Abstract
High species diversity, hybridization potential, broad geographical dispersal range and ornamental characteristics (i.e., attractive size, shape, structure, flowers, and evergreen) have fetched a good international market for Rhododendron. However, most species are restricted to specific geographic areas due to their habitat specificity in acidic soil and cold climates, resulting many species being classified under threat categories of the IUCN. In this review, advances in research on Rhododendron for improvement to floral display quality and stress resistance have been described. The low genetic barrier among species has created opportunities for extensive hybridization and ploidy alteration for introducing quality and adaptive traits during the development of new varieties. Recent technological advances have supported investigations into the mechanism of flower development, as well as cold tolerance and pathogen resistance mechanisms in the Rhododendron. However, most of the species have limited adaptability to drought, line-tolerance, pathogen resistance, and high-temperature conditions and this resistance ability present in few species largely remains unexplored. Additionally, the available genetic diversity and genomic information on species, and possibilities for their application in molecular breeding have been summarized. Overall, genomic resource data are scarce in the majority of the members of this genus. Finally, various research gaps such as genetic mapping of quality traits, understanding the molecular mechanism of quality-related traits and genomic assortment in Rhododendron members have been discussed in the future perspective section.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13205-024-04006-6.
Keywords: Cold adaptation, Genomic resources, Genetic improvement, Ornamental plant, Quality improvement
Introduction
The Rhododendron genus (including azaleas) belonging to the Ericaceae family is gaining ornamental, medicinal, and ecological attention because of its striking flowers, rich species diversity (> 1000 taxa), resistance to cold, high hybridization potential, and specific geographical distribution. Rhododendron species are used in landscaping due to their attractive flowers, size, shape, structure, long blooming period, and evergreen life (Esen et al. 2004; Sharma et al. 2020a). The flower color of Rhododendrons largely varies from white to pink, bright red, yellow, orange, magenta, and blue are widely used for ornamental purposes. Rhododendron species have reached 110 million USD international market for essential oils, perfumes, foods, and medicines dominated by countries like Belgium (44.1 M USD), Netherlands (37.4 M USD), Germany (16.0 M USD), France (14.8 M USD) Finland (11.4 M USD) and China (10.98 M USD) followed by the notable value in Denmark, Britain, Japan, USA, etc. (OEC 2023).
Rhododendron species have a long history of domestication as R. hirsutum was the first recorded species used for cultivation in 1650 BC in Britain. R. ponticum, a native species of Portugal and southern Spain and mainly found in Pontus Mountains and Black Sea was introduced in Britain in 1763 BC. Later, R. canescens Michx., R. nudiflorum L., R. viscosum L., R. maximum L., R. ferrugineum L., R. camtschaticum, R. dauricum, and R. chrysanthum were kept in plantations in Asian and Southern European gardens in eighteenth century (Magor 1990). In the Himalayas, R. arboreum was the first species identified near Srinagar (Jammu and Kashmir) in 1796 BC and introduced in gardens in 1817 BC (Turner 2015). R. mole, a deciduous azalea was introduced in Europe from China in 1823 BC and more than 6 species were introduced into cultivation from China in the nineteenth century (Leach 1961).
In the recent past, the breeding of Rhododendron has received increasing attention for obtaining varieties with better floral display, and cold and pathogen resistance. International Rhododendron Register and Checklist has listed more than 28,000 Rhododendron and azalea cultivars in 2018. The flower color is characterized by the presence of anthocyanins and anthoxanthins localized in the vacuole and carotenoids in the cytoplasm of petal cells (Xia et al. 2022). Inflorescences commonly called as flower trusses can be small with a single flower or may be arranged in a ball or pyramid-like structure (diameter ranged from 1 and 15 cm) comprising a few to > 50 flowers. Corollas often have separated or fused petals formed by flashy architecture or picotees with bright and distinct colors to attract the pollinator. Many species are used as traditional medicines all over the world due to the presence of pharmacological properties directed by certain biologically active molecules such as rhodojaponin III (antihypertensive effect), daurichromenic acid (anti-HIV and anti-inflammatory) and andromedotoxin (bradycardia and hypotension) (reviewed by Popescu and Kopp 2013).
Rhododendron species are well adapted to diverse habitats, but they mostly prefer open, loose, aerated, acidic, and moist habitats with humus-rich soils with low nutrients like nitrogen, phosphorus, calcium, and magnesium. They also grow in forest bases, stream sides, marshes, glades, rocks and boulders, open meadows, or epiphytes with the association of mosses. Roots associated with symbiotic ericoid mycorrhizal fungi improved the nutrient availability to the hosts, and their evergreen life improved soil nutrient-deficient adaptation leading to consistent photosynthesis and reduced nutrient loss (Moore 1980). Roots found as a fine and fibrous mat at the upper soil surface did not have drought-resistant properties. Most of the Rhododendron species exhibited strong iron-deficiency chlorosis symptoms on calcareous soil, except a few highly lime-tolerant species (Chaanin 1998).
Rhododendron contributes substantially to biodiversity, particularly in the timberline-alpine ecotone, therefore considered as keystone taxa of extreme cold climate, where temperature remains between 20 °C and −20 °C. Adaptability to below-freezing temperatures and tolerance for frost or hailstorms has been considered as a driving factor for its distribution towards northern latitudes and higher altitudes (Vetaas 2002). Various Rhododendron species have been kept under different threat categories of IUCN such as extinct in the wild (1 species), critically endangered (02 species), endangered (05 species), vulnerable (18 species), and near threatened (03 species) (IUCN 2022). Thus, Rhododendron species have been targeted as candidates for adversely affecting natural forest dynamics and developing forest management strategies.
Rhododendrons are receiving attention in research for expansion in their distribution, better adaptability, floral display, and longevity. Due to the perennial life form, different approaches have been followed for variety development. In this review, global diversity in genetic resources, traditional hybridization efforts with alteration on the ploidy level, advances in variety improvement including, recent genomic resources, together with chloroplast and genome sequencing achievements to unravel the molecular secrets of complex biological processes have been discussed.
Diversity in genetic resources and their evolution
Rhododendron comprising more than 1000 species has been categorized into eight sub-genera and further many sections and subsections (Fig. 1). Among these, species of subgenus Hymenanthes (elepidotes, with 1 section and 302 species) comprises evergreen shrubs to very large trees (over 40 m) with small to huge leaves and few to numerous flowers (5 to 40 in numbers) at terminal trusses. However, the subgenus Rhododendron (Lepidotes, with > 500 species under three sections) found in sub-Himalaya, arctic, tropical South-East Asia and Australia, consists of smaller bushes and above-ground stem parts covered with scaly hairs and smaller leaves and flowers with aromatic fragrance. Subgenera Pentanthera (30 species under 4 sections), Tsutsutsi (117 species under 2 sections) and Azaleastrum (34 species under 2 sections) are generally termed as azaleas and have scale-less, thin, soft and pointed leaves with long straight hairs parallel to the leaf surface and a terminal single flower per stem with five or six stamens. Three remaining subgenera (Therorhodion, Mumeazalea, and Candidastrum) comprised only four species (Chamberlain et al. 1996).
Fig. 1.
Diversity in the flower color and shape of Rhododendron of central part of Indian Himalayan region. Species are: a R. arboretum, b R. barbatum, c unidentified species, d R. campanulatum, e R. niveum, f R. dalhousiae, g R. campylocarpum, h R. wightii, i R. grande, j R. cinnabarinum, k R. ciliatum, l unidentified species, m R. maddeni, n R. decipiens, o R. thomsonii
Broadly distributed in the temperate regions of the northern hemisphere, centers of Rhododendron dispersal are the Sino-Himalayan region (Eastern Himalaya and Western China) to south-eastern China, and islands between Asia and Australia, predominantly New Guinea. The genus is expected to originate in temperate deciduous broad-leaved forests of northern-most-regions during the beginning of the Palaeocene era and diversified rapidly after the Miocene era (Shrestha et al. 2018). The distribution and temporal trend indicated that the Sino-Himalayan region had the highest diversification rate of Rhododendron species during the last 18 million years (Wen et al. 2014). Species diversity has also been recorded higher in China, Myanmar, Thailand, Indonesia, Malaysia, Philippines and New Guinea. A rich diversity is represented by China with 571 species (409 endemic), North-East India (87 species) and New Guinea (155 endemic species). In addition, the Korean peninsula, Japan, extended to the Alaska region and North America, Caucasus and southern Europe also represent significant Rhododendron species diversity (Mingyuan et al. 2005; Shrestha et al. 2018). In these areas, the founder effect for Rhododendron diversification exhibited by extreme topographical variability due to close proximities of mountain ranges and alpine pastures with subtropical river valleys created rapid range fragmentation and geographical barrier for species isolation by inhibiting gene flow among populations (Wen et al. 2014).
Traditional breeding approaches for quality improvement
Hybridization and traditional breeding
Natural hybridization in Rhododendron has been observed in the species of Sino-Himalayan and south-western China due to the low degree of genetic barrier and hereditary discrimination among species, which dynamically altered the evolutionary process for species diversification (Milne et al. 2003; Yan et al. 2017). Few natural tetraploids (4n = 52), hexaploids (6n = 78), octoploids (8n = 104), and dodecaploids (12n = 156) species and hybrids have been identified in Rhododendron (Jones et al. 2007). Over the million years, American species have conserved their fundamental number of chromosomes with only minor changes in their functionality. Thus, the complete pairing of chromosomes between American and Asian hybrid species has been reported (Krebs 1997). The first artificial hybrid of Rhododendron was developed in Thompson’s Nursery near London with the crossing between R. potenticum and an Azalea species in the 1800s. Hybrid varieties developed from American species (e.g., R. catawbiense and R. maximum) and Asian species (e.g., R. causasicum, R. arboreum and R. ponticum) have good ornamental and horticultural values. Chinese species, R. discolour and R. fortunei are also used as potential parents for the development of garden varieties with high ornamental value. Among the Azalea group, R. calendulaceum, R. nudiflorum, R. viscosum and R. occidentale of American; R. luteum of Eurasian, and R. molle and R. japionicum of the Oriental region are frequently used for hybridization (Cox and Cox 1997).
