Introduction
The genus Hydrangea, belonging to the family Hydrangeaceae, thrives naturally in the temperate regions of Eastern Asia and Northeastern America, as well as in subtropical regions of Middle and South America, and exhibits significant morphological variation within shrubs (McClintock 1956;Pilatowski 1982). This genus encompasses more than 25 species and numerous subspecies, many of which are regarded as ornamental flowering shrubs worldwide (Dirr 2004) because of their large, bright-colored inflorescences ranging from white to pink, purple, and blue (De Smet et al. 2015;Khaing et al. 2016). Hydrangea breeding programs began in the late 19th century in Europe and the United States, focusing on developing new cultivars for the market as flowering-potted plants (Lawson-Hall and Rothera 2005).
Hydrangeas are widely cultivated and have become one of the most popular ornamental flowering shrubs in the United States, with more than 10 million plants sold annually, accounting for 13.5% of the annual U.S. shrub sales (Fulcher et al. 2016). Several species, including Hydrangea macrophylla, H. paniculata, H. quercifolia, H. arborescens, and H. anomala ssp. petioelaris, are predominantly produced for use as landscape plants in nursery cultivation (Dirr 1998). To enhance their characteristics, distant hybridization with related species is commonly employed to introduce genetic variability for commercial purposes. Notably, producing big-leaf appearance is popular for indoor and outdoor cultivation, engaging breeders to utilize these traits to develop new cultivars, resulting in the successful creation of over 1,000 cultivars, many of which retain their original growth habits, floral attributes, and disease resistance (Dirr 2002;van Gelderen and van Gelderen 2004). As a result, numerous sterile cultivated species of Hydrangea genus have become popular ornamental plants in China and Japan (Samain et al. 2010), posing challenges for breeding programs.
The great ornamental value of Hydrangea has encouraged breeders to generate novel cultivars, which has led to challenges in identifying their phylogenetic relationships. Both morphological and molecular studies have been conducted to analyze the relationships among species within the Hydrangea genus (Hufford et al. 2001;Soltis et al. 1995). McClintock (1956) used plant habit, leaf structure, and inflorescence to divide the Hydrangea genus into Hydrangea and Cornidia. However, morphological classification becomes complicated in terms of taxonomy, especially when numerous varieties are created and named as distinct species similar to those in the Hydrangea genus (Cerbah et al. 2001). Employing the matK marker, Samain et al. (2010) suggested that Hydrangea is polyphyletic and comprises eight groups of Hydrangea. Other studies have utilized 13 highly variable plastid markers to divide this genus into 16 sections (Mendoza et al. 2013;Raman et al. 2023). Despite numerous investigations aimed at clarifying the relationships among the species of this genus, the position of H. arborescens remains unresolved, indicating its polyphyly within certain groups (De Smet et al. 2015;Mendoza et al. 2013). In contrast, Yang et al. (2023) employed 81 plastomes and proposed that some sections of Hydrangea are paraphyletic. De Smet et al. (2015) recently proposed two approaches to address phylogenetic relationships within Hydrangea. The first strategy suggested dividing the Hydrangea genus into at least seven new genera, two containing only one species each. The second option involved establishing eight satellite genera, resulting in a comprehensive monophyletic group. However, these approaches may prove less practical for distinguishing morphologically similar taxa and pose challenges when morphological variations occur within the expanded Hydrangea (Raman et al. 2023).
Breeding approaches involving intraspecific hybridization within the Hydrangea genus and closely related species have been used for years to create desirable traits, including improved environmental tolerance (Rinehart et al. 2006). However, hybridization remains a challenge due to a lack of germplasm classification, which has been observed in the crossing of H. macrophylla (Wu and Alexander 2019). Hybrids between H. macrophylla and wild cultivated species such as H. arborescens and H. paniculata typically yield sterile and non-vigorous plants (Reed et al. 2001, 2008). Conversely, hybrids between closely related species like H. macrophylla crossed with H. angustipetala or with Dichroa febrifuga, have resulted in successful cultivars (Kardos et al. 2009;Reed et al. 2008). Furthermore, despite improvements in Hydrangea breeding programs, genetic diversity among cultivars has decreased, leading to the loss of beneficial traits. This is because most cultivars are derived from a limited number of plants domesticated in Europe (Uemachi et al. 2014). In addition, the conventional breeding process of hydrangeas requires a long generation time, primarily focusing on novel flower type selections (Rinehart et al. 2006;Wu et al. 2021).
