fragrans genome (Yuan et al. 2015). Highly polymorphic DNA markers resulting from these DNA was isolated from Osmanthus accessions using efforts are ideal for breeding applications including modified alkaline lysis (Alexander 2016). Primers 123 Genet Resour Crop Evol Table 1 Osmanthus Genus Species Cultivara Sourceb germplasm used to assess cross-transferability of 70 Osmanthus armatus Jim Porter Nurseries Caroliniana SSR loci Osmanthus armatus Longwood Nurseries Caroliniana Osmanthus armatus US National Arboretum Osmanthus 9 burkwoodii US National Arboretum Osmanthus delavayi US National Arboretum Osmanthus 9 fortunei Fruitlandi Nurseries Caroliniana Osmanthus 9 fortunei San Jose Nurseries Caroliniana Osmanthus 9 fortunei US National Arboretum Osmanthus fragrans Apricot Echoc Nurseries Caroliniana Osmanthus fragrans Asiaticd US National Arboretum Osmanthus fragrans Aurantiacusc US National Arboretum Osmanthus fragrans Beni Kin Mokuseic Nurseries Caroliniana Osmanthus fragrans Fodingzhud Nurseries Caroliniana a Missing cultivar name Osmanthus fragrans Thunbergii Nurseries Caroliniana indicates an unimproved Osmanthus heterophyllus Goshiki Nurseries Caroliniana species Osmanthus heterophyllus Hariyama Nurseries Caroliniana b Germplasm was received from Nurseries Caroliniana Osmanthus heterophyllus Head-Lee Fastigate Nurseries Caroliniana as plants in 1 or 3 gallon Osmanthus heterophyllus Ilicifolius US National Arboretum containers; germplasm from Osmanthus heterophyllus Kembu US National Arboretum the US National Arboretum Osmanthus heterophyllus Ogon Nurseries Caroliniana was received as cuttings. All plants were repotted or Osmanthus heterophyllus Purpureus US National Arboretum rooted in pine bark medium Osmanthus heterophyllus Rotundifolius Nurseries Caroliniana c O. fragrans Aurantiacus Osmanthus heterophyllus Sasaba US National Arboretum group Osmanthus heterophyllus US National Arboretum d O. fragrans Asiaticus Syringa 9 oblata Betsy Ross US National Arboretum group were selected from three sources: Osmanthus considered a duplicate if it showed at least 99% enriched-library (OEL) screening (Zhang et al. identity to the query sequence and an Expect value, or 2011), Chionanthus enriched-library (CEL) screening E-value, of 0.0. A sequence was considered to be genic (Arias et al.
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