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We graciously thank the Daniel P. Haerther Charitable Trust for its support of The Morton Arboretum' s New Plant Development program, and the Arnold Arboretum of Harvard University and the Chicago Botanic Garden for supplying germplasm for this study. Acknowledgments References Genome Sizing and Ploidy Estimations of Weigela Species and Cultivars Erin L. Pfarr¹, Michael S. Dosmann², Andrew C. Bell³, and Joseph J. Rothleutner¹ ¹The Morton Arboretum, ²The Arnold Arboretum of Harvard University, ³Chicago Botanic Garden Contact: [email protected] 630-719-4885 Doležel J., J. Greilhuber, and J. Suda. 2007. Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols. 2: 2233-2244. Duron M., and L. Decourtye. 1990. ‘In Vitro Variation in Weigela.’ In Somaclonal Variation in Crop Improvement I, ed. Y. P. S. Bajaj, Springer, pp. 606-623. Knecht D. 2011. Carnaval Weigela. Available from <http://knechts.net/weigelas-are-blooming/>. [17 August 2015] Dewitte A., K. Van Laere, and J. Van Huylenbroeck. 2012. ‘2n Gametes in Plant Breeding.’ In Plant Breeding, ed. I. Y. Abdurakhmonov, InTech. pp. 59-86. Beck W.T., and T.G. Ranney. 2014. ‘Ploidy Levels and Interploid Hybridization in Panicle Hydrangea (Hydrangea paniculata).’ SNA Research Conference 59: 181-187. A.P. Sokolovskaya and N.S. Probatova. 1985. Chromosome numbers in the vascular plants from the Primorye territory, Kamchatka, region, Amur valley and Sakhalin. Botanicheskii Zhurnal SSSR. 70: 997-999 Weigela is a genus of spring flowering shrubs native to China, the Korean peninsula, and Japan. In the last 20 years, weigela have regained popularity in the green industry thanks to a surge of new cultivars with characteristics including reblooming, larger flowers, smaller growth habit, fall color, and interesting foliage color and variegation. One reblooming cultivar with pink, red and white flowers on the same plant, Weigela 'Courtalor' Carnaval®, is widely advertised as being a reblooming polyploid. In some plant species, variations in ploidy between individuals exists. Polyploid plants can have altered phenotypes such as broader and thicker leaves, larger flowers, more compact stature, greater tolerance to environmental stress and disease, sterility, and flowering time variation (Dewitte et al., 2012). In other ornamental species like Hydrangea paniculata, it appears that polyploids have been preferentially been selected for use in horticulture (Beck and Ranney, 2014). In this project we investigated 1) Does polyploidy occur naturally in wild weigela populations? 2) Are plant breeders unknowingly selecting for polyploids when selecting for novel traits such as reblooming? Introduction and Objective Significant differences in genome size were observed between accessions by one-way ANOVA test (P < 0.05). A Fisher’s LSD test was used for means separation (P < 0.05). ‘Courtalor’ Carnaval® was the only plant that was separated from all other accessions by the Fisher’s LSD test. Carnaval® has a genome size of 3.03 pg of DNA and appears to be a triploid with (2n=3x=54), while all other accessions tested, including W. praecox and W. ‘Vanicek’, ranged from 