Clematis Genetic Diversity and Hybrid Identication using ISSR Markers

Background Clematis taxa are diverse, with high ornamental value. However, these plants have a complicated genetic background and a long growth period. Thus, molecular identifications are necessary to shorten the breeding cycle. Results Here, the genetic diversity of 17 parental taxa (five wild species and 12 Texas cultivars) were analyzed using inter simple sequence repeat (ISSR) markers. We obtained 108 alleles using 12 ISSR primers (an average of 9 alleles per primer). Genetic parameters, including the number of alleles (Na), the effective number of alleles (Ne), Nei's genetic diversity (H), and Shannon's diversity (I), suggested that these 17 taxa were highly diverse. Phylogenetic analysis recovered the 17 taxa in two large clades: one cluster included all of the cultivars, as well as Clematis pinnata, C. brevicaudata, and C. tubulosa; the second cluster included C. fusca and C. reticulata. The pairwise genetic distances between all cultivars were 0.421–2.368, suggesting that these cultivars may have derived from closely related species. We next performed five crosses between parental taxa and used ISSR markers to validate the authenticity of the 15 hybrid offspring. ISSR primers amplified bands specific to the male parents of each cross. Male parent-specific bands were identified in 11 of the 15 offspring; these 11 offspring were identified as true hybrids. The remaining progeny were considered self-hybrids due to the absence of male parent-specific bands. Our results demonstrated that ISSR molecular markers may be useful tools for the verification of true Clematis hybrids. ISSR-based genetic diversity analyses, early hybrid identification, and marker assisted selection of Clematis taxa may improve breeding efficiency, excavate key genes associated with important traits, promote the development of new varieties, and shorten the breeding cycle.

with morphological variability and the evolutionary history of the morphotypes [17]. ISSR markers are also simple to use, have good reproducibility, and can distinguish among similar genotypes [18]. Therefore, ISSR markers are ideal for genotype identification, map construction, gene tagging, and genomic and cDNA fingerprinting [19].
Clematis ISSR markers have been widely used for studies of genetic diversity and evolutionarily relationships [20]. For example, Yu Weijun [21] investigated genetic diversity among wild Clematis, horticultural varieties, and local populations using ISSR markers, showing that ISSR molecular markers could be used to identify, and differentiate between, horticultural Clematis varieties and wild species. ISSR markers were also used to show that the main parental populations of Clematis in China, as well as their derived offspring, had high levels of genetic diversity, and that most of the genetic variation was among varieties [22].
However, due to the long growth cycles of some Clematis, there are relatively few studies of hybrid populations, especially artificial hybrid populations. Studies demonstrating the molecular identification of Clematis hybrid progeny are also lacking.
To address this knowledge gap, we designated 17 Clematis taxa (five wild species and 12 Texas cultivars) as parents and conducted artificial crosses to obtain five hybrid combinations and 15 F1 hybrids. We then used ISSR markers to verify the true Clematis hybrids, and to analyze the molecular relationships among the wild species and the cultivars. The specific objectives of this study were: (1) to assess the feasibility of ISSRs for Clematis research, (2) to use ISSR markers to quantify genetic diversity among the 17 clematis (5 wild species and 12 cultivars) (3) to use ISSR markers to visualize phylogenetic relationships among species to provide a framework for subsequent classification and analysis of Clematis species and varieties, and (4) to use ISSR markers to identify true Clematis hybrids. Our results may provide a framework for the development of new, high-quality, highly ornamental Clematis varieties via uniparental inheritance. In addition, the use of molecular markers to clarify the genetic relationships among Clematis taxa is of great importance for the cultivation of new varieties.

Hybrid identification
For each combination, only the primers that successfully amplified male parent-specific bands were used to verify hybrid purity. Nine primers (U824, U844, U845, U841, U815, U834, U835, U840, and U899) verified that both of the offspring of combination X1 ('Bode' ♀  C. fusca ♂), A-75 and A-83, were true hybrids. Similarly, all 12 primers verified that six of the eight offspring of combination X2 (C. pinnata ♀  C. tubulosa ♂) were true hybrids due to the presence of 1-3 male parent-specific bands in these progeny: D-105, D-101, E-53, E-51, E-25, and E-31. None of the ISSR primers amplified male parent-specific bands in offspring E-34 and E-52, indicating that these progeny were not true hybrids. Seven primers (U836, U844, U845, U841, U834, U835, and U899) amplified male parent-specific bands in the single offspring of combination X3 ('Bode' ♀  C. reticulata ♂), D-81. Two primers (U845 and U841) verified that one of the three progeny of combination X4 (C. tubulosa ♀  C. brevicaudata ♂), E-73, was a true hybrid. The other two offspring of this cross, E-64 and E-62, were not true hybrids. Finally, male parent-specific bands were amplified in the single offspring of combination X5 ('Anisa' ♀  C. reticulata ♂), E-95, by six primers (U836, U845, U866, U834, U840, and U899).  (Table 3) in parent plants and their offspring for all the crosses included in this study (X1-X5; Table 2). Note:Each parent combination + offspring is grouped in the gel image using vertical lines. In each group, the first two lanes are the female and male parents (as indicated by the symbols ♀ and ♂), and the remaining lanes correspond to the offspring. Combination X1, Lane 1-2:

Phylogenetic positions of the 17 parental Clematis taxa
We successfully used ISSR molecular markers to genotype five wild Clematis and 12 Clematis cultivars, suggesting that ISSR markers are suitable for genetic clustering and distance analyses in this genus. The five wild clematis were recovered in two distinct clusters: C. pinnata, C. tubulosa, and C. brevicaudata formed a clade, corresponding to Sect. Clematis. C. fusca and C. reticulata, both of which fall into the Clematis subgenus Urophylla, also formed a clade. These placements were thus consistent with traditional systematic taxonomy [6]. In combination, molecular and morphological characters may more accurately reflect phylogenetic relationships among taxa. The species C. pinnata belongs to the Subsect. Pinnatae of the Sect. Tubulosae. C. pinnata is a woody vine with ternately compound leaves or one to two pinnately compound leaves; the sepals of this plant extend obliquely, with tubular blue-white flowers. C. brevicaudata, which is also a woody vine, belongs to the Subsect. Vitalbae of the Sect. Clematis [6]. C. brevicaudata has pinnate or twice-pinnate compound leaves, with extended sepals and round white flowers. Both C. pinnata and C. brevicaudata fall into the Subgen. Clematis [6]. In contrast, the status of C. pinnate is unclear as this plant is morphologically ambiguous. Xie et al [28] pointed out that "erect vs. spreading sepals" is not a stable character in C. pinnata, because in this plant the sepals are first erect but later become spreading or bent backwards. Thus, this character cannot be used to distinguish C. pinnata. Although C. pinnata is morphologically unlike C. brevicaudata, a recent revision placed C. pinnata in the Sect. Tubulosae [29], which was consistent with our results, as well as those of a previous molecular phylogenetic study [30]. In addition, C. crotula, which, like C. pinnata, is a semi-shrub with blue-purple sepals and tube-shaped flowers, also falls into the European Clematis subgenus and has also been placed in the Sect. Tubulosae. This result was consistent with the conclusion of Shi Jinghua [31] that C. pinnata is the product of interspecific hybridization.
C. fusca and C. reticulata are woody vines [32]. C. fusca, which falls into the Sect. Viorna, has pinnately compound leaves, sepals covered with brown hairs, and lilac, bell-shaped flowers. C. reticulata has yet to be systematically classified. This species, which has pinnately compound leaves and white-purple bell-shaped flowers, is morphologically very similar to C. fusca. This suggests that C. reticulata may fall into in Sect. Viorna with C. fusca. Thus, our results were consistent with systematic taxonomy based on morphological characters.
All 12 of Clematis cultivars included in this study belong to the Texas group, with morphologically similar bell-shaped flowers. Consistent with the morphological characters, our molecular results suggest that these cultivars are closely related. Here, 12 Clematis from the Texas group clustered with the three wild Clematis from Sect. Clematis, indicating a close genetic relationship and hinting that the hybrid parents may originate from Sect. Clematis, providing a theoretical basis for the cultivation and breeding of new varieties.

Identification of true Clematis hybrids
When breeding hybrid Clematis, it is very important to verify hybrid authenticity early in the growth process, as Clematis grow slowly and are difficult to keep alive. Early identification of true hybrids helps to prevent wasted time and effort. In addition, it is critical to accurately predict whether offspring are more likely to favor the male or female parent in order to determine whether or not to continue a given breeding program. Traditionally, hybrids in a variety of plant groups have been identified based on phenotype [33]. However, phenotyping is time-consuming and requires extensive growing areas. In addition, morphological characteristics are easily affected by external environmental factors, compromising phenotype-based identifications [34]. Thus, there is a need for hybrid-verification methods that are more sensitive, less affected by external factors, and applicable earlier in the growth process.
To address this need, several studies have investigated DNA-based molecular markers in a variety of plants [35,36,37]. However, studies using ISSR markers to authenticate true Clematis hybrids are rare. Here, we used ISSR molecular markers to authenticate 15 offspring of five Clematis crosses; 11 of the 15 offspring (73.33%) exhibited male parent-specific bands and were thus identified as true hybrids. Our results demonstrated that ISSR molecular markers can be used to rapidly and simply verify true Clematis hybrids, which can then be used for map construction and further cross-breeding programs.
We found that the ISSR primer U845 successfully identified true hybrids in every combination we tested. Thus, U845 might represent a potential universal primer for the identification of true Clematis hybrids.