Cytogenetics, a fusion of cytology and genetic studies, focuses on mitotic and meiotic cells to study chromosome number, structure, and behavior, and has been widely employed to provide relevant information for creating hybrids and improving crops (Bennett 2010;Griffiths et al. 2000). With a growing horticultural industry importing and exporting flowering ornamental plants every year, chromosome data on gametic and somatic cells have helped in the identification and differentiation of wild and domesticated species, as well as providing viability and stability of the crossed plants (Kiihl et al. 2011). Therefore, understanding their genetics through the identification of chromosome numbers, ploidy levels, and genome size is useful for assessing the relationship between wild and cultivated Hydrangea species.
This study aimed to provide a comprehensive review of cytogenetic studies conducted in the genus Hydrangea, focusing on conventional karyotyping and molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH) and flow cytometric analysis, as well as future directions for cytogenetic research in Hydrangea.
Cytogenetic studies in Hydrangea and their importance
Hydrangeas possess numerous characteristics that make them valuable ornamental plants in horticultural markets. While Asia and Europe primarily focus on developing flower colors, shapes, and types, breeders from the United States emphasize improving environmental traits and disease resistance (Khaing et al. 2016). Flower architecture is a key trait in developing Hydrangea cultivars. Enhancing flower structures improves their decorative values; however, cultivating double-flower phenotypes often results in sterility due to the loss of stamen, posing challenges for breeding (Suyama et al. 2015). Intraspecific and intergeneric hybridization within Hydrangeaceae offer opportunities to further develop cultivars with improved traits but, these processes frequently produce hybrids that are weak, sterile, or have reduced fertility, resulting in little to no commercial value (Kardos et al. 2009). The poor viability of interspecific hybrids seems to result from cytogenetic incompatibility between species (Reed et al. 2001). Interspecific crosses are unsuccessful when there are significant chromosomal and DNA differences between species, such as variations in chromosome number, ploidy level, and chromosome structure (Badger 1988;Stebbins 1971).
The genetic makeup of Hydrangea is intricately influenced by differences in chromosome numbers and levels of polyploidy, which significantly affect genome size, intraspecific diversity, and the identification of different varieties (Taniguchi et al. 2024). Woody ornamental plants, such as hydrangeas, are often distinguished by their small genomes and small chromosomes, which also possess weak and tiny roots with a low mitotic index, making them more difficult to handle (Van Laere et al. 2018). These challenges hamper the application of cytogenetic techniques; hence, only a few chromosomal studies have been conducted on this genus (Cerbah et al. 2001;Mortreau et al. 2010;Van Laere et al. 2008), as presented in Table 1.
Conventional Cytogenetics
The primary goals of cytogenetics include the identification of chromosomes and ploidy levels and the construction of karyotypes. Chromosome counting, a conventional and established method for ploidy level determination, has proven to be efficient and reliable (Hwang et al. 2020). Metaphase chromosomes are typically used for this cytogenetic technique, and several chemical pretreatment methods, such as colchicine, 8-hydroxyquinoline, and α-bromonaphthalene, were developed to accumulate more metaphase chromosomes. Young root tips are the best plant tissue for metaphase chromosome visualization. On the other hand, flower bud tissue could also be used to visualize chromosomes, and it allows simultaneous observation of both mitotic and meiotic chromosomes in a single squash preparation, which provides a more comprehensive overview. For example, a few mitotic metaphase chromosomes were observed in the flower buds of Hydrangea, as shown in Figure 1A (unpublished data). The squash method is widely used by plant cytogeneticists to prepare chromosomal samples. However, they are more effective in plants with large chromosomes (Fukui and Nakayama 1996). On the other hand, the “SteamDrop” protocol for chromosome preparation, which results in high-quality slides of chromosomes, has been useful in species with small chromosomes, such as hydrangeas (Kirov et al. 2014;Van Laere et al. 2018). Various staining methods, such as the use of acetocarmine, Feulgen, aceto-orcein, and Giemsa C-banding, aid in clearly observing chromosomes under a microscope (Hiremath and Chinnappa 2015).