In Rhododendrons, breeding efforts are required for the development of cold, warm summer, drought, nutrients and moisture deficiency-resistant hybrids. Fortunately, a broad distribution range supported the functional diversity of adaptive character required to triumph over these challenges. For example, lepidote species (Subgenus Rhododendron) found in the alpine mountains of the Sino-Himalayan region, Northern Europe, and North America are extremely cold-hardy plants. On the contrary, most elepidote species (section Ponticum) rich in ornamental traits are low or moderately cold hardy. Inter-specific hybridization among these groups has been largely practiced in Rhododendron breeding for the expansion of apparently superior and cold hardy hybrids (Susko et al. 2016).
R. catawbiense and R. maximum found in North America, and R. brachycarpum and R. yakushimanum found in Asia are the hardiest (> −25 °C for buds and leaves) elepidote germplasm for cold resistance breeding. While R. minus from North America and R. dauricum and R. mucronulatum from northern Asia are the hardiest lepidote species. R. viscosum, R. arborescens and R. canadense found in North America are the hardiest (flower bud hardiness −30 °C) deciduous azaleas (section Pentanthera) (Uosukainen 1992). In a few azaleas buds are hardy up to below −37.2 °C and their acclimatized leaf can survive at −50 °C also (Sakai et al. 1986; Wei et al. 2005a). R. brachycarpum ssp. Tigerstedtii, R. catawbiense var. album, R. yakushimanum, R. smirnowii, R. forrestii, and R. ‘Dr. H.C. Dresselhuys’ are good genetic resources for hybridization with hardy progeny, but have limited availability of flower color (Uosukainen 1992). The R. ‘Northern Lights’ hybrid of deciduous azaleas is the most frost-tolerant Rhododendron in the world (Susko et al. 2016). In previous traditional breeding efforts of Rhododendron, inter-specific crossing of multiple species was used at many generations. Crossing among different species without backcross resulted in a high degree of genome complexity among hybrids. Hardy yellow R. ‘Capistrano’ hybrid of David Leach contains genetic architecture from more than ten species including, North American (02 species) and Asian (07 species) along with a few other uncharacterized ancestral species.
The intra-sectional hybridization generated high genetic variations for breeding due to several mechanisms including, pollen tube incompatibilities, embryo abortion and many others. Some inter and intra-sectional hybrids may have higher adaptive fitness for specific habitats than their parents and may choose distinct preferred habitats from either parent. Hybrids present in intermediate habitats than parents indicated interaction of genes for trait expression (Zha et al. 2010). Sometimes, hybridization appears only to the F1 generation and does not express further generations with a reason of low fertility at F1 level or superiority of F1 hybrid over other generations or any other unknown reasons (Milne et al. 2003). However, a long juvenile period and complicated genome structure cause restricted data on the inheritance of important quality-related traits in Rhododendron, which ultimately leads to difficulties in specific variety development and genetic improvement efforts.
Manipulation in ploidy level
Altering the ploidy level to overcome the barriers to wide hybridization has been targeted for introducing improved floral characters and adaptive traits in the past few decades. Due to the occurrence of additional sets of genes (termed as gigas) polyploids are considered a superior material for breeding due to their altered physiological activities, including larger cell size, expanded leaf, flowers and fruits, larger guard cells pollen size, a fewer number of stomata, decreased cell divisions rate, slower growth, and improve desired non-metric ornamental traits, like healthy stems and leaves, and better floral display (Jones et al. 2007; May et al. 2023). The polyploids also have the ability of heterozygosity for buffering effects for gene redundancy on mutations and facilitated asexual or self-fertilization. In addition, polyploids have higher chances of altered functions for stresses, growth, and development because of the availability of multiple copies of genes thus, having higher isozyme diversity and flexibility in machinery for biosynthesis of secondary metabolites (May et al. 2023).
Inter-sectional crosses between diploid elepidote and tetraploid deciduous azaleas produced sterile azalea-dendrons hybrids with similar features to deciduous azalea parents due to gene dosage effects. Superior offspring can be only achieved in crossing between both tetraploid parents such, as artificial tetraploid lepidote R. minus (also known as R. ‘Epoch’) and tetraploid elepidote cultivars (Jones et al. 2007). On the contrary, tetraploid hybrids R. NSB and R. CW4 have less freezing tolerance in terms of electrolyte leakage under controlled freezing conditions, than their diploid hybrids of R. PJM and R. Cinninggham’s White (Vainola and Repo 1999).
Additionally, artificial polyploids are characterized by lower branch growth, higher water content, higher stomatal size, declined stomatal density, reduced transpiration rate and derisory chromosomal segregation (Maherali et al. 2009). However, artificial tetraploids were found comparatively less cold-hardy than their diploid parents due to larger xylem vessels present in the leaf or stem causing higher occurrence of freezing–thawing mediated freezing injuries (Lipp and Nilsen 1997). Somatic doubling produces duplicate alleles, which cause inbreeding depression resulting in increased homozygosity, which ultimately may lead to the expression of harmful and lethal genes.
Modern advances in variety improvement
Flower development and ornamental characters
The plant flowering process is tightly regulated by an integrated network of genes of which, FT, SOC1 and LFY genes have been recognized as key genes, regulating multiple pathways of flowering. A comparative transcriptome identified six transcripts homologous to FT, one transcript homologous to SOC1, one transcript homologous to LFY, 4 genes homologous to DELLA protein and 1 gene homologous to LRY gene family regulating the flowering process in R. pulchrum. Among these, FILC (flowering initiation regulation) and FT (flowering control) genes have been reported to regulate numerous flowering pathways identified through co-expression analysis (Wang et al. 2018a). Phase transition towards reproduction has been regulated by a compact gene network stimulated by temperature and photoperiod. At the onset of flowering, the expression of cold-hardiness related responsive genes acts as a stimulus for ‘common flowering trigger’ in Rhododendron. Ethylene played a central role during cold resistance and its combination with auxins and jasmonic acid triggered the expression of many ethylene-responsive factors during phase transition from vegetative to flowering phase (Choudhary et al. 2019). Initial low non-freezing temperature exposure followed by sub-freezing exposure helped Rhododendron to acclimatize for winter and flowering initiation. Flowering processes are induced by the leaf during the reproductive phase. A comparative transcriptome study identified the involvement of genes e.g., FCA, FCA-γ like, FRI, FRI-like, PHYE, CRY1, CRY2, PIF3 and others for reproductive organ development during flowering (Choudhary et al. 2018a). Expression of genes like, ASHH, CPR, EID, FPF, GRP7, HUB2, PHP and WNK5 have been found important for phase transition from vegetative to the reproductive stage of meristem tissue; while LIF2, GNC, GNL/CGA1, YABBY, CLF, dsRBD-like and HUA1 floral genes have been found as floral repressors in leaf tissues (Choudhary et al. 2019). Flower initiation and flower color controlling genes such as LFY, TFL1, AP3, and anthocyanin biosynthesis pathway have been cloned and characterized in many Rhododendron (Nakatsuka et al. 2008; Cheon et al. 2011, 2017; De Keyser et al. 2013), however, experimental validation of many other such genes and in silico miRNA-target interactions remained unexplored.
Furthermore, ornamental characters like flower size and color remained unpredicted by the Mendelian model as they have many intermediate morphotypes and complex genetic regulations. Anthocyanins as a major color-determining pigment of Rhododendron, is dominated by cyanidin, peonidin, delphinidin and malvidin (Mizuta et al. 2014). Typically, anthocyanin biosynthesis pathways have been unrevealed in few other Rhododendron species, and biosynthetic (CHS, CHI, F3H, F3H, F35H, DFR, ANS, and flavonol synthase), modification, transportation (AT, OMT, & GST), regulation (e.g., R2R3-MYB unigene) and catabolism and degradation (BGLU, PER, & CAD) related genes have been identified in few species (Mizuta et al. 2014; Jia et al. 2020; Liu et al. 2021a; Ye et al. 2021; Sun et al. 2022). In blooming flowers of R. pulchrum 149 derivatives of glycosylated and methylated flavonoids, including flavone, flavonol, flavanone and isoflavone have been detected (Wang et al. 2021a). Comparative transcriptome analysis of distinct colored flowers revealed high expression levels of homologous to F3′H, FLS, and F3H unigenes in R. pulchrum (purplish red flower). Similarly, higher expression of unigene homologous to CHI in R. mariesii (pink flower), homologous to ANR and LAR in R. simsii (red flower), and early biosynthetic genes F3′5′H and minimum expression of LDOX in R. fortune (light pink flower) has been identified as color determining factors (Li et al. 2021a, b). In R. simsii, expression of CHS increased at the bud development, peaked at the flower bud opening, and then decreased sharply during the flowering stage. However, expression of CHS and DFR genes in eight azalea cultivars cloned from R. simsii hybrids revealed no significant correlation with flower color phenotype (Schepper et al. 2001). Also, yellow color controlling pigment, β-carotene biosynthesis pathway coding genes including PSY, PDS, LCYB, and LCYE, have been characterized in some Rhododendron species (Xiao et al. 2018; Li et al. 2021a, b; Sun et al. 2022) and over-expression of regulation gene RmLCYB from R. molle has been identified for increased carotenoid (Zhou and Zhu 2020). However, great diversity of flower color, pigment content and gene-expression analysis relationship can further lead to deeper molecular insights of flower color control in Rhododendron.
