The genus Hydrangea comprises ≈30 species that are widely distributed in America and Asia (McClintock, 1957). Famous for their large inflorescences surrounded by showy sepals, several Hydrangea species have been cultivated and are now some of the most popular ornamental flowering shrubs worldwide (Dirr and Dirr, 2004). Hydrangeas are one of the most economically important nursery crops in the United States, with sales exceeding $120 million in 2014 (U.S. Department of Agriculture, 2014). Five species of Hydrangea (H. macrophylla, H. paniculata, H. quercifolia, H. arborescens, and H. anomala ssp. petiolaris) are widely cultivated in the United States as landscape plants (Dirr, 2009). Among them, H. macrophylla, also known as bigleaf hydrangea, is the most commonly cultivated species. One of the remarkable traits of bigleaf hydrangea is the aluminum (Al)-dependent flower color; plants may have pink or blue flowers in the presence of Al and a favorable substrate pH (Ma et al., 1997). Bigleaf hydrangeas are popular for their utility as landscape plants, potted plants, and cut flowers; therefore, traits such as flower and foliage color, sepal size and shape, and leaf characteristics have been enhanced through conventional breeding efforts that have led to more than 1000 named cultivars (van Gelderen and van Gelderen, 2004). These breeding efforts have focused largely on novel floral traits rather than disease resistance or environmental tolerance, and much room for improvement exists for a number of environmental and ornamental traits.
Improvements in environmental tolerance and combinations of desirable traits can be achieved through breeding approaches such as crosses within H. macrophylla or interspecific hybridization between H. macrophylla and closely related species (Rinehart et al., 2006). Limitations to hybrid breeding of H. macrophylla include lack of precise germplasm classification, difficulty recovering favorable trait combinations, and a long juvenile period before selection can occur. The taxonomy classification of the two most important H. macrophylla subspecies, ssp. macrophylla and ssp. serrata, has been disputed due to the lack of strong classification tools and convincing results (Reed and Rinehart, 2007). Most Hydrangea species are diploid, with a chromosome number of 2n = 36 and genome size of 2.0 Gb (Cerbah et al., 2001); however, different ploidy levels among hydrangea species and within bigleaf hydrangea have been reported (Alexander, 2017; Jones et al., 2007; Sax, 1931; Schoennagel, 1931). Cultivars of H. macrophylla that are resistant to powdery mildew (Erysiphe polygonii) infection have been identified and include ‘Veitchii’, which has been widely used as a parent in crosses (Li et al., 2009; Windham et al., 2011). However, ‘Veitchii’ is not completely resistant to powdery mildew; it has lacecap flowers and ordinary foliage, which often make seedlings undesirable as new cultivars. Hybrids between Dichroa febrifuga, which is semi-evergreen and tropical, and H. macrophylla showed improved drought and sun tolerance but lacked the showy sepals that appeal to consumers (Jones and Reed, 2006; Reed et al., 2008). Closely related species with ploidy differences have produced fertile interspecific hybrids with H. macrophylla, suggesting that this breeding strategy could significantly expand hydrangea genetics over the long term, even when just using the species identified by Rinehart et al. (2006). Hybridizations between H. macrophylla and hydrangea species outside the group of closely related taxa have only been accomplished with embryo rescue, and offspring were not vigorous or fertile (Kudo and Niimi, 1999; Kudo et al., 2002; Reed, 2004; Reed et al., 2001).
Releasing hydrangea cultivars requires screening of hundreds or thousands of seedlings over multiple generations to breed cultivars with the combination of desirable floral traits that consumers expect. As an obligate out-crossing species, H. macrophylla has high heterozygosity, and breeding is further hampered by long generation times typical of woody plants (Rinehart et al., 2016). Modern breeding methods that use molecular markers to identify desirable traits during the plant seedling stage could effectively reduce the breeding time, investment, and effort of cultivar release. However, unlike agronomic crops, little is known about the H. macrophylla genetic background because prior breeding efforts were mostly based on open-pollinated hybridizations rather than marker development. Simple sequence repeats markers (SSRs) have been developed and used to describe germplasm resources, parentage/pedigree, and intraspecific and interspecific hybrids (Rinehart et al., 2006, 2010). A phylogenetic tree of Hydrangea species was established to study the possibility of interspecific hybridization by using 13 highly polymorphic chloroplast markers (Mendoza et al., 2013). Recently, Waki et al. (2018) screened 768 SSR primer pairs in 93 F2 progeny to identify markers linked to double-flower and hortensia (mophead) traits in H. macrophylla; they found that both traits are each controlled by a single recessive gene. Many SSRs were developed through transcriptome sequencing and used for genetic mapping of powdery mildew resistance, remontancy, and flower type in hydrangea, but they were insufficient for linkage mapping due to the lack of marker density (Rinehart et al., 2018).
Accelerated breeding of new H. macrophylla cultivars directly helps the nursery industry because consumer interest is driven, in part, by the release of new and novel plants. Conventional breeding can be vastly improved by investigating genetic availability in germplasm collections and increasing breeding accuracy and efficiency using molecular markers. In this regard, genotyping-by-sequencing can be used because of its utility for detecting large numbers of single-nucleotide polymorphism loci and rapidly genotyping diverse accessions. The goals of this research were to apply genotyping-by-sequencing to discover SNPs for bigleaf hydrangea cultivars, investigate the genetic diversity and population structure of H. macrophylla cultivars, and determine the disputed taxonomic classification of H. macrophylla ssp. serrata.
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