1.91-2.32pg of DNA and all appear to be diploid (2n=2x=36). Data Analysis and Results Photo 1. Weigela ‘Courtalor’ Carnaval® (Knecht 2011) Methods 74 accessions representing 11 species and 48 cultivars of weigela were sampled from The Morton Arboretum, the Chicago Botanic Garden, and the Arnold Arboretum. Genome sizes were determined by using a flow cytometer (CyFlow® Ploidy Analyser; Partec. Münster, Germany) and with materials and protocols from CyStain PI absolute P Test Kits (Partec. Münster, Germany). Tissue samples were collected from expanding leaves and co-chopped with an internal standard, Pisum sativum ‘Ctirad’, which has a known genome size of 8.76 pg (Doležel et al., 2007). After chopping, the sample solution was filtered through a 30-micron mesh filter (CellTrics®; Partec. Münster, Germany) and then stained with the Propridium Iodide from the test kit. Samples were then immediately loaded and analyzed by the flow cytometer. Data was collected until at least 5000 nuclei were counted for the weigela, at least 3000 nuclei were counted for the internal standard, and CVs were maintained at < 5%. Three replications were performed per genotype tested. In literature review, W. praecox and W. ‘Vanicek’ were found to be diploids with 2n=2x=36 chromosomes (Sokolovskaya and Probatova, 1985; Duron and Decourtye, 1990). These two taxa would be used to confidently infer ploidy level from genome size. 0 5 10 15 20 25 30 35 Number of individuals Picograms of DNA Genome sizes of weigela cultivars and wild collected accessions Discussion • All surveyed wild collected and cultivated species of Weigela were diploid. If polyploidy does occur naturally in Weigela it is not common. Although the Weigela collections at the Arnold Arboretum and The Morton Arboretum are impressive, it would be informative to be able to sample a greater number of populations over each species’ native range. • Of the cultivars investigated, only one, Carnaval® was triploid. In our literature review we discovered that it was bred by crossing a colchicine-induced tetraploid with a diploid (Duron and Decourtye, 1990). • It also appears that plant breeders are not preferentially selecting for polyploidy in Weigela. The novel traits that many of the new cultivars possess, including reblooming, are not due to polyploidy. z Genome sizes with letters in common were not observed to be different by means separation, Fisher’s LSD (P<0.05). Accession source indicated by accession number; y The Morton Arboretum, x The Arnold Arboretum, w The Chicago Botanic Garden Florescence Range Number of Nuclei W. ‘Courtalor’ Carnaval® W. ‘Bokrafive’ Merlot Rose Pisum sativum ‘Ctirad’ Table 1. Genome size and estimated ploidy levels for Weigela cultivars and species. Accession # ID Genome sizeᶻ Ploidy Accession # ID Genome sizeᶻ Ploidy 359-2015 ct ʸ W. 'Courtalor' Carnaval® 3.03 a 3x 898-1998 ʷ W. 'Olympiade' Briant Rubidor 2.01 defghij 2x 354-2015 ct ʸ W. middendorffiana 2.32 b 2x 61-2012 ʷ W. 'Java Red' Sport 2.01 defghij 2x 53-200*1 ʸ W. decora 2.28 bc 2x 04R5293*03 ʷ W. 'Dark Horse' 2.01 defghij 2x 1317-84-A ᵡ W. japonica 2.23 bcd 2x 1317-2004*1 ʸ W. 