In epigenetic regulation of gene functioning, miRNA-mediated degradation of target RNA controls the flowering development and circadian rhythm by regulating the phenological and developmental responses according to their external environment (Luo et al. 2013). In R. arboretum, miR156 and miR172 pair has been observed as the master regulator of vegetative to reproductive transition. Also, verbalization controlling protein VIN3-like protein (VIL1) has been targeted by the expression of miRNA211, which has been regulated by annual temperature pattern and thus any shift in mean temperature causes a change in flowering time (Choudhary et al. 2018a).
Flower symmetry in angiosperms determines ornamental quality and pollinator attraction capacity by regulating floral display. In radially symmetric R. taxifolium Merr. and bilaterally symmetric R. beyerinckianum Koord., CYCLOIDEA orthologs genes are expressed differentially in longer ventral petals, contributing to the abaxial curving of the corolla tube and positioned floral organs. However, RADIALIS and DIVARICATA expressed uniformly in both species (Ramage et al. 2021). Further, the floral development progress needs to be validated experimentally for further studies.
Cold resistance
Cold hardiness breeding has been accelerated with increased knowledge of physiology, genetics and genomics (Fig. 2). American Rhododendron Society categorized and prioritized 1775 taxa of Rhododendron for various cold-hardy zones of USA. Based on electrolyte leakage, flower bud hardiness was recorded up to −37.2 °C in a few deciduous azaleas (Susko et al. 2016). Structural changes like cuticle wax and additional palisade layer in leaves of R. catawbiense and R. ponticum were identified as potential adaptation mechanisms for preventing light penetration during winter to down-regulate photosynthesis (Wang et al. 2008). Also, water availability in different tissues leads to increase or decrease in the turgor pressure of epidermal cells resulting in thermonastic leaf curling (Nilsen and Tolbert 1993). Rhododendron (e.g., R. ferrugineum) adapted to winter drought generally have low optimal net photosynthetic rate, low stomatal conductance, and less stomatal density to achieve hardening, but stomata get activated after the rise in leaf temperatures up to 10 °C (Ranney et al. 1995; Lipp and Nilsen 1997).
Fig. 2.
Tolerance mechanism and response of Rhododendrons against cold and associated stresses at structural, physiological, molecular and metabolic level
Leaves of R. catawbiense plants possess ectodormancy, as naturally cold-acclimated plants restarted photosynthesis within a day after transferring to warmer growth conditions (Harris et al. 2006). Transferring high-intensity light-treated plants to normal light conditions causes rapid recovery by de novo synthesis of damaged protein, but prolonged exposure to light intensity along with cold temperature causes degradation of pigments (Sakai and Larcher 1987). High irradiation at higher altitudes on Rhododendron during the winter season causes a high risk of photo-inhibition due to excess excitation energy of sunlight. The electron transport system of photosystem-II (PS-II) deactivation leads to retardation of photosynthesis by the reduction in the maximum quantum yields for CO2 uptake and O2 evolution (Nilsen et al. 1988). Rhododendrons having low carbon metabolism efficiency exhibited stronger photo-inhibition than other sun-adapted plants (Krause 1988). Dehydration mediated by photo-inhibition in R. ferrugineum during entire winters protects the plants by improving the efficiency of PS-II and maintaining the water imbalance (Neuner et al. 1999). R. English Roseum developed the ability to acclimate to extreme irradiance conditions by accumulating water-soluble phenolics in the leaves on exposure to UV-B treatment (Dunning et al. 1994).
Thermonastic leaf curling and uncurling have been sensed in R. catawbiense at below sub-freezing temperature, whereas no leaf curling has been recorded in R. ponticum. Here, early light-inducible proteins (ELIPs) found in thylakoid membranes of chloroplasts worked as light trapping Chlorophyll a/b-binding proteins. Reduced photo-inhibition with stronger antioxidant systems and thermonastic leaf movements has been reported in R. ponticum, while up-regulated early light-inducible proteins (ELIPs) expression and the antioxidant system has been reported in R. catawbiense (Wang et al. 2008). Seven ELIPs characterized in R. catawbiense were found distinctly up-regulated during the cold stimulation and indicated its adaptive response to cold and radiation stress in winter-adapted Rhododendron leaves (Peng et al. 2008a). ELIPs (reducing photo-oxidative stress), LEA, dehydrins (accumulate in response to dehydration and desiccation stresses), cytochrome P450 (photosynthetic adjustments), and β-amylase (regulate osmotic pressure) genes/ gene families were found highly expressed in winter tissue of R. catawbiense leaves (Wei et al. 2005a). Also, RcPIP2s (Plasma Membrane Intrinsic Proteins), an aquaporin protein that facilitates the transport of water molecules across cell membranes in the leaves of a thermonastic (R. catawbiense) and non-thermonastic species (R. ponticum) revealed no differential expression after exposure with different temperature conditions (Peng et al. 2008a). Similarly, photosynthetic metabolism has been down-regulated by reduced expression of five genes related to photosynthesis (e.g., light-harvesting chlorophyll a/b-binding protein, RuBisCO small subunit precursor, RuBisCO activase, plastidic fructose bisphosphate aldolase, and chloroplast precursor of plastocyanin) during over-wintering leaves of R. catawbiense (Wei et al. 2005a). Besides, auxin response factors (ARFs) and ethylene-responsive factors (ERFs) of AP2/EREBP family, and TFs of bZIP, C2H2, MYB, NAC and WRKY families were major responsive factors in temperature resistance in R. arboreum. Genes related to the biosynthesis of secondary metabolites, CYPs, hemicelluloses, lignins, pectins, spermine, and simple sugars provided a membrane-stabilizing effect under frost during the flowering phase in R. arboreum (Choudhary et al. 2018b).
Besides absolute temperature, cold resistance of buds, flower buds, and leaves depends on many other factors such as the rate of temperature decrease, photoperiod and drought stress due in frozen soils, and previous exposure to cold acclimation. Photoperiod has been identified as a limiting factor for the distribution of Rhododendron species towards the polar region (Vainola and Junttila 1998; Vainola and Repo 1999). Furthermore, flowering in Rhododendron is highly dependent on photoperiod as species growing in long-day conditions may produce several flushes (Collin et al. 1996). Critical photoperiod induces cold acclimation by influencing physiological processes like decreased water level, and drought tolerance capacity, regulates biosynthesis of structural membrane proteins and membrane lipids and low-molecular-weight compounds protected freezing in extremely cold conditions accumulate in hardy plants (Sakai and Larcher 1987). In the process of achieving complete hardiness through photoprotection, the plant accumulates and converts its starch to sugars during cold acclimation through the upregulation of circadian rhythm-related genes (Liu et al. 2022a).
Biochemical parameters e.g., soluble protein content along with qualitative changes in their gene expression have been reported as consequences of cold acclimation (Thomashow 1999). The proportion of phospholipids and di-unsaturated fatty acids is increased to enhance the fluidity of membranes in the chilling atmosphere (Sakai and Larcher 1987). In R. Jean Marie-de-Montague, the concentration of phospholipids and galactolipids increased significantly in roots during cold acclimation along with the concentration of unsaturated fatty acids such as linolenic acid increased (Loubaresse et al. 1991). During cold acclimatization, expression of various proteins associated with chilling stress tolerance, carbohydrate and lipid metabolism pathway, transactional regulation, signaling pathways, secondary metabolite pathway, cell membrane permeability and photosynthesis has increased and expression of various proteins involved in photosynthesis has been decreased (Die et al. 2017). Hardening has also been determined by the rate of temperature change and winter severalty with the support of kinetics of dehydrin proteins in various genotypes of azalea (Kalberer et al. 2007). Recently, a comparative proteomic study revealed that proteins like cold shock dehydrin erd-10-like protein; two photo-oxidation inhibiting monodehydro-ascorbate reductases proteins of chloroplast stroma; various proteins related to carbohydrate metabolic pathways, remodeling of the cell wall and cell membrane permeability and two chlorophyll a/b (CAB) proteins were found up-regulated in cold-acclimated tissues of R. catawbiense (Die et al. 2017). CAB gene family has quickly expressed members of ELIPs, which are supported to reduce oxidative stress induced by solar energy absorbed by evergreen Rhododendron leaves during the winter (Peng et al. 2008a). In alpine species R. chrysanthum, a quantitative proteomics and phospho-proteomics study revealed that photosynthesis was inhibited and various ROS scavenging pathways and calcium-mediated signaling were initiated to control the damages of cold stress (Liu et al. 2022b).
Dehydrin protein (25 kDa) has been identified as a biochemical and genetic marker abundantly expressed in cold-acclimated leaf tissue in the super-hardy North American species R. catawbiense than the less-hardy Asian species R. fortunei (Lim et al. 1999). While, 11 dehydrins have been detected ranging from 25 to 73 kDa in other Rhododendron taxa within sections Ponticum and Rhododendron, and among these, a sequence of 25 kDa dehydrin was found conserved across the other species (Marian et al. 2004). In vitro water loss assays using purified recombinant RcDHN5 protein showed that it has strong dehydration stress protection activity (Peng et al. 2008b). Further, dehydrin expression constitutively increased the cold/freezing tolerance in many transgenic plants (Peng et al. 2008c; Qiu et al. 2014). Phylogenies of Rhododendron CT1sHSP (a heat-shock protein protected against environmental stress) nucleotide revealed that two types of CT1sHSP displayed greater diversity at N-terminal region flanking to α-crystallin domain (ACD), which influenced chaperon activity due to altered efficiency of the substrate binding and substrate specificity. Both types of CT1sHSP evolved long ago with the mutations in the ACD (Liao et al. 2010). Thus, overall, various mechanisms involved in different species towards cold tolerance need further genomic and proteomic studies for confirmation and validation of phenotypic expression in Rhododendrons.
