Genome size variation can be used to investigate biodiversity, genome evolution, and taxonomic relationships among related taxa. Plant breeders use genome size variation to identify parents useful for breeding sterile or improved ornamentals. Lilacs (Syringa) are deciduous trees and shrubs valued for their fragrant spring and summer flowers. The genus is divided into six series: Syringa (Vulgares), Pinnatifoliae, Ligustrae, Ligustrina, Pubescentes, and Villosae. Reports conflict on genome evolution, base chromosome number, and polyploidy in lilac. The purpose of this study was to investigate genome size and ploidy variation across a diverse collection. Flow cytometry was used to estimate monoploid (1Cx) and holoploid (2C) genome sizes in series, species, cultivars, and seedlings from parents with three ploidy combinations: 2x x 2x, 2x x 3x, and 3x x 2x. Pollen diameter was measured to investigate the frequency of unreduced gametes in diploid and triploid Syringa vulgaris cultivars. Three triploids of S. vulgaris were observed: ‘Aucubaefolia’, ‘Agincourt Beauty’, and ‘President Grévy’. Across taxa, significant variations in 1Cx genome size were discovered. The smallest and largest values were found in the interspecific hybrids S. ×laciniata (1.32 ± 0.04 pg) and S. ×hyacinthiflora ‘Old Glory’ (1.78 ± 0.05), both of which are in series Syringa. Series Syringa (1.68 ± 0.02 pg) had a significantly larger 1Cx genome size than the other series. No significant differences were found within series Pubescentes (1.47 ± 0.01 pg), Villosae (1.55 ± 0.02 pg), Ligustrina (1.49 ± 0.05 pg), and Pinnatifoliae (1.52 ± 0.02 pg). For S. vulgaris crosses, no significant variation in 2C genome size was discovered in 2x x 2x crosses. Interploid crosses between ‘Blue Skies’ (2x) and ‘President Grévy’ (3x) produced an aneuploid population with variable 2C genome sizes ranging from 3.41 ± 0.03 to 4.35 ± 0.03 pg. Only one viable seedling was recovered from a cross combination between ‘President Grévy’ (3x) and ‘Sensation’ (2x). This seedling had a larger 2C genome size (5.65 ± 0.02 pg) than either parent and the largest 2C genome size currently reported in lilac. ‘Sensation’ produced 8.5% unreduced pollen, which we inferred was responsible for the increased genome size. No unreduced pollen was discovered in the other diploids examined. Increased ploidy may provide a mechanism for recovering progeny from incompatible taxa in lilac breeding.
Jason D. Lattier and Ryan N. Contreras
Jason D. Lattier and Ryan N. Contreras
Lilacs (Syringa sp.) are a group of ornamental trees and shrubs in the Oleaceae composed of 22–30 species from two centers of diversity: the highlands of East Asia and the Balkan-Carpathian region of Europe. There are six series within the genus Syringa: Pubescentes, Villosae, Ligustrae, Ligustrina, Pinnatifoliae, and Syringa. Intraspecific and interspecific hybridization are proven methods for cultivar development. However, reports of interseries hybridization are rare and limited to crosses among taxa in series Syringa and Pinnatifoliae. Although hundreds of lilac cultivars have been introduced, fertility and cross-compatibility have yet to be formally investigated. Over 3 years, a cross-compatibility study was performed using cultivars and species of shrub-form lilacs in series Syringa, Pubescentes, and Villosae. A total of 114 combinations were performed at an average of 243 ± 27 flowers pollinated per combination. For each combination, we recorded the number of inflorescences and flowers pollinated as well as number of capsules, seed, seedlings germinated, and albino seedlings. Fruit and seed were produced from interseries crosses, but no seedlings were recovered. A total of 2177 viable seedlings were recovered from interspecific and intraspecific combinations in series Syringa, Pubescentes, and Villosae. Albino progeny were produced only from crosses with Syringa pubescens ssp. patula ‘Miss Kim’. In vitro germination was attempted on 161 seed from interseries crosses, resulting in three germinations from S. pubescens Bloomerang® x Syringa vulgaris ‘Ludwig Spaeth’. None survived, yet cotyledons produced callus for future efforts to induce embryogenic shoots. This study is a comprehensive investigation of lilac hybridization, and the knowledge gained will aid future efforts in lilac cultivar development.
