Abstract
Denphal-type Dendrobium is the most popular orchid for cut flower and potted plant trade. To improve commercial traits, many novel cultivars have been produced through hybridization by commercial breeders. However, the genetic relationship of most cultivars is unclear, thus hindering the progress of Denphal-type Dendrobium breeding programs. Therefore, the development of molecular markers is encouraged to identify different cultivars. In this study, based on the transcriptome database of the Denphal-type Dendrobium ‘Red Bull’, the polymorphisms expressed sequence tag-derived simple sequence repeat (EST-SSR) were developed from 100 pairs EST-SSRs that were randomly selected from the EST-SSR database. The genetic relationship of 42 Denphal-type Dendrobium cultivars was analyzed according to the developed EST-SSRs. Then, the transferability of EST-SSRs was analyzed by performing a relationship analysis of 40 Dendrobium species. The results showed that a total of 5174 potential EST-SSR markers were identified with 4486 unigene sequences, and 5289 primer pairs were successfully designed. Of the selected 100 pairs of EST-SSRs, a total of 86 pairs produced the expected polymerase chain reaction products of the primary screening, 58 pairs produced the expected fragment size, and 20 pairs showed polymorphisms. Furthermore, the dendrogram of 42 cultivars showed that at a genetic distance of 0.15, the cultivars collected were grouped into five clusters of three major clusters and two minor clusters; all these clusters had the same characters of each cluster. The transferability analysis showed that 18 of the 20 EST-SSR markers among the 40 Dendrobium species were polymorphic. Overall, this study developed EST-SSR markers and will be valuable to facilitating genetic diversity in Denphal-type Dendrobium cultivars and Dendrobium species.
Orchids belong to the largest and most diverse family, Orchidaceae, and consists of more than 25,000 to 30,000 species and more than 150,000 artificial hybrids (Hsiao et al. 2011). The Dendrobium is one of the most important genera in Orchidaceae, with more than 1000 species. Dendrobium plants are distributed among tropical and subtropical Asia and North Australia, and most occur as epiphytic life forms. Members of the Dendrobium have high economic potential as ornamental plants and for medicinal purposes. Dendrobium flowers vary greatly in form, size, color, and fragrance. Dendrobium has been used as a commercial cut flower and potted plants. Although many morphological variations of Dendrobium exist, there are two groups of the Dendrobium suited for ornamental sale: the Nobile-type and Denphal-type Dendrobium. The Nobile type produces inflorescences and flowers that are distributed over the pseudobulbs, and the Denphal type produces one or more terminal inflorescences from the pseudobulbs (Cardoso 2012). The Denphal-type Dendrobium has great value as cut flowers and potted plants, and they are some of the most popular orchids worldwide because of their rapid growth, floriferous flower sprays, wide range of colors, sizes, shapes, year-round availability, and long flowering life of several weeks to months (Kuehnle 2007). Denphal-type Dendrobium is the most popular orchid for the cut flower and potted flower trade of Asia. Denphal-type Dendrobium is important to Thailand’s economy. In 2012, the cut flower and potted plant exports were valued at $63.6 billion and $17.8 million, respectively, with the Denphal-type Dendrobium comprising 94.73% and 51.40% of the values in Thailand (Thammasiri 2015).
To improve commercial traits, many novel cultivars of Denphal-type Dendrobium have been produced through hybridization by commercial breeders. However, commercial breeders generally choose parents for breeding based on favorable characteristics and experience. When hybrids are fertile and used for further breeding, the identity of progeny could become untraceable. Moreover, the use of abbreviated and trade names and the sometimes intentional nondisclosure of parentage for commercial considerations further complicate genetic relationships between hybrids. Additionally, the Dendrobium cultivars on the market are diverse and complex, and it is difficult to distinguish one cultivar from another because of their similar outward appearance before flower development. To avoid mistakes in supply and demand in the market and further commercial breeding, it is important to identify cultivars and characterize the genetic relationships of Dendrobium cultivars and species. Traditionally, comparative vegetative anatomy and plant systematics are common strategies for assessing the relationship of the taxa Dendrobium (Adams et al. 2006; Morris et al. 1996). However, these strategies are insufficient to distinguish Dendrobium cultivars because morphological characteristics are quantitatively inherited, and their expression is affected by environmental factors. Therefore, a rapid and robust DNA marker technique has been used to identify Dendrobium cultivars.