Heat and temperature resistance
Heat stress has been recognized as a major constraint for the distribution and adaptability of Rhododendron in the southern hemisphere. R. hyperythrum, (Sec. Ponticum) and R. catawbiense are among the few heat-tolerant species found in Taiwan and Louisiana, respectively and are preferred breeding materials for warm-adapted hybrid development. Among these, R. hyperythrum exhibited high heat tolerance represented by higher net photosynthesis and stomatal conductance at 40 °C (Ranney et al. 1995). Foliar spray of CaCl2 and salicylic acid in alpine R. lapponicum seedlings growing in a warmer chamber (40 ℃ day/32 ℃ night) increased the heat tolerance due to the accumulation of malondialdehyde and free proline in the young leaves that delayed the degradation of chlorophyll content (Zhao et al. 2010; Li et al. 2017a). High-temperature stress tolerance has been recorded in seedlings of R. simiarum Hance and R. jinggangshanicum Tam. (Subgenus Hymenanthes) mediated by improved membrane lipid peroxidation, high malondialdehyde, hydrogen peroxide, proline and soluble protein content and low ascorbic acid content in leaves (Zhang et al. 2009). Heat-sensitive R. hybridum exhibited decreased stomatal opening, chlorophyll and carotenoid contents, and increased malondialdehyde superoxide dismutase, peroxidase enzymes and hydrogen peroxide enzymes, xylem vessels, leaf ultrastructure and chloroplast, osmotic adjustment by small molecules for maintaining cell membrane stability and reducing cellular ROS level assured heat tolerance in Rhododendron cultivars (Shen et al. 2017).
In R. hainanense, 2658 heat-responsive genes have been identified together with transcription factors (ABR1, IAA26, NAC29, NAC72, OBF1, LUX, SCL3, DIV and TCP3 families) regulating the heat-stress tolerance process (Zhao et al. 2018). Similarly in R. obtusum Yanzhimi leaves, relatively lower Fv/Fm and Fv’/Fm’ indicated the blockage in electron transfer at the acceptor side of PS-II under heat stress. Heat shock protein (HSFA3) homologous genes (C63903, C63904, C63905, and C63906) were found upregulated during heat treatment. Also, up-regulation of homologous unigene AtHSFA2 (C80422) provides heat tolerance to R. obtusum by regulating the heat shock proteins like chloroplast-localized HSP21 and protect the chloroplasts by repairing the damage of thylakoid membrane (Fang et al. 2017). Various transcriptome studies validated through real-time quantitative PCR analysis revealed the role of various small heat shock protein (CPsHSP) and MADS-box genes for regulating high temperature or heat stress (Wu et al. 2007; Liao et al. 2010; Huo et al. 2021). Much work is needed to perform to develop a clear understanding about the regulatory mechanisms of the heat stress response in Rhododendrons.
Arbuscular mycorrhizal fungi improve the heat tolerance in Rhododendron by preventing the reduction of soluble sugar, free proline content, soluble protein and chlorophyll in leaves and maintain cell membrane permeability under heat stress (Lin et al. 2023). Heat stress influenced the growth of soil microorganisms leading to altered soil nitrogen availability, while endophytic bacteria influenced the ability of roots to absorb nitrogen, thus regulate the physiological processes of R. hainanense. Such findings will contribute to develop improved thermo-tolerance cultivars of Rhododendron.
Salt and salinity resistance
Naturally occurring few Rhododendron (e.g., R. balfourianum, R. cuneatum, R. hirsutum and few others) on limestone rock in Yunnan are used as genetic material for salinity-tolerant variety development. Crossing between R. micranthum and R. hirsutum produced lime tolerant R. Bloombux hybrid, that can survive up to soils with pH 7.5 (Chaanin 1998). Among the 200 Rhododendron species and hybrids treated with lime revealed that most of the taxa preferred low pH (pH 4.2) soil with low lime levels, however, R. micranthum, R. occidentale, and R. schlippenbachii can tolerate extreme salinity (Chaanin 1998). Indica azalea group is considered as the extreme salt-tolerant evergreen cultivars of Rhododendron. Satsuki hybrid group originated from R. indicum and R. eriocarpum (syn. R. tamurae) and can grow on the island of Yakushima south of Kyushu, and R. eriocarpum and R. indicum are found in the hot and humid coastal environment (Tagane et al. 2008). Accumulation of micronutrients such as iron and manganese have been reported without any pH variations. However, plants growing over a pH 4.0–8.0 range did not increase calcium and magnesium levels in leaves tissues with different pH and micronutrient levels of soil (McAleese and Rankin 2000).
Activities of superoxide dismutase, peroxidase and glutathione reductase, and antioxidant enzymes such as (SOD), (POD) and ascorbate and glutathione were found higher in the cytosol of NaCl treated three Rhododendron cultivars, however, malondialdehyde level in leaves of R. Yanzhi Mi did not show any significant change in the three subcellular compartments under salt stress as compared to other sensitive species (Liu et al. 2020a). Thus, salt and salinity tolerance has been evidenced in a few Rhododendron species, however, the much work is needed to understand the molecular mechanism behind the salt and salinity tolerance and its genetic control in Rhododendrons.
Pathogen and disease resistance
Root rot disease caused by Phytophthora cinnamomi is a common disease in ericaceous plants, while few Rhododendron (e.g., Indica cultivars) possess some resistance against it. Around 6–8% of Rhododendron showed high to moderate resistance to P. cinnamomi (Krebs and Wilson 2002). Southeast Asian origin of the P. cinnamomi pathogen and the presence of several taxonomically diverse resistant species with diverse geographical regions indicated its co-evolutionary history. A few lepidote species, R. simsii and R. hyperythrum showed resistance to P. cinnamomi and are used as a genetic resource for hybridization to generate resistant plants with ornamental value. While, R. pseudochrysanthum, R. formosanum, and R. morii have demonstrated higher resistance to P. cinnamomi as well as heat-resistant properties (Chung et al. 2007). The use of Piriformospora indica, an endomycorrhizal-like fungus can improve biological protection against Phytophthora cinnamomi and P. plurivora in two rhododendron cultivars ‘Nova Zembla’ and ‘Alfred’ (Trzewik et al. 2020). Petal blight disease caused by Alternaria sp. causes upregulation of genes associated with H2O2 accumulation, defense-related genes (PR and FRK), and secondary metabolites indicated its possible defense response (Zhang et al. 2023). Salt stress and pathogen resistance are jointly regulated by abscisic acid and salicylic acid-mediated defense signaling pathways against abiotic and biotic stresses. Abscisic acid worked as antagonistic to salicylic acid, thus during the stress condition increased abscisic acid level suppresses the production of salicylic acid, which has been considered as very essential for the expression of defense-related genes in the host plant (Yasuda et al. 2008). However, molecular diagnosis of pathogen and resistance mechanism in Rhododendron requires further investigation.
Drought resistance
Drought as the most common consequence of climate change is an expected threat to the distribution of Rhododendron in the future. Nonetheless, few species have been identified with some tolerance to drought through morphological and physiological changes (Fig. 3). R. hirsutum found in the alpines exhibited drought-induced vessel blockage and controlled stomata opening, thus having the ability to grow on the edges of the dry limestone site (Mayr et al. 2010). In R. catawbiense Boursault, exposure to continuous or periodic water stress can increase cold hardiness. The effectiveness of the periodic water stress depends on the timing of application, which reflects the dependence on the stage of the cold acclimation process (Anisko and Lindstrom 1996a). Both regulated deficit irrigation and partial drying during flowering initiation time promote the flower numbers at inflorescence, but prolonged drought initiates premature floral development and repression in floral initiation as a result of the inhibitory action of abscisic acid transported from stressed roots to the apical meristem (Anisko and Lindstrom 1996b). However, in R. fortunei, net photosynthetic rate, transpiration rate, and stomatal conductance decreased with the increase in drought stress under the optimum light and temperature conditions, while water use efficiency improved under mild stress, but became lower under moderate and severe stress. In R. fortunei, light compensation point and stomatal resistance increased, and light net photosynthesis, Fv/Fm and transpiration rate declined along the water stress gradients (Ke and Yang 2007a). In addition, the dark respiration rate increased under light and moderate stress but showed a reverse trend with heavy stress (Ke and Yang 2007b).
Fig. 3.
Potential mechanism of drought tolerance of Rhododendron delavayi with the application of external brassinosteroids
(Modified from Cai et al. 2021)
Exogenous application of 24-epibrassionlide in R. delavayi improved the photosynthesis rate and reduced the negative effects of drought-induced membrane peroxidation and severe oxidative stress by increasing net photosynthetic rate, stomatal conductance, transportation rate, light-saturated photosynthetic rate, light compensation point, light saturation point, photochemical quenching and electron transport rate (Cai et al. 2020). Brassinosteroids-mediated response in R. delavayi against drought involved different mechanisms including cell detoxification through improving antioxidant enzymes; improved photosynthesis through controlling stomatal conductance, light utilization efficacy, photochemical efficacy, light harvesting chlorophyll-protein complex, Photosystem-I and II, photosynthetic electron transport system, and ATP synthetase; relive oxidative stress by increasing peroxidases and glutathione antioxidant system; adjust osmatic pressure by controlling soluble sugar and soluble proteins; increase the cellular synthesis of protein; control energy metabolism and regulate activity of Heat shock proteins, glycerol kinase and lipoxygenases (Cai et al. 2021). Furthermore, In R. pulchurum var. ‘Baifeng 4’, different draught and water treatment exhibited differential expression of 34 to 161 transcriptional factors mainly combining effect of ERF, bHLH and MYB genes families. However, WRKY, bZIP, PLATZ were up-regulated under drought stress and GATA was upregulated on rehydration (Wang et al. 2018b). Controlled water stress can substitute for pruning as a means of manipulating growth and enhancing final plant quality in Rhododendron. These results demonstrated that resistance is available to a certain extent in the species and these species/ cultivars can be a genetic resource for the development of different drought stress resistance cultivars and varieties for expansion of the drier regions of the world.