'Red Prince' 2.00 efghij 2x 414-2007-B ᵡ W. hortensis 2.20 bcde 2x Q4R5295*7 ʷ W. 'Walweigeye' Eye catcher® 1.99 efghij 2x 221-2014*2 ʸ W. 'Sunset' My Monet® Sunset 2.16 bcdef 2x 317-2001-C ᵡ W. subsessilis 1.99 efghij 2x 333-85*1 ʸ W. × incarnata 2.14 bcdefg 2x 79-1999*5 ʷ W. 'Dart's Pink Lady' 1.99 efghij 2x 1019-90-rA ᵡ W. floribunda 2.13 bcdefgh 2x 785-2005*1 ʸ W. 'Brigela' French Lace™ 1.99 efghij 2x 348-2015 ct ʸ W. 'Carlton' Ghost™ 2.13 bcdefgh 2x 82-2010-A ᵡ W. florida 1.98 efghij 2x 214-2007*2 ʸ W. 'Verweig' My Monet® 2.12 bcdefghi 2x 360-2015 ct ʸ W. 'Kolmagira' Rainbow Sensations™ 1.98 efghij 2x 357-2015 ct ʸ W. 'Bokratwo' Merlot Pink 2.09 bcdefghij 2x 64-2012*10 ʷ W. 'Rumba' 1.97 efghij 2x 1007-80*1 ʸ W. 'P. Duchartre' 2.08 cdefghij 2x 382-2001*8 ʷ W. 'Kosteriana Variegata' 1.97 efghij 2x 426-2001*5 ʸ W. 'Alexandra' Wine and Roses® 2.08 cdefghij 2x 587-53-A ᵡ W. subsessilis 1.97 fghij 2x 353-2015 ct ʸ W. 'Bristol Snowflake' 2.08 cdefghij 2x 167-97-B ᵡ W. maximowiczii 1.97 fghij 2x 559-71*1 ʸ W. 'Argento-marginata variegata' 2.07 cdefghij 2x 554-79*11 ʸ W. praecox 1.97 fghij 2x 1078-2004 ʸ W. 'White Knight' 2.07 cdefghij 2x 249-2008-A ᵡ W. subsessilis 1.96 fghij 2x 164-2008 ʸ W. 'Bramwell' Fine Wine® 2.07 cdefghij 2x 178-85*2 ʸ W. hortensis 1.96 fghij 2x 905-62*1 ʸ W. florida 'Variegata' 2.06 cdefghij 2x 1202-2013*1 ʷ W. 'Verweil-4' Sonic Bloom® Red 1.96 fghij 2x 358-2015 ct ʸ W. 'Bokraspiwi' Spilled Wine® 2.06 cdefghij 2x 612-2012*5 ʷ W. 'Java Red' 1.96 fghij 2x 236-1992 ᶻ W. 'Pink Delight' 2.05 defghij 2x 961-2013*3 ʷ W. 'Bokrasopin' Sonic Bloom™ Pink 1.95 fghij 2x 564-71*1 ʸ W. 'Groenewegenii' 2.05 defghij 2x 481-2003 ʷ W. 'Suzanne' 1.95 fghij 2x 639-2012 ʷ W. ' Bokrashine' Shining Sensation™ 2.05 defghij 2x 132-96-B ᵡ W. florida 1.94 fghij 2x 709-2003*6 ʷ W. 'Victoria' 2.05 defghij 2x 422-93-A ᵡ W. florida 1.94 fghij 2x 355-2015 ct ʸ W. ' Bokrafive' Merlot Rose 2.05 defghij 2x 817-84-B ᵡ W. florida var. venusta 1.94 fghij 2x 89-75*1 ʸ W. 'Pink Princess' 2.04 defghij 2x 957-1991*1 ʷ W. 'Foliis Purpurius' 1.94 fghij 2x 356-2015 ct ʸ W. 'Bokrafour' Flamingo pink® 2.04 defghij 2x 319-94*1 ʸ W. florida 1.94 fghij 2x 65-2012*3 ʷ W. 'Samba' 2.04 defghij 2x 279-84-B ᵡ W. hortensis 1.94 fghij 2x 330-85*2 ʸ W. 'Centennial' 2.03 defghij 2x 966-85-D ᵡ W. praecox 1.93 fghij 2x 81-90-A ᵡ W. decora 2.03 defghij 2x 191-2013*1 ʷ W. 'Sunny Princess' 1.93 ghij 2x 171-2003*1 ʷ W. 'Candida' 2.03 defghij 2x 638-2003*3 ʷ W. 'Styriaca' 1.92 ghij 2x 501-2010 ʷ W. 'Elvera' Midnight Wine® 2.03 defghij 2x 1423-2002*2 ʷ W. looymansii 'Aurea' 1.90 hij 2x 906-77-E ᵡ W. subsessilis 2.02 defghij 2x 843-84-B ᵡ W. preacox 1.90 ij 2x 423-58*1 ʸ W. coraeensis 2.02 defghij 2x 404-86-B ᵡ W. florida 1.90 ij 2x 1004-80*1 ʸ W. 'Bristol Ruby' 2.02 defghij 2x 76-1999 ʷ W. 'Abel Carriere' 1.90 ij 2x 1009-80*3 ʸ W. 'Vanicek' Newport Red 2.02 defghij 2x 125-2003-B ᵡ W. florida 1.89 j 2x 66-2012*2 ʷ W. 'Tango' 2.02 defghij 2x 30-2001-C ᵡ W. hortensis 1.88 j 2x 1178-2014*4 ʷ W. 'Bokrasopea' Sonic Bloom™ Pearl 2.01 defghij 2x 482-2003*6 ʷ W. praecox 'Korean sunrise' 1.87 j 2x Graph 2. Histogram of the genome sizes of tested weigela Graph 1. Flow cytometry output histogram Photo 2. Weigela ‘Verweig’ My Monet®