During the past two decades, several types of molecular markers have been successfully used to characterize the genetic diversity of Dendrobium species and cultivars, including restriction fragment length polymorphism, amplified fragment length polymorphism, random amplified polymorphic DNA, and intersimple sequence repeat markers (Ding et al. 2013; Peyachoknagul et al. 2014; Shen et al. 2006; Wang et al. 2009; Zheng et al. 2012). However, these markers have the common shortcomings of poor repeatability and dominance. Moreover, these markers are time-consuming and expensive to use. Simple sequence repeat (SSR) markers have been used for germplasm management and marker-assisted selection of plants because of their advantages of being highly polymorphic, highly reproducible, and readily transferable to other populations within the same species and related species and genera (Kang et al. 2015; Lu et al. 2013; Tsai et al. 2015). Recently, a more informative co-dominant type of marker (i.e., SSRs) has been developed to identify Dendrobium and genetic linkage maps of Dendrobium (Liu et al. 2014; Lu et al. 2012a; Lu et al. 2012b; Lu et al. 2014. However, these studies were most focused on medical Dendrobium and Nobile-type Dendrobium plants. Therefore, fewer SSRs have been developed for Denphal-type Dendrobium. Moreover, the developed SSR markers did not provide adequate coverage and were not evenly distributed across the Denphal-type Dendrobium genome because of the limited number of markers. Therefore, it is still essential to develop more effective SSR markers for Denphal-type Dendrobium.
According to the origin of the sequences used for the initial identification, SSRs can be divided into genomic SSRs (derived from random genomic sequences) and expressed sequence tag-derived SSRs (EST-SSRs). Compared with genomic SSRs, EST-SSRs are tightly linked with functional genes that may influence certain important agronomic characteristics. Currently, EST-SSRs are widely used in studies of plant genomes, such as genetic mapping, comparative mapping, evaluation of genetic diversity, germplasm identification, and phylogenetic and evolutionary studies (Wu et al. 2014). However, only a few Denphal-type Dendrobium EST sequences are available in public databases. In this study, the transcriptome of Dendrobium ‘Red Bull’ was obtained by Illumina sequencing (Illumina, Inc., San Diego, CA, USA) to validate and characterize SSR markers. Based on the databases, thousands of SSR loci were used to design SSR primers. A sample of these primers was further developed to estimate the genetic relationship of Denphal-type Dendrobium cultivars and Dendrobium species.
Materials and Methods
Plant materials.
Newly grown leaves of 42 Denphal-type Dendrobium cultivars and 40 species of Dendrobium were sampled from the orchid germplasm resource garden of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science, Hainan, China.
Marker loci detection and SSR primer pair design.
A total of 63,101 EST sequences were obtained from the Dendrobium ‘Red Bull’ transcriptome sequences of flower bud (transcriptome data were unpublished). The microsatellite identification web tool (Beier et al. 2017) was used for SSR mining and identification. The minimum numbers of repeats used for selecting SSRs were nine for dinucleotide repeats, seven for trinucleotide repeats, six for tetranucleotide repeats, five for pentanucleotide repeats, and four for hexanucleotide repeats.
Primers were designed to flank the SSRs using the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The parameters for primer design were as follows: primer length, 18 to 24 bp (21 bp was the optimum); polymerase chain reaction (PCR) product size, 100 to 300 bp; annealing temperature, 59 to 62 °C (60 °C was the optimum); and GC contents, 45% to 55% (50% was the optimum). Primers were synthesized by Shanghai Sangon Biological Engineering Technology (Shanghai, China).
DNA extraction and EST-SSR marker amplification.