Genomic resource availability and molecular breeding
Molecular markers for germplasm characterization
The experimentally variable individuals tagged with molecular markers are used for determining frequency of recombination and linkage distance during breeding programs. Thus, molecular markers have been frequently used in diversity analysis, phylogenetics, genetic mapping, marker-assisted breeding, genomic analysis, genetic conservation and genotyping of Rhododendron. Before the availability of genomic resources, diverse types of non-sequence specific genetic marker systems (e.g., morphological, protein-based and arbitrary DNA primer-based) have been applied for the classification of genotypes or taxa or germplasm characterization. Random Amplified Polymorphic DNA (RAPD) analyses using arbitrary primers for PCR amplification of genomic DNA were the initial techniques for diversity assessment and germplasm characterization in Rhododendron. RAPD marker based analysis revealed the grouping of species according to their subgenus and quantifying genetic diversity and differentiation of R. schlippen-bachin Maxim (Ji et al. 2010), R. nilagiricum (Jayanthi and Seeni 2000), and R. arboreum populations (Jain et al. 2000; Kuttapetty et al. 2014). Similarly, in ISSR marker based analysis, arbitrary and longer-size primer have been used to increase reproducibility (Rawat et al. 2017) and were successfully applied to assess the genetic diversity in the natural populations of R. fortunei (Zexin et al. 2006), R. shanii (Zhao et al. 2013), and many other species (Xiao et al. 2015). Also, AFLP marker system has been successfully used in determining genetic diversity and genetic differentiation of R. ferrugineum populations (Escaravage et al. 1998; Pornon et al. 2000), R. concinnum (Bing et al. 2012), R. protistum var. giganteum (Wu et al. 2015), R. ferrugineum (Wolf et al. 2004) and many other species (Wang et al. 2018c). Besides, RAPD in combination with Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers has been used for diversity characterization in many Rhododendron species (Xu et al. 2017). Overall, a high level of genetic diversity was reported in most of the species of Rhododendron (Supplementary Table 1). Relatively, a good of genetic diversity has been estimated in endangered and threatened Rhododendrons with very narrow distribution, such as R. jinggangshanicum, R. pubicostatum, R. rex, R. longipedicellatum, R. meddianum and R. protistum var. giganteum (Li et al. 2015; Wu et al. 2015, 2017; Zhang et al. 2020a, b, 2021; Cao et al. 2022). In species like R. boninense, no genetic diversity was recorded due to the only presence of clonal propagules mother plants in the world (Isagi et al. 2020).
SSR marker development and their applicability
The importance of simple sequence repeats (SSRs) or microsatellite markers in constructing genetic maps, marker-assisted selection, genetic diversity and gene targeting is gaining momentum, due to their high frequency, co-dominance, multi-allelic nature, reproducibility, extensive genome coverage, and easiness in detection (Sharma et al. 2020a). SSR markers have been developed in different Rhododendron species using different methods (Table 1) and are widely used for genetic characterization. Various approaches have been used in Rhododendron including from enriched genomic libraries prepared by ligation linker/adapter of fragmented genomic DNA, or primer extension using SSR specific markers, non-enriched genomic libraries through random library construction by cloning and transformation of restricted DNA fragments, cloning and sequencing of RAPD/ ISSR/ AFLP amplicon, next generation sequencing of whole cell RNA (transcriptome) or whole genome or organellar DNA, and use of transferable SSRs among closely related species. Using these approaches, microsatellite has been developed for R. delavayi (Wang et al. 2010a), R. arboretum (Choudhary et al. 2014), R. longipedicellatum (Li et al. 2018a), R. amagianum (Yoichi et al. 2017), R. simsii (Tan et al. 2009), R. ovatum (Liu et al. 2017), R. aureum (De Keyser et al. 2009), R. brachycarpum (Kwak et al. 2015) and many other species of Rhododendron genus (Wang et al. 2010b). Microsatellites revealed significant efficacy in differentiating two varieties of R. protistum var. giganteum found in China and Nepal (Li et al. 2018b)., while a high bottleneck was found in populations of R. protistum var. giganteum collected from Yunnan Province, China (Wu et al. 2017). Also, these markers revealed differential genetic diversity levels in populations of the Alps and Pyrenees of R. ferrugineum (Charrier et al. 2014), in Mt. Kamakuraji Hiroshima populations of R. metternichii var. hondoense (Naito et al. 1999), in Central-Eastern Alps and Maritime Alps populations of R. ferrugineum (Bruni et al. 2012), and in Mount Jinggangshan, (China) populations of R. jinggangshanicum (Li et al. 2015).
Table 1.
Existing information on the development of genomic resources (SSRs) and their validation and application in different Rhododendron species using traditional and next-generation sequencing approaches
| S.no. | Species | No. of markers developed | Data created through/ acquired from | Validation | Application and other information | Reference |
|---|---|---|---|---|---|---|
| 1 | R. amagianum | 19 polymorphic EST-SSR developed | Developed from EST libraries of R. amagianum and R. hyugaense | HO ranged from 0.000 to 0.931 and He from 0.000 to 0.904 | These 19 SSRs shown to be polymorphic across the four species of Rhododendron and will help in evolutionary processes | Yoichi et al. (2017) |
| 2 | R. arboretum | 41 microsatellite markers | Designed from an enriched DNA library | PIC value varied from 0.104–0.911 (average—0.464); HO from 0.167–0.933 (average—0.523) and HE from 0.422–0.917 (average—0.723) | Some of the loci showed significant deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium was detected | Choudhary et al. (2014) |
| 3 | R. arboretum | 35,419 microsatellite regions identified | Flowers and leaves—Illumina’s NextSeq500 platform | The PIC value i varied from 0.343 to 0.773 (average 0.6) in tested 43 polymorphic loci. HO was found 0.364 and HE was 0.650. Also, 36,921 SNPs were located | 719 transcripts had 811 high-quality SNPs variants with a minimum coverage of 10, a total score of 20, and SNP% of 50 | Choudhary et al. (2018b) |
| 4 | R. aureum and R. brachycarpum | 19 microsatellite markers | Low-depth next generation sequencing | All 19 loci were polymorphic. Number of alleles varied from 4–25 (average—15 alleles per locus) | Three R. aureum loci showed departure from HWE and one R. brachycarpum locus deviated from HWE | Kwak et al. (2015) |
| 5 | R. calendulaceum | Forty-eight primer pairs designed | Paired-end Illumina MiSeq data | 15 primer pairs were polymorphic | Successfully cross-amplified in all other members of sect. Pentanthera, suggesting their utility across the entire clade | Thompson et al. (2020) |
| 6 | R. dauricum and R. mucronulatum | 57,951 microsatellites identified | Specific-locus amplified fragment sequencing | Among the 40 primers tested, 36 (90%) give clear polymorphic results generating 274 alleles | Ar and Hs was 1.508 and 0.511 for R. dauricum and 1.445 and 0.444 for R. mucronulatum, respectively | Wang et al. (2021b) |
| 7 | R. delavayi | 22 microsatellite | Designed from an enriched DNA library | HE was between 0.036–0.709 and HO between 0.026–0.951 based on 34 individuals of five populations | 15 pairs showed polymorphism across populations of R. delavayi and 6 pairs in R. decorum and 9 pairs in R. agastum | Wang et al. (2010a) |
| 8 | R. ferrugineum | 18 nobel microsatellite markers | Pyrosequencing of enriched DNA libraries | HE ranged from 0.07 to 0.79 and HO from 0.05 to 0.85 | Cross-species amplified in 22 other Rhododendrons and 4 other Ericaceae species | Charrier et al. (2014) |
| 9 | R. ferrugineum | 102 novel microsatellite loci | 454 FLX Titanium pyrosequencing of RNA of leaf | Nine of the 24 loci were polymorphic. Mean HO and HE ranged from 0 to 0.76 and from 0.03 to 0.66 | Cros-species amplified in 13 Rhododendrons and two other species from the Ericaceae family (Vaccinium myrtillus and Erica scoparia) | Delmas et al. (2011) |
| 10 | R. Genus | 38 microsatellites | Rhododendron delavayi Franch. and R. decorum Franch | HO and HE per locus varied from 0.07 to 0.65 and 0.44 to 0.81, respectively | 16 pairs were amplified successfully in all species. 9 loci were polymorphic across six examined species | Wang et al. (2010b) |
| 11 | R. lapponicum | 64,327 potential SSRs identified | SMRT sequencing technology | The PCR products of 150 primer gave an amplification efficiency rate as 84.67% | – | Jia et al. (2020) |
| 12 | R. latoucheae Franch | 14,415 EST-SSRs identified | RNA sequencing of six tissues—stems, leaves, shoot tips, flower buds, flowers & fruits | Among of 80,660 unigenes, 43.22% were annotated against public protein databases. Among the 200 primer pairs, 58 primer pairs amplified successfully | Among 58 primer pairs, 37 primer pairs were polymorphic in 37 Rhododendrons and 30 R. latoucheae individuals | Xing et al. (2017) |