Conclusions
This study is the first to use ISSR molecular data to authenticate true Clematis hybrids. Our results showed that ISSR markers are a powerful and efficient approach to hybrid identification in this genus. The simple, low-cost molecular method of true Clematis hybrid verification demonstrated herein might be applicable to Clematis breeding programs worldwide, while the specific ISSR primers developed for this study might be useful for the identification, registration, and protection of Clematis taxa. Finally, by classifying the 17 Clematis taxa included in this study and resolving the genetic relationships among the wild Clematis species, between the wild species and the cultivars, and among the cultivars, our results help to clarify the phylogenetic positions of Clematis taxa within the genus and provide reliable background data for future hybrid breeding programs. Genetic diversity analysis, early hybrid identification, and marker-assisted selection in Clematis using ISSR markers may help to improve breeding efficiency, excavate key genes associated with important traits, perform gene cloning and transgenesis, breed new varieties, and shorten the breeding cycle.

Plant materials and treatments
A total of seventeen Clematis accessions (five wild and 12 cultivars) and their offspring were used in this study (Tables 1 and 2). The five wild Clematis had different geographical origins, and were genetically and morphological diverse [20]. The 12 cultivars used were all bell-shaped varieties in the Texas group.
In our preliminary work, we performed many forward and backward crosses among 5 wild Clematis species and 12 horticultural varieties. However, the long breeding cycle of this genus and the low survival rate of the hybrids presented severe challenges. Finally, 5 hybrid combinations were successfully obtained. Due to their slow growth rates, the 15 hybrid progeny required 6 months of cultivation before they could be accurately identified. Single genotypes from each parental accession were hand pollinated between June and August 2020 to generate the F1 hybrids. A total of 15 F1 individuals were derived from five cross ( Table 2). F1 seeds were harvested from the female parents in October 2020. The F1 individuals were grown in a greenhouse at approximately 22°C under a 16 h photoperiod for 12 weeks and then transplanted into an experimental field at the Agricultural University, Baoding, Hebei, China (115.49058, 38.817921). 'Nazawa' Cultivar

DNA extraction
DNA was extracted from the 17 Clematis parental taxa and young leaves of the 15 6-month-old F1 offspring using plant genomic DNA extraction kits (CW0531S, Kangwei Century, Beijing China), following the manufacturer's instructions. The quality and quantity of the genomic DNA were estimated by measuring the A260/A280 ratio using a UV spectrophotometer and by performing gel electrophoresis [23]. DNA concentrations were adjusted to 40 ng/μL to facilitate polymerase chain reaction (PCR) amplification. DNA samples were stored at −20°C until use.

Primer selection and PCR amplification
After preliminary screening and re-screening of 100 pairs of ISSR primers (previously developed by our research group; [20]), effective polymorphic ISSR primers were selected and used to genotype the 17 parental taxa and the 15 F1 offspring. We tested which of the selected ISSR primers amplified characteristic paternal bands. We then used the identified primers to amplify the DNA of the 15 F1 plants; plants with characteristic paternal ISSR bands were identified as true hybrids [24].

Data analysis
Several genetic diversity metrics, as well as genetic distance, were calculated for the 17 clematis using Popgene 1.32 and NTSYS 2.10 [25]. including the observed number of alleles (Na), effective number of alleles (Ne), Shannon's information index (I), and expected heterozygosity (He). Unweighted pair-group method with arithmetic means (UPGMA) cluster analysis of the 17 clematis was performed based on Nei's genetic distance data using NTSYS 2.10. We determined and analyzed the molecular weight of each amplified DNA fragment based on the gel images [26].

Availability of supporting data
Not applicable

Competing interests
Not applicable

Funding
This work was supported by Collection of Germplasm Resources of Clematis in Hebei Province and Innovation of New Germplasm (20326339D)

Authors' contributions
WANG Xin Complete all experiments, perform data analysis and wrote the manuscript; LI Mingyang helped perform the analysis with constructive discussions; TIAN Lin performed the experiment; LIU Dongyun contributed to the conception of the study.

Acknowledgements
Thanks to all the authors for completing this study and the project of Collection of Germplasm Resources of Clematis in Hebei Province and Innovation of New Germplasm for funding.

Authors' information
WANG Xin, Male gender, Read master's degree in Hebei Agricultural University, Master Degree Candidate, Research interest is molecular plant breeding and genetic diversity analysis of flowers.