Jason D. Lattier, Hsuan Chen and Ryan N. Contreras
Althea (Hibiscus syriacus) is a shrub prized for its winterhardiness and colorful summer flowers. Altheas are tetraploids (2n = 4x = 80); however, breeders have developed hexaploids and octoploids. Previous studies report anatomical variation among polyploids, including stomata size. The purpose of this study was 4-fold. First, identify genome size and ploidy variation in cultivars via flow cytometry and chromosome counts. Second, create a ploidy series consisting of 4x, 5x, 6x, and 8x cytotypes. Third, investigate the ploidy series for variation in stomatal guard cell lengths, stomatal density, and copy number of fluorescent ribosomal DNA (rDNA) signals. Fourth, investigate segregation patterns of rDNA signals in a subset of pentaploid seedlings. Flow cytometry revealed most cultivars to be tetraploid with holoploid 2C genome sizes from 4.55 ± 0.02 to 4.78 ± 0.06 pg. Five taxa (‘Aphrodite’, ‘Pink Giant’, ‘Minerva’, Azurri Satin®, and Raspberry Smoothie™) were hexaploids (6.68 ± 0.13 to 7.05 ± 0.18 pg). Peppermint Smoothie™ was a cytochimera with tetraploid cells (4.61 ± 0.06 pg) and octoploid cells (8.98 ± 0.13 pg). To create pentaploids, reciprocal combinations were made between hexaploid ‘Pink Giant’ and tetraploid cultivars. To create octoploids, seedlings were treated with agar solutions containing 0.2% colchicine or 125 μM oryzalin. Guard cell lengths were significantly different among the four cytotypes: 4x (27.36 ± 0.04 μm), 5x (30.35 ± 1.28 μm), 6x (35.59 ± 0.63 μm), and 8x (40.48 ± 1.05 μm). Measurements of stomatal density revealed a precipitous decline in average density from the 4x cytotype (398.22 ± 15.43 stomata/mm2) to 5x cytotype (194.06 ± 38.69 stomata/mm2) but no significant difference among 5x, 6x, and 8x cytotypes. Fluorescent in situ hybridization (FISH) revealed an increase in 5S and 45S rDNA signals that scaled with ploidy: 4x (two 5S + four 45S), 6x (three 5S + six 45S), and 8x (four 5S + eight 45S). However, pentaploid (5x) seedlings exhibited random segregation of rDNA signals between the 4x and 6x cytotypes, including all six possible combinations (two 5S, three 5S) × (four 45S, five 45S, six 45S).
Justin A. Schulze, Jason D. Lattier and Ryan N. Contreras
A tissue culture protocol was developed to germinate immature Prunus lusitanica seeds in vitro. The study was conducted by first identifying the best media for germination, followed by investigating effects of seed conditioning. In Expt. I, seeds were collected 12 weeks after pollination (WAP) ± 1 week and placed on media after removing the pericarp. Eight different MS media (Murashige and Skoog, 1962) were tested (M1–M8) containing two concentrations each of 6-benzylaminopurine (BA), gibberellic acid (GA3), and sucrose. The longest shoots resulted from M4 (1.45 µm GA3, 6 µm BA, and 30 g·L−1 sucrose), followed by M1 (0 µm GA3, 3 µm BA, and 30 g·L−1 sucrose). Radicle and shoot emergence was greater than or equal to 90% for M1, M3, and M4 after a stratification treatment. In Expt. II, M1 was used to test root and shoot emergence at 6, 9, and 12 WAP, with and without cold stratification. Little success was seen 6 and 9 WAP, with only callus development in 6 WAP, nonstratified seed. Cold stratification increased shoot emergence in the 12 WAP group from 4% to 28%, appearing to be critical for shoot emergence. If the cotyledons are retained on the seed, future efforts to expedite breeding of P. lusitanica using in vitro germination should not be collected before 12 WAP and will benefit from cold stratification before germinating on M1 or M4. Chemical names: 6-benzylaminopurine (BA), gibberellic acid (GA3).