| 13 | R. longipedicellatum | 102 primer sets were designed | transcriptome sequences | HO and HE for 15 loci varied from 0.255 to 0.913 and from 0.306 to 0.851, respectively | Among 102 primer sets, 48 were successfully amplified, 15 showed polymorphisms and cross amplified in R. molle and R. simsii | Li et al. (2018b) |
| 14 | R. molle G. Don | 8266 simple sequence repeats (SSRs) detected | RNA sequencing of Flowers at four developmental stages | – | – | Xiao et al. (2018) |
| 15 | R. micranthum | 479,724 SSR markers were identified | Illumina HiSeq XTen sequencing of leaf DNA | Among 100 primers, 71 gave polymorphism | – | Zhou et al. (2020) |
| 16 | R. ovatum | 23 microsatellite markers | Developed by biotin-streptavidin captured method | 16 microsatellites were polymorphic and produce 4–30 locus in 89 individuals from 3 populations of China. HO varied from 0.103–0.933, while HE ranged from 0.102 to 0.954 | 12 primers (75%) were found to be functional in R. simsii | Liu et al. (2017) |
| 17 | R. obtusum ex Wats. variety ‘Yanzhimi’ | 37,777 EST-SSRs identified | RNA sequencing of Fresh leaf samples | Among the 60 screened EST-SSRs, 30 were polymorphic generating 196 alleles (6.53 band per locus). NA and NE ranged from 3–17 & 1.28–6.75, (average—6.53 & 2.98) | Frequent gene flow occurred with the mean value of 3.89 | Fang et al. (2021) |
| 18 | R. pubicostatum | 66,229 microsatellites identified | MiSeq Benchtop sequencer | Among 100 primers developed, 76, 71 and 64 primers successfully amplified in R. pubicostatum, R. bureavii and R. sikangense, respectively | – | Zhang et al. (2020a) |
| 19 | R. rex Levl | A total of 15,314 potential EST-SSRs identified | RNA sequencing of Fresh leaf samples | Among 164,242 unigenes (70.07%) unigenes were annotated in public databases. The PIC ranged from 0.154–0.870, (mean—0.482 | Among the 100 primer, 36 primer pairs were polymorphic in 20 individuals from 4 R. rex populations. High transferability (58.33–83.33%) recorded among the 6 subgenera | Zhang et al. (2017a) |
| 20 | R. shanii | 25,564 SSRs identified | Two terminal sequencing of leaf cDNA | HO ranged from 0.000 to 1.000, and He ranged from 0.000 to 0.918 with 24 primer sets | 24 primer sets for cross-amplification in R. annae, R. chihsinianum, R. decorum, R. denudatum, R. fortunei, R. neriiflorum, R. rex, and R. simiarum | Pan et al. (2019) |
| 21 | R. simsii | 8 microsatellite loci | – | The average allele number was 7.1 per locus, ranging from 6 to 9. The HO and HE were 0.278–0.944 and 0.556–0.871, respectively | These markers were useful for genetic structure, gene flow, and the phylo-geography of R. simsii | Tan et al. (2009) |
| 22 | R. simsii | 44,205 SSR identified | SMRT sequencing of pellet tissue RNA | – | – | Liu et al. (2022a, b) |
| 23 | R. simsii hybrid ‘Flamenco’ and the deciduous R. luteum | 127 EST SSR based primer set generated | Sequencing of cDNA fragments from flowers | R. simsii hybrids (‘Lara Rood’, ‘Coelestine’ and ‘Flamenco’), and 3 species (R. luteum, R. simsii and R. noriakianum) gave 93% amplification | PIC was 35% (45 polymorphic markers). 33 markers segregated in the offspring of at least one crossing population. R. luteum-based markers scored better than ‘Flamenco’ (77 vs. 72%) | Keyser et al. (2009) |
Abbreviations: pp percentage polymorphism, HO observed heterozygosity, HE expected heterozygosity, AR allelic richness, NE Nie’s gene diversity, I, Gst genetic differentiation, Nm gene flow among population, Na observed number of alleles, Ne effective number of alleles, He Nei’s gene diversity, I Shannon’s information index, PIC polymorphism information content, AMOVA analysis of molecular variance, Ht total gene diversity, Hs population gene diversity
Expressed sequence tags (EST) are short single pass sequences of cDNA that represent expressed parts of DNA. EST libraries from cold-acclimated and non-acclimated leaves of R. catawbiense identified 308 cold-responsive cDNA transcripts in cold-acclimated tissue and a very little overlap (6.3%) observed in total unique transcripts between acclimated and non-acclimated libraries (Wei et al. 2005b). ESTs can be mapped to specific chromosome locations using physical mapping techniques. To date (31 December 2023) details on available EST in the public database are given in Supplementary Table 2, which can be used as genomic resource for the development of a large number of markers. SSR markers developed from these EST sequences (EST-SSRs) recognized as functional markers are highly informative for trait identification (Sharma et al. 2020a). A total of 19 polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense (Yoichi et al. 2017).
EST-SSR markers showed good transferability among related species due to the convergent evolution of genes in different species. In R. longipedicellatum, among the 102 primer sets, 46% provided good amplification, and 15% exhibited polymorphism in 150 individuals from five populations (Li et al. 2018a). Among the 38 EST-ESRs, 59.38 to 93.75% cross-species transferability was observed in 15 Himalayan species (Sharma et al. 2020b). Similarly, 58.33 to 83.33% transferability has been found in different members of Rhododendron in primer pairs developed from transcriptome analysis of R. rex (Zhang et al. 2017a). Similarly in R. latoucheae, 15% EST-SSR primers were found polymorphic and exhibited 100% transferability across the 37 species of evergreen or deciduous Rhododendron from Hymenanthes, Rhododendron, Tsutsusi and Azaleastrum subgenera (Xing et al. 2017).
Next-generation sequencing (NGS) platforms revolutionized the process of genomic resource data generation and now millions of sequences can be produced at a lower cost. It has accelerated the development and utilization of sequence-specific markers for genetic mapping and molecular breeding of Rhododendron. Transcriptome sequencing of leaf samples of R. rex (Lévl.) generated 15,314 potential EST-SSRs (one EST-SSR per 5.65 kb), of which 36% SSRs were found polymorphic in genotypes of R. rex (Zhang et al. 2017a). Similarly, in transcriptome sequencing of different plant tissue of R. latoucheae (Franch.) 14,415 EST-SSRs were identified from 16,019 unigene sequences (one SSR per 2.87 kb). A total of 18.5% polymorphism (from 200 tested primers) was detected in 37 Rhododendron species and 30 genotypes of R. latoucheae (Xing et al. 2017). In another study, a total of 8266 simple sequence repeats (SSRs) were detected in the floral transcriptome sequencing of R. molle G. Don. (Xiao et al. 2018). In R. catawbiense, highly polymorphism (61%) intron-flanking EST-SSRs were obtained after sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs (Wei et al. 2005b). NGS technology has now facilitated the generation of genomic resource data creation in many other species in recent years such as R. longipedicellatum (Li et al. 2017b), R. fortune (Xu et al. 2019), R. hybridum (Cheng et al. 2018), and R. pulchrum cv. Baifeng (Wang et al. 2018d), R. obtusum ex Wats. variety ‘Yanzhimi’ (Fang et al. 2021), R. dauricum (Wang et al. 2021b), R. mucronulatum (Wang et al. 2021b), R. lapponicum (Jia et al. 2020), R. pubicostatum (Zhang et al. 2020a), R. calendulaceum (Thompson et al. 2020) and R. micranthum (Zhou et al. 2020).
SNP characterization
Single-nucleotide polymorphism (SNP) is a nucleotide site having a high substitution probability among individuals in a population and nowadays it has become an important tool for accurate and faster genome mapping and molecular breeding due to its higher density in the genome and high throughput obtained in a shorter time (Liu et al. 2020b). RNA-sequencing identified SSRs, SNPs and InDels in R. fortunei (12,756, 38,313 and 3174, respectively), R. simsii (13,294, 136,590 and 6258), R. mariesii (15,724, 44,942 and 4126) and R. molle (10,214, 77,829 and 3416), dominated (30.475–34.99%) by mutation of C:G → T:A SNP type (Wang et al. 2018e). In the case of threatened endemic (to the Cangshan Mountains in Yunnan, China,) species R. cyanocarpum, 6584 SNPs were identified using the double digest restriction-site-associated DNA-sequencing (ddRAD-seq) technique and these SNPs exhibited high genetic diversity (π = 0.070 and He = 0.067) and low population differentiation (Fst: 0.031–0.045). Also, these SNPs were able to identify temporal changes in effective population size of the species over the last 150,000 years and revealed a bottleneck incident of 60,000 years ago, followed by recovery of effective population size over a quick period (Liu et al. 2020b). In R. arboretum, sequencing of cDNA libraries from floral and foliar tissues of identified 35,419 microsatellite regions with 811 high-quality single-nucleotide polymorphic (SNP) variants were detected from 719 flowering-related transcripts (Choudhary et al. 2018a, b). While in R. ripense, 12,463 high-confidence SNPs were detected from a mapping population of 136 plants and it generated 13 linkage groups corresponding to the number of chromosomes in the species.
Currently, double digest restriction-site-associated DNA sequencing (ddRAD) based SNP discovery and subsequent diversity analysis has been successfully utilized in R. meddianum, R. ripense, R. kiyosumense, R. pentaphyllum and R. quinquefolium (Shirasawa et al. 2021; Yoichi et al. 2021; Zhang et al. 2021). However, applications of SNPs in domestication and ornamental trait improvement remain unexplored in Rhododendron.