Newly grown leaves (5 g) from the tested individuals were collected and used for genomic DNA extraction. Genomic DNA was isolated by a Super Plant Genomic DNA Kit (Polysaccharides & Polyphenolics-rich; Tiangen, Beijing, China). DNA was quantified by comparison of known concentrations of DNA following electrophoresis in a 1% agarose gel and and a visible spectrophotometer (NanoVue; GE, Chicago, IL, USA). The working concentration of DNA was adjusted to 20 ng⋅μL−1.
Eight Dendrobium cultivars (Tongchai Gold, Burana Charming, Enobi Purple, Sunny Red, Aridang Blue, Coerulea Blue, Bangkok Green, and Candy Stripe) were selected to screen EST-SSR markers for polymorphisms. The PCR experiments were performed in 20-μL reaction volumes containing 40-ng template DNA, 1 × Taq buffer, 0.4 μL dNTP (10 mm), 0.15 Mm⋅L−1 Mg2+, 1.0 μL of each primer (10 μmol), and 1.0 μL Taq DNA polymerase (1 U) (Fermentas, Burlington, ON, Canada). The PCR reaction routine was as follows: DNA denaturation at 95 °C for 3 min; 36 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s; and 72 °C for 10 min as a final extension. Finally, the products were examined on a 2% agarose gel. Amplified products that showed a band of the expected size were mixed with loading buffer [2.5 mg⋅mL−1 bromophenol blue, 2.5 mg⋅mL−1 diphenylamine blue, 10-mm EDTA, and 95% (volume/volume) formate] and denatured for 10 min at 94 °C, followed by 5 min on ice. The amplified DNA was analyzed using a DNA analyzer (ABI 3730xl; Thermo Fisher Scientific, Waltham, MA, USA). Allele sizes were assessed using GeneMarker2.2 (Thermo Fisher Scientific). To confirm the reproducibility of the results, PCR amplification with each primer pair was performed three times.
Data processing and genetic analysis of Denphal-type Dendrobium cultivars.
According to the primer screening results, the optimized polymorphic primers were used to analyze the genetic diversity of 42 Denphal-type Dendrobium cultivars. Each allele was scored as present (1) or absent (0) for each of the SSR loci, and two binary qualitative data matrices were constructed. A dendrogram of the samples was established using the neighbor-joining method with the MEGA5 software (Tamura et al. 2011).
Cross-amplification and genetic analysis of Dendrobium species.
According to the primer screening results, the optimized polymorphic primers were used to analyze the genetic diversity of 40 Dendrobium species. Each allele was scored as present (1) or absent (0) for each of the SSR loci, and two binary qualitative data matrices were constructed. A dendrogram of the samples was established using the neighbor-joining method with MEGA5 (Tamura et al. 2011).
Results
Distribution and frequency of SSR markers.
A total of 63,101 sequences in a mean length of 902 bp were obtained to identify perfect SSRs, which represented ∼56.95 Mb. Using the SSRIT tool, 5174 potential EST-SSRs were identified from 4486 sequences (7.11%), of which 552 sequences (12.30%) contained more than one SSR. On average, one SSR was identified for every 11.01 kb or corresponded to one SSR for every 14.07 sequence in the Dendrobium ‘Red Bull’ transcriptome. The EST-SSRs contained five diverse types of repeat motifs, and there was an uneven distribution of EST-SSRs. Among the motifs identified (Table 1), dinucleotide repeats were the most abundant type, with a frequency of 57.21% (2960), followed by trinucleotide (36.92%; 1910), hexanucleotide (3.13%; 162), tetranucleotide (1.49%; 77), and pentanucleotide repeats (1.26%; 65). There were large proportions of both dinucleotide and trinucleotide motifs, whereas the others were less than 2.16%. The number of SSR repeats ranged from 4 to 15, and SSRs with six repeats were most abundant (26.67%; 1380), followed by those with five (21.65%; 1120), seven (16.60%; 859), and eight tandem repeats (9.57%; 495) (Table 1). Motifs that showed more than 11 reiterations were rare, with a frequency less than 0.48%. SSR loci with a length of 15 bp were the most frequent (20.39%; 1055), followed by those with 18 bp (17.07%; 883), 12 bp (16.76%; 867), and 14 bp (10.92%; 565) The longest SSR locus was 33 bp. Within the developed SSR motifs, a total of 132 SSR motifs were identified. Among these, there were 4, 10, 18, 22, and 78 motifs containing dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats, respectively. The AG/CT dinucleotide repeat was the most abundant motif detected in EST-SSRs (43.08%; 2229), followed by AT/AT (11.75%; 608), AAG/CTT (9.84%; 509), ATC/ATG (4.91%; 54), AGG/CCT (4.89%; 253), CCG/CGG (4.23%; 219), AAC/GTT (3.96%; 205), and AGC/CTG (3.63%; 189). The frequency of the remaining 124 types of motifs accounted for 13.68% (Table 1).