Jason D. Lattier, Hsuan Chen and Ryan N. Contreras
Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.
Hsuan Chen, Jason D. Lattier, Kelly Vining and Ryan N. Contreras
Lilacs (Syringa sp.) have been used as ornamental plants since the mid-16th century and remain important in modern gardens due to their attractive and fragrant flowers. However, a short flowering season is a critical drawback for their ornamental value. Breeders have identified remontancy (reblooming) in dwarf lilac (Syringa pubescens), and have tried to introgress this trait into related species by interspecific hybridization. Molecular tools for lilac breeding are limited because of the shortage of genome sequence knowledge and currently no molecular markers are available to use in breeding for remontancy. In this study, an F1 population from crossing Syringa meyeri ‘Palibin’ × S. pubescens ‘Penda’ Bloomerang® Purple was created and subjected to genotyping-by-sequencing (GBS) analysis and phenotyped for remontancy. Plants were categorized as remontant, semi-remontant, and nonremontant based on the relative quantity of inflorescences during the second flush of flowers. A total of 20,730 single-nucleotide polymorphism (SNP) markers from GBS were used in marker-trait association to find remontant-specific marker(s) without marker position information. Two SNP markers, TP70580 (A locus) and TP82604 (B locus), were correlated with remontancy. The two loci showed a partial epistasis and additive interaction effects on the level of remontancy. Accumulation of recessive alleles at the two loci was positively correlated with increased reblooming. For example, 87% of aabb plants were remontant, and only 9% were nonremontant. In contrast, 100% of AaBB plants were nonremontant. These two SNP markers associated with remontancy will be useful in developing markers for future breeding and demonstrate the feasibility of developing markers for breeding woody ornamental taxa that lack a reference genome or extensive DNA sequence information.
Jason D. Lattier, Thomas G. Ranney, Paul R. Fantz and Tony Avent
Liriope Lour. and Ophiopogon Ker Gawl., collectively known as liriopogons, represent important evergreen groundcovers grown throughout the world for their ornamental features and medicinal qualities. As a result of the diversity of desirable traits and evidence of wide hybridization, there is considerable potential for breeding and improvement of liriopogons. However, confusion over taxonomy and proper identification and lack of information on ploidy levels and cytogenetics of individual clones and cultivars have constrained breeding efforts. Objectives of this study were to validate the identification and nomenclature and determine genome sizes and ploidy levels for an extensive reference collection of species and cultivars of liriopogons. Identification was accomplished using existing keys, nomenclature was corrected, and numerous accessions were reassigned based on morphology. Genome sizes were determined by flow cytometry. Ploidy levels for each species were confirmed by traditional cytology. Results confirmed a basic chromosome number of x = 18 for liriopogons with aneuploidy, polyploidy, and cytochimeras found in some cases. The Liriope examined included diploids (L. graminifolia, L. longipedicellata, L. minor, and some of the L. platyphylla), tetraploids (L. muscari and the remaining L. platyphylla), and hexaploids (L. exiliflora and L. spicata). The Ophiopogon studied included diploids (O. intermedius, O. jaburan, O. planiscapus, and O. umbraticola) and a tetraploid/hypotetraploid species (O. japonicus). Monoploid (1Cx) genome sizes varied by genus and species with 1Cx values ranging from 4.27 pg in L. exiliflora to 8.15 pg in O. jaburan. These results clarify nomenclature and taxonomy and provide specific information on genome sizes and ploidy levels of cultivated liriopogons. This information and associated reference collection will aid future taxonomic revisions and enhance efforts to develop new cultivars of liriopogons.