QTL mapping and linkage mapping
Modern genome-wide association mapping techniques can contribute significantly to Quantitative trait locus (QTL) mapping in perennial Rhododendron. Flower color is the most important plant ornamental trait for selection in the breeding program, which appears only in later stages of cultivation. QTL map has been developed in R. simsii hybrids through genotyping of 4 genetically diverse mapping populations using dominant AFLP (364 per population) in combination with myeloblastosis (MYB)-based markers (15 nos.), co-dominant SSR (23 nos.) and EST markers (12 nos.). A total of 16 stable linkage groups were obtained for the 4 populations, which was higher than the basic chromosomal number of the species (n = 13) (De Keyser et al. 2010). Pink flower color can be obtained as a result of a gene-dosage effect and two major QTLs have been identified for pink flower color along with some minor QTLs. However, only a few QTLs were identified for leaf color, and some major QTLs were identified for leaf shape and size. These QTLs can be key candidates for breeding programs for quality improvement in azalea (De Keyser et al. 2013).
Similarly, a molecular linkage map has been developed for leaf and flower architecture of Rhododendron using a segregating population of inter-species cross. Parent-specific maps constructed through 239 RAPD, 38 RFLP and two microsatellite markers generated 13 linkage groups in the male parent (R. Cunningham’s White) in accordance to its basic chromosome and 18 linkage groups in the female parent (R. Rh 16’). Also, 2 QTLs exhibited a considerable role in leaf chlorosis resistance and 2 chromosomal regions were found responsible for flower color development (Dunemann et al. 1999). After the identification of QTLs, trait-controlling genes and their regulators can be detected. With the increasing information on genomic resources, large-scale screening of diversity of wild populations using such mapping tools can be utilized for the detection of other quality traits in Rhododendron.
Genome assemblies
To date, draft genome/ whole genomes have been available for five Rhododendron species (Table 2). Genome size varied from 506.7 (R. ripense to 695.09 Mb) (R. delavay var. delavayi). The draft genome of R. delavayi var. delavayi is represented by 32,938 genes of which, 25,560 genes are classified into families and consist of a high density of transposable elements (51.77%) and long terminal repeat elements (LTRs: 37.48%) (Zhang et al. 2017b). The genome of R. williamsianum consists of 23,559 genes having an average gene length of 4628 bp with a gene density of 0.044 genes/kb. Multiple round whole-genome duplication was observed in the species in the syntenic region consisting of more than one homolog. Transposable elements account for 26% of the genome and total repetitive content contributes 59% of the genome (Soza et al. 2019). While, the genome assembly of R. simsii predicted the presence of a total of 34,170 genes. The average length of genes was estimated as 5089.2 bp including 1416.3 bp transcript length, 1288.7 bp coding sequence (CDS) length, 259.7 bp exon sequence length, and 403.1 bp intron sequence length. A total of 954,329 repeat elements (47.48% of the genome) and a considerable number of transposable elements (known 25.56% and uncharacterized 19.24%) were identified. Among the total genes, 424 genes related to flowering-time control were identified by querying the Flowering Interactive Database, FLOR-ID. Also, 58 genes encoding enzymes for the carotenoid biosynthesis pathway and 125 genes encoding enzymes for anthocyanin and flavonol biosynthesis were identified. Transcription factors controlling, flowering time, flower color change and metabolic pathways for anthocyanins and carotenoids were reconstructed in the R. simsii (Yang et al. 2020). Recently, a total of 34,606 genes were predicted in the R. ripense genome, of which, 35,785 flower and 48,041 leaf transcript isoforms were identified (Shirasawa et al. 2021). Chromosomal level genome of R. griersonianum is represented by 38,280 genes with low heterozygosity (0.18%) and 57% repeated sequences (Ma et al. 2021a).
Table 2.
Comparative presentation of available whole genomes information on different Rhododendron species
| Name of species | R. delavayi var. delavayi (Zhang et al. 2017b) | R. williamsianum (Soza et al. 2019) | R. simsii (Yang et al. 2020) | R. ripense (Shirasawa et al. 2021) | R. kiyosumense (Shirasawa et al. 2021) | R. griersonianum (Ma et al. 2021b) |
|---|---|---|---|---|---|---|
| Total size of genome | 695.09 Mb | 532.12 Mb | 525 Mb | 506.7 Mb | 591.6 Mb | 677 Mb |
| Total genes | – | 23,559 | 34,170 | 34,606 genes | – | 38,280 |
| Gene prediction | 32,938 genes | 86% of total genes | 32,999 genes | 84.6% of total genes (BUSCOs) | – | – |
| Contribution of transposable elements | 51% | 26% | 25.56% | 51.4% | – | 57% |
| Contribution of repeat elements | 37.48% | 58.8% | 47.48% | 15.2% | – | – |
| Species specific genes | 1097 genes | – | – | – | – | – |
| Detailed description | Divergence time between R. delavayi and Actinidia chinensis was estimated to be in the range of 56.1–120.8 million years ago | Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralogous gene pairs, which identified whole-genome duplications | 424 genes related to flowering-time control identified by querying the Flowering Interactive Database, FLOR-ID. Also, 58 genes encoding enzymes for the carotenoid biosynthesis pathway and 125 genes encoding enzymes for anthocyanin and flavonol biosynthesis were identified | A high sequence similarity at the chromosome segment level with those of Actinidia chinensis, Actinidia eriantha, and Diospyros lotus | The obtained Iso-Seq reads were clustered into 35,785 and 48,041 transcript isoforms (mean length: 2243 bp) in flowers and leaves, respectively | – |
The emerging sequence data in Rhododendron initiated ‘The Rhododendron Plant Genome Database (RPGD)’ as a comprehensive online database for Rhododendron genomics information. It contains all the information of genome sequence assemblies of 3 species (R. delavayi, R. williamsianum and R. simsii) along with gene expression profiles derived from published RNA-Sequencing data, functional annotation, gene family, transcription factor, homolog gene information, simple sequence repeats and chloroplast genome assemblies (Liu et al. 2021c). The availability of the genome sequence can now facilitate de novo genome assembly, and allow investigating more complex traits and interactions between environmental factors and phenotypic expression. The transcript levels are usually proportional to the structural gene dosage, but in a few cases, gene expression has been greater or less than expected in polyploids. These genomic sequencing efforts have made significant contributions to understanding the flowering process, flower color development, stress adaptation mechanisms, and many more. However, more studies on other species having other unique characteristics (e.g., adaptability to specific habitats, abiotic and biotic stress tolerance, medicinal properties, flower display character, etc.) need to be sequenced with longer sequence reads using Single Molecule Real Time (SMRT), MinION sequencing with improved contiguity of the reference genome. Continuous improvement in genomic assembly is also required for comparative genomic or pan-genomics analysis within and among the species of Rhododendron.
Organelle genomics sequencing
Sequencing of organelle provides information on plant evolution, speciation history and phylogenetics. Typically, the chloroplast genome of Rhododendron species comprises a quadripartite cycle, composed of a pair of inverted repeat regions (IRa & IRb) separated by a large single-copy region (LSC) and a small single-copy region (SSC). However, in R. pulchrum, it was reported as non-quadripartite cycle due to lack of inverted repeat regions. Chloroplast genome size ranged between 190,698 (R. datiandingense) to 230,777 bp (R. kawakamii), while it was 146,941 bp in R. pulchrum (Shen et al. 2022). A quadripartite structure of chloroplast consists of 93 to 150 genes including protein coding genes, ribosomal genes and tRNA genes (Table 3). As codon utilization rates in organelles vary greatly among different species, R. delavayi represented 65 codons for 20 amino acids (Li et al. 2020).
Table 3.