Frequency of different repeat motifs among the expressed sequence-tagged simple sequence repeat markers of Denphal-type Dendrobium cultivar.
Development of EST-SSR markers.
Based on the flanking sequences of each SSR locus, the primer pairs were designed; 5289 primer pairs were designed successfully. A total of 100 primer pairs were randomly selected for SSR PCR using eight cultivars. Overall, 86 pairs produced the expected PCR products of the primary screening; of these 86 primer pairs, 58 produced the expected fragment size, accounting for 67.44%. Of the 58 primer pairs, 20 pairs showed polymorphisms among the eight cultivars (Table 2).
Primer list, repeat patterns, and allele size of the 20 developed expressed sequence-tagged simple sequence repeat (EST-SSR) markers.
Genetic diversity of 42 Denphal-type Dendrobium cultivars.
The 20 optimized SSRs were used to analyze the genetic diversity of 42 Denphal-type Dendrobium cultivars, generating 235 alleles with 158 polymorphic loci (67.23%), which were treated as informative loci for dendrogram construction. The number of SSR alleles was 4 to 22 for one primer pair, and each primer pair had an average of 11.75 alleles.
The dendrogram (Fig. 1) shows that at a genetic distance of 0.15, the cultivars collected here were grouped into five clusters of three major clusters (I, II, and III) comprising multiple cultivars and minor clusters (IV and V) comprising only single cultivars. Major cluster I consisted of 33 cultivars and was divided into three subclusters, I-A, I-B, and I-C. The subcluster I-A contained 21 cultivars with the common traits of a tall plant, rounded and flattened petals, and white flower background color. The subcluster I-B contained seven cultivars with the common traits of a tall plant, slender and flattened petals, and purple-red flower color. The subcluster I-C contained five cultivars with the common traits of a tall plant, slender petals, and pink flowers. The second major cluster contained two cultivars with the common traits of a medium plant size and twisted petals. The third major cluster contained five cultivars with the common traits of a shorter plant, slender petals, and pink flowers. Cluster IV contained one cultivar with the traits of a small plant, large flower number, small flower, and fragrant flower. Cluster V contained one cultivar with the traits of a medium plant size, a large number of flowers, and twisted petals. The five clusters divided had the same traits within clusters and different traits between clusters, which indicated that these 20 pairs of primers can be used for the analysis of genetic diversity and related relationships of Denphal-type Dendrobium cultivars.
Phylogeny of 42 Denphal-type Dendrobium cultivars based on developed expressed sequence-tagged simple sequence repeat (EST-SSR) markers.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05250-22
Transferability of EST-SSR markers and application to the genetic diversity of wild Dendrobium species.
The transferability of 20 optimized Denphal-type Dendrobium cultivar EST-SSR markers were explored for 40 Dendrobium species. Eighteen markers were successfully amplified in at least one of the 40 species, and six EST-SSR markers (SS46, SS94, SS18, SS82, SS72, and SS27) could produce the anticipated SSR bands in all tested species. Of the 18 primer pairs generating 213 alleles, 141 polymorphic loci (66.20%) showed high polymorphism. Figure 2 shows that the Dendrobium species collected were grouped into eight clusters with three major clusters (I, II, and III) containing multiple cultivars and five minor clusters (IV, V, VI, VII, and VIII) containing only a single species at a genetic distance of 0.28. The first major cluster consisted of 29 species and was divided into four subclusters I-A, I-B, I-C, and I-D. Subcluster I-A consisted of 13 species collected from Yunnan province in China, and all belong to Sect. Dendrobium. Subcluster I-B consisted of four species collected from India. Subcluster I-C consisted of eight species collected from Vietnam. Sub-cluster I-D consisted of four species collected from Hainan in China. Cluster II consisted of four species collected from Australia. Cluster III consisted of two species collected from Guangxi in China. The clusters IV, V, VI, VII, and VIII consisted of one species collected from Guizhou in China, Thailand, Malaysia, and Indonesia, respectively.