In the Hydrangea genus, most species are reported to have a diploid chromosome number of 2n = 2x = 36 (Sax 1931;Schoennagel 1931), such as H. macrophylla (Haworth-Booth 1984). However, species classified into the subspecies Asperae also presented 2n = 2x = 30 and 34 chromosomes (Cerbah et al. 2001). Furthemore, Kudo and Niimi (1999) reported 52 chromosomes in H. macrophylla ssp. macrophylla ‘Blaumeise’. The Asiatic species Hydrangea involucrata has 2n = 2x = 30, whereas the American species H. arborescens has 2n = 2x = 38; however, the former has twice the nuclear DNA content of the latter, even though both are diploids (Zonneveld 2004). Polyploids are also present in the same species, such as H. paniculata individuals with chromosome numbers 2n = 36, 54, 72, and 108 (Funamoto and Tanaka 1988). Sixteen taxa of Hydrangea were also analyzed for their chromosomal composition using Feulgen staining, which showed varied chromosome numbers of 2n = 30, 34, and 36 (Cerbah et al. 2001). Polyploidy in plants commonly results in larger cell size due to genome duplication, a process termed the gigas effect. This can lead to polyploid individuals having larger organs like roots, leaves, tubers, fruits, flowers, and seeds when compared to diploids. Other important consequences of polyploidy include buffering of deleterious mutations, increased heterozygosity, and hybrid vigor (Sattler et al. 2016).
Different staining methods used for chromosome preparation produced varying results in the karyotyping of H. macrophylla. Using Feulgen staining, Cerbah et al. (2001) observed a karyotype containing six metacentric, four submetacentric, and eight subtelocentric chromosomes. In contrast, 4′,6-diamidino-2-phenylindole (DAPI) staining was used by Van Laere et al. (2008) to observe a karyotype consisting of six metacentric, eight submetacentric, and four subtelocentric chromosomes. Although effective in other species, Feulgen staining can produce weak staining results in plants with small chromosomes, such as Hydrangea. Fluorescence staining with DAPI is more suited to this species (Maluszynska 2003). Fig. 1A shows the DAPI-stained metaphase chromosomes of H. serrata ‘Odoriko Amacha’ with 2n = 2x = 36 chromosomes, while the meiotic metaphase 1 chromosomes of H. serrata ‘Kai Kyo’ with 18 bivalents are presented in Fig. 1B (unpublished data). The small chromosomes of hydrangeas make it difficult to arrange them in pairs; hence, constructing a karyotype with molecular markers makes individual chromosomes more distinguishable.
Molecular Cytogenetics
Knowledge of the exact locations of specific genes on chromosomes enhances the efficiency of breeding and selection. Molecular cytogenetic techniques, such as FISH, have made it possible to target the chromatin regions of individual chromosomes based on DNA sequence information, in addition to their morphological characteristics (Schubert et al. 2001). FISH is an efficient method for constructing detailed karyotypes and visualizing specific chromosomal DNA sequences (Hwang et al. 2020). It is particularly useful for identifying sites with highly repetitive genes, such as ribosomal DNAs (rDNAs) genes, which are challenging to map using other techniques (Leitch and Heslop-Harrison 1992). The most frequently used DNA probes for FISH karyotyping are 5S and 45S rRNA, which consist of tandem repeated sequences that are conserved across plant species.