Comparison of different full chloroplast genomes available in Rhododendron species
| Species name | Genome size (bp) | Number of genes | GC content | Other important feature | Reference |
|---|---|---|---|---|---|
| R. calophytum | 200,196 bp | 110 genes, including 77 protein-coding genes, 29 tRNA genes, and 4 rRNA genes | 35.9 | Quadripartite cycle, 2 IRs of 44,494 bp separated by a LSC (108,602 bp) and a SSC (2606 bp) region | Ma et al. (2022) |
| R. concinnum | 207,236 bp | 93 genes, including 59 protein-coding genes, 30 tRNA genes, and 4 rRNA genes | 35.87 | Quadripartite cycle, 2 IRs of 47,154 bp separated by a LSC (110,326 bp) and a SSC (2602 bp) region | Zhou et al. (2023) |
| R. datiandingense | 190,689 bp | 110 genes, comprising 77 protein-coding genes, four ribosomal RNA genes, and 29 transfer RNA genes | 36.06 | Quadripartite cycle, a pair of IRs (7020 bp) separated by LSC (190,689 bp) and SSC (2582 bp) region | Wang et al. (2021c) |
| R. delavayi | 193,798 bp | 123 unique genes, including 80 protein genes, 35 tRNAs and 8 rRNAs | 35.99 | Quadripartite cycle, 2 IRs of 15,494 bp separated by LSC (160,234 bp) and SSC (2576 bp) region | Liu et al. (2020c) |
| R. fortunei | 200,997 bp | 147 genes including 99 protein-coding genes, 42 tRNA, and 6 rRNA genes | 41.23 | Quadripartite cycle, 2 IRs with length of 44,619 bp separated by LSC (109,151 bp) and SSC (2604 bp) region | Xiao et al. (2023) |
| R. henanense subsp. lingbaoense Fang | 208,015 bp | 119 genes, including 86 protein-coding genes, four rRNA genes, and 29 tRNA genes | 35.81 | Quadripartite cycle, 2 IR of 47,408 bp separated by a LSC (110,593 bp) and a SSC (2606 bp) region | Zhou et al. (2021, 2023) |
| R. huadingense | 198,952 bp | 113 genes including 79 protein-coding genes, 30 tRNA, and 4 rRNA genes | 35.89 | Quadripartite cycle, 2 IRs with length of 45,171 bp separated by LSC (108,557 bp) and SSC 53 bp) region | An et al. (2022) |
| R. kawakamii | 230,777 bp | 110 genes including, 77 protein-coding genes, 29 tRNA genes, and four rRNA genes | 35.10 | Quadripartite cycle, 2 IRs of 6270 bp each separated by LSC (146,155 bp) and SSC (72,082 bp) region | Wang et al. (2021d) |
| R. mariesii | 203,480 bp | 151 genes, including 98 protein-coding genes, 45 tRNA genes and 8 ribosomal RNA genes | 39.52 | Quadripartite cycle, 2 IRs of 40,918 bp separated LSC (113,715 bp) and SSC (7953 bp) region | Li et al. (2023) |
| R. micranthum, | 207,233 bp | 92 genes, including 58 protein-coding genes, 30 tRNA genes, and 4 rRNA genes | 35.91 | Quadripartite cycle, 2 IRs of 47,118 bp separated by a LSC (110,376 bp) and a SSC (2621 bp) region | Zhou et al. (2023) |
| R. molle | 208,878 bp | 149 genes, including 97 protein-coding, 44 tRNA, and 8 rRNA genes | 36.00 | Quadripartite cycle, 2 IRs of 1117 bp separated LSC (198,019 bp) and SSC (629 bp) region | Liu et al. (2021a,b), Xu et al. (2023) |
| R. platypodum | 201,047 bp | 143 genes, including 93 protein-coding genes, 42 transfer RNA genes and 8 ribosomal RNA genes | 35.90 | Quadripartite cycle, 2 IRs of 44,650 bp separated by a LSC (109,134 bp) and a SSC (2613 bp) region | Ma et al. (2021b) |
| R. przewalskii subsp. przewalskii | 201,233 bp | 142 genes, including 91 protein-coding genes, 43transfer RNA genes and 8 ribosomal RNA genes | Quadripartite cycle, 2 IRs of 45,266 bp separated by aLSC (108,077 bp) and a SSC (2624 bp) region | Wang et al. (2023) | |
| R. pulchrum | 146,941 bp | 119 genes, including 81 protein-coding genes, 34 tRNA genes, and 4 rRNA genes | 35.80 | No inverted repeat regions | Shen et al. (2022) |
| R. shani | 204,170 bp | 150 genes, including 95 protein-coding genes, 47 tRNA genes, and 8 rRNA genes | 35.80 | Quadripartite cycle, 2 IRs of 4715 bp separated by a LSC (107,189 bp) and a SSC (2615 bp) region | Yu et al. (2022) |
| R. simsii | 206,912 bp | 93 genes, including 59 protein-coding genes, 30 tRNA genes, and 4 rRNA genes | 35.35 | Quadripartite cycle, 2 IRs of 47,036 bp separated by a LSC (110,234 bp) and a SSC (2606 bp) region | Zhou et al. (2023) |
The phylogenetic relationships suggested that the Rhododendrons are closely related to Ericaceae members such as Vaccinium oldhamii Miq. and Vaccinium macrocarpon. However, the relationship of species does not follow an exact pattern of classification up to subspecies or sections (Fig. 4). Among the large set of available repeated sequences, 371 SSRs were identified dominated by 233 mononucleotide SSRs (Li et al. 2020). Also, higher numbers of microsatellites were identified in R. henanense subsp. Lingbaoense (351 SSRs) (Zhou et al. 2021), and R. molle (273 SSRs) (Xu et al. 2023), While it was comparatively lower in R. mariesii (70 SSRs (Li et al. 2023), R. datiandingense (77 SSRs) (Wang et al. 2021c) and R. huadingense (75 SSRs) (An et al. 2022). Likewise, 77 SSRs were identified in R. fortunei and among the 29 microsatellites distributed in the gene coding region, 22 were found in the rpl16 gene, while the other 7 repeat motifs were detected in the genes matK, ndhA, rpoA, rps7, rps8, ccsA, and cemA. These genomic resources might be helpful for studying evolutionary process, phylogenetics, germplasm characterization and other purposes.
Fig. 4.
Phylogenetic tree constructed from whole chloroplast genomes sequences of different Rhododendron species
Future prospective and conclusion
Rhododendron known for ornamental value are widely distributed throughout the northern half of the globe. Studies have exhibited that flower color development is associated with petal tissue structure, pigment types and its distribution, which are regulated by environmental factors and genetic architecture. Restricted distribution to the moist and cold climate created an opportunity to identify/ develop genotypes or variety for the tropical and coastal regions of the southern parts. Stress induced by heat, high salt, drought and pathogens are the key limiting factors for its distribution. Multiple abiotic stress tolerances are currently an area of research as species frequently face combined environmental and biotic stresses. Salt and salinity exposure at higher level increases pathogen susceptibility in many Rhododendrons. Additionally, current climate change has increased the stress levels beyond the ability of plants to adapt. A species might not be able to adapt to abiotic stresses together with heat, flooding, drought, and high salt concentrations, cause plants to be more susceptible to biotic stresses, a phenomenon known as disease predisposition.
With good hybridization potential of Rhododendrons, functional marker development for stress tolerance could improve the accuracy and efficiency of indirect selection. However, long generation times hamper the use of traditional breeding approaches practiced for crops (Harfouche et al. 2012). Currently, few species of polyploids Rhododendron are preferred as a potential resource by breeders. Marker-Assisted Selection (MAS) can reduce the selection cycles in Rhododendron as it can be conducted with leaf development during the seedling stage. However, breeding and QTL identification for quality-related traits in such tree species using MAS can be impacted due to the small range of alleles and weak linkage disequilibrium (Resende et al. 2012). Further, high heterozygosity and weak linkage disequilibrium (LD) along a large number of low frequencies of rare alleles is the challenging feature of tree species for breeding (Quesada et al. 2010). The Genomic Selection (GS) tool could be a substitute for MAS that uses genotypic and phenotypic information to predict the genomic landscape of future progeny in a predictive model. Modern investigations on forest plants through GS revealed possibilities for developing the predictive models, even with a small number of markers and small effective breeding size populations which are comparable to conventional sized populations (Resende et al. 2012).
Molecular markers, like ESTs (expressed sequence tags) based on functional molecular markers and genetic linkage maps are essential for rapid genetic progress in the breeding of a species. The discovery of genes and functional genomics studies become possible due to availability of large-scale transcriptome data in NGS platform and these newly developed expression-based EST-SSR markers can be employed in various Rhododendrons. Therefore, the advancement of phenotypic selection by Rhododendron breeders can be performed apart from gene expression alone. The selection at the whole genome level using genome-wide association studies (GWAS) is the most important tool for tree breeders (Resende et al. 2012). Traits variation detected through GWAS, are best matched to QTL or individual candidate genes and frequent alleles, but can classically only recognize a small fraction of the actual difference in individuals (Elshire et al. 2011).
Proteomics and metabolomics for adaptation, stress resistance or display related traits have huge potential and are not well characterized due to their complexity (Nilsson et al. 2010). These approaches help to produce data on structural conformation and differences in proteins after post translational modification or quantitative traits like stability and tissue-specific protein deposition and can be used to forecast the level of mRNA of gene products and post-translational alteration events (Baginsky et al. 2010). Combining transcriptomics, proteomics, and metabolomics into breeding programs will complete the already vibrant functional genomics toolbox for the development of superior cultivars with ornamental value. Finally, mixing the data collected from breeding populations recorded through GS or allele variants from GWAS investigations, complete information from the incorporation of several types of data from the genetic to cell chemistry will increase the productivity and flowering quality of cultivars developed by Rhododendron plant breeders. Several genes associated with ornamental character-related pigments e.g., anthocyanin biosynthesis pathways (for example, chalcone synthase, chalcone isomerase, flavanone hydroxylase, leucoanthocyanidin synthetase) and other structural genes and transcription factors related to these pathways play important role in flower color development (Zhao and Tao 2015). Among others, physical factors like temperature, light, photoperiod and edaphic factors like environmental pH, irrigation water pH, mineral nutrient pH and plant hormones are closely associated with the flower color and need to be explored in Rhododendron.
An investigation conducted in Himalaya revealed that excessive cold temperatures might represent a hard boundary for the survival of certain tree species (Vetaas 2002). Thus, enhanced temperature resulting from global warming may not cause a dramatic die-back of Rhododendron trees. This supports the hypothesis that several tree species may stay alive in global warming in-situ due to high-temperature tolerance. Even though the trees will live under enhanced temperatures, the long-term consequence of their regeneration is doubtful as frosts are requisite for the seasonal growth rhythm of the species. Rich germplasm resources and large distribution make Rhododendron candidates for the study of cold resistance, flower development, genetic interaction for speciation, etc. Various existing biological phenomena are well studied in the species however, heat tolerance, pathogen resistance, salt-resistance varieties with the more attractive, large and longer life of flowers need to be developed for their dispersal to tropical countries.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We thank the Director of GBPNIHE for providing facilities and encouragement. We also thank colleagues in the Sikkim Regional Centre for their support and help. Partial financial support received from Institute as In-house Project (Project No-02) and DBT (project grant no. BT/PR41722/NER/95/1886/2021) is deeply acknowledged.
Author contributions
SR: conceptualization, data curation & collection, of figures and tables, designing, writing manuscript, and correspondence. AKJ: literature compilation, writing, reviewing & editing of the manuscript. HS: Literature compiltion and manuscript editing. All the authors read and approved the final version of the manuscript.
Declarations
Conflict of interest
All the authors declared that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethics approval
There is no ethical standard related to the present review article.
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