Phylogeny of 40 Dendrobium species based on developed expressed sequence-tagged simple sequence repeat (EST-SSR) markers.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05250-22
Discussion
The frequency of SSRs in SSR containing ESTs can accurately reflect the density of SSRs in the transcribed region of the genome. During this research, a total of 63,101 potential unique EST sequences (∼56.95 Mb) were used for the SSR search, and 4486 ESTs (7.11%) contained SSR motifs, generating 5174 unique SSRs. This SSR frequency was consistent with the range frequencies that were reported for dicotyledonous plant species (2.65%–16.82%) (Kumpatla and Mukhopadhyay 2005) and was similar to that of Medicago sativa (7.20%) (Wang et al. 2014), Dactylis glomerata (7.0%) (Bushman et al. 2011), and Paphiopedilum concolor (7.58%) (Li et al. 2015). Moreover, the frequency of the occurrence of EST-SSRs was one SSR in every 11.01 kb. It showed that the distribution density or frequency of occurrence of Dendrobium ‘Red Bull’ was higher than that in Ananas comosus (1/13 kb) (Ong et al. 2012), Nelumbo nucifera (1/13.04 kb) (Pan et al. 2010), Linum usitatissimum (1/16.5 kb) (Cloutier et al. 2009), and Boehmeria nivea (1/19.3 kb) (Liu et al. 2013). However, this value was low compared with that of Ricinus communis (1/1.77 kb) (Qiu et al. 2010), Gossypium herbaceum (1/2/4 kb) (Jena et al. 2012), Hevea brasiliensis (1/3.39 kb) (Feng et al. 2009), Colocasia esculenta (1/5.90 kb) (You et al. 2015), Ipomoea batatas (1/7.1 kb) (Wang et al. 2011), Glycine max (1/7.25 kb) (Xin et al. 2012), M. sativa (1/7.47 kb) (Wang et al. 2014), and Paeonia suffruticosa (1/9.24 kb) (Wu et al. 2014). These results indicated that the abundance estimation and frequency of SSRs in EST sequences were highly variable among plant species. Frequency variations were induced by different species and were dependent on the SSR search criteria, the size of the dataset, and the database-mining tools for different studies (Varshney et al. 2005).
Until recently, both dinucleotide and trinucleotide repeats have been found to be predominantly repeat types in many plants, whereas the dominant repeat motifs were usually different (Varshney et al. 2005). In this research, dinucleotide repeat motifs were the most frequent repeat type of Dendrobium, as in many plants, such as P. suffruticosa (Wu et al. 2014), C. esculenta (You et al. 2015), M. sativa (Wang et al. 2014), and P. concolor (Li et al. 2015). Furthermore, the shorter motifs appeared more frequently than longer motifs, except for hexanucleotides, which were more frequent than tetranucleotides and pentanucleotides in the SSRs. The most dominant dinucleotide repeat motif was AG/CT (43.08%), followed by AT/AT (11.75%); the same results were found for C. esculenta (You et al. 2015), B. nivea (Liu et al. 2013), and I. batatas (Wang et al. 2011). Previous studies showed that the GC/CG motif was a rarity for many plants (Kumpatla and Mukhopadhyay 2005; Victoria et al. 2011; Wu et al. 2014). During this research, the GC/CG motif was the least common in the dinucleotide repeat motifs (0.41%), which was in agreement with recent studies of P. suffruticosa (0.4%) (Wu et al. 2014), Coffea (Aggarwal et al. 2007), and Epimedium sagittatum (Zeng et al. 2010). The most abundant trinucleotide repeat motif was AAG/CTT (9.84%), which is consistent with the recent results obtained for E. sagittatum (Zeng et al. 2010), Caragana korshinskii (Long et al. 2015) and B. nivea (Liu et al. 2013). Previous studies suggested that the trinucleotide CCG/CGG was a common motif for monocots, such as Hordeum vulgare, Triticum aestivum, Zea mays, Sorghum bicolor, and Oryza sativa (Kantety et al. 2002; La Rota et al. 2005). During this research, the CCG/CGG repeat motif was the fourth most dominant trinucleotide repeat motif, and the trinucleotide CCG/CGG was also common to Denphal-type Dendrobium cultivars, as with other monocots.