FISH analysis has been employed in some Hydrangea species. FISH, associated with fluorochrome staining, was used to examine the distribution of the 5S and 18S rDNA gene families and GC- and AT-rich DNA regions in H. involucrata and the four subspecies of H. aspera, which showed important differences between these two species. Moreover, B chromosomes have been observed in some accessions of the H. aspera Villosa Group, which constituted the first description of B chromosomes in this genus (Mortreau et al. 2010). A comparative FISH karyotype analysis of three popular Hydrangea species, namely H. macrophylla, H. paniculata, and H. quercifolia, was also conducted using 45S rDNA as the probe. The results revealed variability in chromosomal morphology and 45S rDNA signal distribution (Van Laere et al. 2008). The efficacy of rDNAs as cytogenetic markers relies on the number of rDNA loci in relation to the total chromosome number, as well as their distribution patterns and distinctiveness (Mortreau et al. 2010). The use of several FISH probes simultaneously is more efficient and has enabled the identification of all or almost all chromosomes in Hordeum vulgare (Leitch and Heslop-Harrison 1992), Arabidopsis thaliana (Murata et al. 1997), and some Brassica species (Hasterok et al. 2001). To date, there has not yet been a specific probe developed for Hydrangea chromosomes partly due to limited available genomic information.
Flow cytometry analysis has also been useful and accurate in confirming ploidy levels and estimating DNA content and genome sizes (Doležel and Bartoš 2005). The “C” value represents the DNA quantity in an unreplicated haploid genome and typically remains consistent among individuals of the same species (Bennett and Leitch 1995). However, differences in nuclear DNA content have been observed across different species within the same genus and across various plant groups (Buitendijk et al. 1997;Dickson et al. 1992) and thus can be a useful taxonomic marker. This information, provided by flow cytometry, is beneficial for breeders and molecular geneticists.
The nuclear DNA content, which represents the genome size of a species, is a crucial factor in utilizing molecular tools to enhance agricultural and horticultural traits (Cerbah et al. 2001), as well as it provides insight into the environmental adaptation strategies. For instance, the DNA content of H. macrophylla subsp. macrophylla which inhabits sea-level environments, was found to be higher than that of H. macrophylla subsp. serrata which grows in the mountainous regions (Iwatsuki et al. 1995). Cerbah et al. (2001) and Zonneveld (2004) conducted cytometric analyses of DNA content in relation to chromosome number to investigate genome size variations and species relationships within the genus Hydrangea. Their results suggested that chromosome number is independent of DNA content and differs significantly among Hydrangea species. Using DAPI-stained nuclei for flow cytometry, 100 diploid and 21 triploid H. macrophylla subsp. macrophylla cultivars were identified. All 23 H. macrophylla subsp. serrata taxa examined were diploid. Their flow cytometric findings were consistent with the chromosome counts, showing 36 chromosomes in diploids and 54 in triploids; however, no details were reported regarding the specific cultivars examined or the number of cells counted (Demilly et al. 2000). The genome size values for H. involucrata and H. aspera reported by Zonneveld et al. (2005) were 8% and 12% higher respectively, compared to the findings of Mortreau et al. (2010). This difference may be partially attributed to the different internal standards used in the flow cytometry analysis.
Future Directions in Cytogenetic Research
The composition of repeats in Hydrangea species varies according to their origin and phylogeny. Species-specific satellite DNAs (satDNAs), identified by Taniguchi et al. (2024), could serve as cytogenetic markers for distinguishing Hydrangea species and cultivars, as well as for determining the parental species of older Hydrangea varieties. These information on repeatomes and cytogenetics enhance the genetic resources available for studying the evolution of hydrangeas and for guiding future breeding efforts.