During this study, 86 of 100 randomly designed primer pairs had successfully amplified products. The amplification efficiency was higher than that of C. esculenta (85%) (You et al. 2015), I. batatas (84.6%) (Wang et al. 2011), Helianthus tuberosus (70.83%) (Yang et al. 2018), Melilotus albus (63.82%) (Yan et al. 2017), and Magnolia sinostellata (51.33%) (Wang et al. 2019). However, this was less than those of Lycium barbarum (Chen et al. 2017) and Elymus sibiricus (Zhou et al. 2016), which were 88% and 87.6%, respectively. It could be attributable to the introns in the genomic sequence. Among the 86 successful primer pairs, 58 produced the expected fragment size, accounting for 67.44%. Nonetheless, 20 of those 58 primer pairs were polymorphic among 8 cultivars. The percentage of polymorphic loci for the tested cultivars was 34.48%, which was higher than the results for E. sibiricus (Zhou et al. 2016), Corchorus (Zhang et al. 2015), and I. batatas (Wang et al. 2011); however, it was lower than that obtained during some previous studies (Kim et al. 2019; Wang et al. 2019; Wu et al. 2014; Yan et al. 2017; Zheng et al. 2013).
Cultivar identification is the greatest concern for Denphal-type Dendrobium growers, breeders, and scientists, and molecular markers are recognized as reliable and indispensable tools. However, to date, few molecular markers have been used for Denphal-type Dendrobium identification; only amplified fragment-length polymorphisms (Wahba et al. 2014; Xiang et al. 2003) and SSRs (Yue et al. 2006) have been reported. During this study, the phylogenetic relationships of 42 Denphal-type Dendrobium cultivars were analyzed by the developed 20 EST-SSR markers. The results showed that the 42 Denphal-type Dendrobium cultivars could be divided into five clusters at a genetic distance of 0.15, and ‘Sonia Hiasakul’ and ‘Caesar’ could be grouped into one cluster, which is in agreement with the results of previous studies (Wahba et al. 2014; Xiang et al. 2003; Yue et al. 2006). Almost all the cultivars tested were registered with the Royal Horticulture Society and could be searched on OrchidRoots (BlueNanta, 2022). The OrchidRoots parentage record was consistent with the genetic relationship derived from our EST-SSR analysis. Caesar was the seed parent of Sonia, and it clustered closely to ‘Sonia’; ‘Emma White’ and ‘Liberty White’ had the same seed parent and were clustered together. This was also observed for ‘Nopporn Pink’ and ‘Nopporn White Diamond’. These 20 EST-SSR markers had high efficiency for cultivar identification of Denphal-type Dendrobium.
To test the versatility of EST-SSR markers derived from Denphal-type Dendrobium cultivars, the 20 EST-SSR primer pairs were used to analyze the genetic diversity of 40 Dendrobium species. Clear bands were generated, and the transfer rate (90%) detected by using these EST-SSR primer pairs was higher than that detected using Lens culinaris (45.1% to 71.3%) (Singh et al. 2019), Olea europaea (46%) (Arbeiter et al. 2017), and E. sibiricus (49.11%) (Zhou et al. 2016), thus indicating the availability of SSR markers for Dendrobium.
Conclusions
The current study provided a set of 20 polymorphic EST-SSR markers of Denphal-type Dendrobium cultivars. These could be useful for studies of the genetic relationship of cultivars. Moreover, they have good transferability to Dendrobium species. Therefore, they could advance Dendrobium research and breeding programs in the future.
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