For most Hydrangea species, there remains a lack of detailed knowledge regarding parental chromosome profiles, including the chromosomal landmarks needed to track chromosome behavior in interspecific hybrids and backcross plants (Van Laere et al. 2008). Another cytogenetic technique called genomic in situ hybridization (GISH) functions similarly to FISH; however, it employs (1) total genomic DNA from one of the parents involved in the hybrid cross and (2) unlabeled DNA from the other parent, also known as blocking DNA, as probes (Silva and Souza 2013). This technique can be applied to hybrids to distinguish chromosomes from different parental genomes in allopolyploid species, interspecific hybrids, and backcross progenies, as well as to trace intergenomic chromosomal rearrangements (Parokonny et al. 1997;Schwarzacher et al. 1989;Takahashi et al. 1997). In Brassica, GISH was successfully employed to confirm the hybrids resulting from the cross between the wild species B. maurorum, and two cultivated species, B. rapa and Brassica napus (Yao et al. 2010). To date, this technique has not been applied to other Hydrangea species.
Traditional ornamental breeding techniques have developed numerous novel cultivars but often take four to six years of field evaluation, which is time-consuming and laborious; therefore, the need to investigate genome sequences expects to provide alternative techniques to reduce the time necessary to create a cultivar (Wu et al. 2023). Marker-assisted selection has also become increasingly popular in plant breeding programs to accelerate the selection process (Van Laere et al. 2018). Mendoza et al. (2013) integrated 13 highly variable plastid markers sequenced from 20 species to identify H. sargentiana, H. seemannii, H. integrifolia, and H. arborescens as promising candidates for future bridge-crossing. Single-nucleotide polymorphism (SNPs) analysis combined with genome wide association (GWA) has revealed that the inheritance of mophead phenotype in F2 populations of H. macrophylla is based on qualitative rather than quantitative changes (Uemachi and Okumura 2012). Genetic maps are also commonly used (Ibitoye and Akin-Idowu 2010) however, they do not accurately represent the actual physical distance between genes or markers because recombination frequencies are unevenly distributed along chromosomes (Ariyadasa et al. 2014;Si et al. 2015). In contrast, physical maps do not depend on recombination frequencies and thus accurately reflect the actual positions of sequences on chromosomes. They could be used alongside genetic maps to gain a more comprehensive understanding of the genome organization. No genetic or physical maps have yet been established for the Hydrangea genus, and no specific cytogenetic markers have yet been identified. Additionally, physical maps are valuable for molecular plant breeding as they aid in the breeding and selection of new crop varieties (Dohm et al. 2014). Identifying plant genes associated with important traits, such as disease resistance and tolerance to abiotic stresses, will improve plant breeding efficiency in constantly changing climates. Physical maps also offer a basis for effective gene cloning and the development of markers that are closely linked to genes of interest (Van Laere et al. 2018).
The modification of crop genomes with foreign genes of high breeding value has become common over the past two decades, offering solutions to the issues associated with conventional breeding approaches. The CRISPR technology, which has revolutionized plant breeding by delivering a more precise, affordable, and speedy tool for introducing favorable traits in plants (Saini et al. 2023), has been utilized in several plant species including hydrangeas. Double flower types, a desirable trait in Hydrangea breeding, have been investigated using CRISPR/Cas9 gene-editing to study AP3 gene, which is involved in calyx formation in double-flowered H. macrophylla ‘Dooley’ (Tong et al. 2024). Although limited research has been conducted using this technology in hydrangeas, CRISPR/Cas9 system has the potential to greatly impact Hydrangea breeding and research, creating new opportunities for genetic improvement in this species.
Conclusion
Despite the high commercial value and extensive breeding efforts of Hydrangea, there is limited published literature on its cytogenetics. Parent selection for interspecific hybridization plays a critical role in breeding programs. As crossbreeding to produce new cultivars typically involves the best varieties, examining cytogenetic data is vital for improving the efficiency of these crosses. Although the aforementioned cytogenetic techniques provide accurate and efficient chromosomal characteristics of plants, they also have limitations. Therefore, integrating cytogenetic studies as a foundation for more advanced fields may further develop cytogenetic and genomic research in Hydrangea.
