Forsythia suspensa (Thunb.) Vahl is a deciduous shrub of Forsythia genus in the family of Oleaceae, mainly distributed in Hebei, Shanxi, Shaanxi, Shandong, Anhui, Henan and other places in China. Forsythia suspensa has high medicinal value, which is recorded in 'Divine Farmer’s Classic of Materia Medica' and 'Compendium of Materia Medica'. Its fruit can be used as medicine, which is divided into 'Qingqiao' and 'Laoqiao' in Chinese. Forsythia suspensa has the effect of clearing heat and removing toxicity, eliminating swelling and dispersing knots, which is the main component of Lianhuaqingwen capsules (Xie et al., 2010). Fruits of Forsythia suspensa contain a large number of flavonoids, lignin, alkaloids and acids, which are commonly used as bulk medicinal materials in China and are widely used in traditional Chinese medicine enterprises. Studies have shown that the fruits of Forsythia suspensa have obvious effects of anti-inflammatory, antibacterial, antiviral and hypolipidemic, which has important development value and application prospect in clinical research and drug development (Jiang, 2015). Forsythia suspensa is full of treasures, in which Forsythia suspensa leaves are rich in nutrients, such as total flavonoids, forsythiaside and phillyrin. And a kind of health care drink of Forsythia suspensa has been developed (Wang et al., 2016).

Forsythia suspensa is an important medicinal plant that has great value to study, the existing research on it is mainly focused on planting and cultivation techniques, determination of extract components and development and utilization of metabolite active components and so on. Forsythia suspensa resources are mainly wild, soil environment, climate conditions and varieties have great influence on the yield and quality of Forsythia suspensa. The wild Forsythia suspensa resources are limited, which are difficult to meet the needs of the market, and it is also easy to destroy the diversity of wild resources. However, the existing artificially cultivated Forsythia suspense is a wild resource without systematic artificial cultivation. It is mixed with various types, and its yield and quality are uneven. The lack of good Forsythia suspensa varieties has become the 'bottleneck' problem of large-scale and industrial production for Forsythia suspensa. Collecting and evaluating the existing Forsythia suspensa germplasm resources and screening high-yield and high-quality Forsythia suspensa germplasm are of great significance to the development of Forsythia suspensa industry. Therefore, the analysis of genetic diversity of Forsythia suspensa germplasm resources and the development of unique molecular markers of Forsythia suspensa are beneficial to the collection and variety identification of Forsythia suspensa germplasm resources.

Forsythia suspensa has strong geographical adaptability and abundant genetic diversity. Fully exploiting genetic information can effectively improve the efficiency of germplasm resource identification and breeding of Forsythia suspense. Molecular markers such as randomly amplified polymorphic DNA (RAPD) and simple repeat sequences (SSR) have been developed for genetic relationship identification and polymorphism analysis of Forsythia suspensa. RAPD markers was used to analysis the Forsythia suspensa resources from different habitats and styles. It was considered that the differences of environmental factors would lead to the genetic diversity of Forsythia suspensa resources (Li et al., 2011, Proceedings of the 10^th National Conference on Medicinal Plants and Phytomedicines, pp.118). Li et al. (2019) screened 13 ISSR primers to analyze 25 Forsythia suspensa germplasm resources. The results showed that the genetic diversity of Forsythia suspensa germplasm resources were related to ecological factors and growth environment. Meng (2014) used 11 pairs of polymorphic SSR primers to detect Forsythia suspensa germplasm resources in 16 regions of China, suggested that SSR molecular markers could be used for the genetic diversity analysis of Forsythia suspensa germplasm resources. Wang et al. (2015) developed SSR molecular markers for Forsythia suspensa based on RNA-seq transcriptome sequencing technology. A total of 3 199 SSR loci were detected, and 12 pairs of polymorphic SSR primers were screened to distinguish Forsythia suspensa resources from different habitats.

Specific-Locus Amplified Fragment Sequencing (SLAF-seq) is a new simplified genome sequencing technology, which has been widely used in germplasm collection, evaluation and genetic diversity analysis. At present, a large number of SNP markers have been developed based on SLAF-seq technology from Manihot esculenta, Helianthus annuus, Camellia oleifera and Perilla frutescens. However, there are few reports on the development of SNP molecular markers using SLAP-seq sequencing technology in Forsythia suspensa. In this study, the SLAF-seq database of Forsythia suspensa was constructed to screen the specific fragments from the database for sequencing. The bioinformatics software was used to analyze the polymorphism SLAF of Forsythia suspensa, so as to develop specific SNP molecular markers and provide reference for the identification and breeding of Forsythia suspensa germplasm resources.

1 Results and Analysis

1.1 Database construction and evaluation

SLAF-predict software was used to predict the reference genome of Olea europaea by electronic restriction. According to the principle of whether the enzyme segments were evenly distributed on the genome and whether the size of the enzyme segments was consistent with the experimental system (Davey et al., 2013), the restriction endonuclease combination was RsaI+Hpy166II. Enzyme digestion efficiency is a key indicator to evaluate SLAF experiment. The experimental results showed that the efficiency of RsaI+Hpy166II double digestion was 90.01%, and the proportion of residual enzyme digestion sites was 9.99%, indicating that the enzyme digestion reaction is normal. In this study, the sequences with the size of 364~414 bp in the enzyme section were identified as SLAF tags, and 129 643 SLAF tags were estimated. To evaluate the effectiveness of the SLAF experiment process and the implementation of the enzyme digestion program, this study used SOAP (Li et al., 2009b) software to compare the sequencing data of Nipponbare (rice) and the rice genome data. The results showed that the paired-end mapped was 98.09%, the singles-end mapped reads was 0.59%, and the unmapped reads was 1.32% (Table 1). The real fragment size of SLAF was calculated based on the position of the sequencing double-terminal comparison sequence on the reference genome (rice), and the distribution of insert fragments of rice sequences was drawn (Figure 1). The results showed that most of the inserted fragments were in the range of 364~414 bp, indicating that the sequencing method used in this study was highly reliable and the SLAF database was basically normal.

1.2 Evaluation of sequencing data

To verify the accuracy of the experimental data analysis, the reads of 126 bp×2 was used as the analysis standard for subsequent data evaluation in this study. The sequencing quality value Q30 was used to evaluate the accuracy of sequencing single base. The base separation was checked by base distribution. The accuracy of the database was evaluated with Nipponbare (rice) as the control. After sequencing on the Illumina platform, the data amount of 0.46 Mb reads was obtained by the control, and a total of 112.28 Mb reads were obtained from 39 Forsythia suspensa samples. The reads data of each sample were between 1 324 860 and 5 911 565. Among them, No.6 (an) of Quzhou City Hebei Province obtained the largest amount of data of 5 911 565 reads. The data amount of Nipponbare (rice) reads was the smallest, which was 457 544 reads. The average Q30 percentage of this experiment was 96.29%, and the average GC percentage was 36.97% (Table 2). The sequencing results showed that the sequencing quality of this study was high, and the data were reliable to meet the sequencing requirements.

1.3 Development of SLAF tag and SNP marker

The results of Forsythia suspensa sequencing data analysis showed that a total of 535 357 SLAF tags were developed from 39 Forsythia suspensa germplasm resources, with an average of 162 683 SLAF tags per Forsythia suspensa sample. The average sequencing depth of tags was 16.20 x, and 262 297 polymorphic SLAF tags were developed. Based on the SLAF tag of Forsythia suspensa, 1 809 741 SNP molecular markers were developed. The number distribution of SNP in samples was 858 739~1 146 427, the hetloci ratio was 7.86%~13.36%, and the integrity ratio of SNP was 47.45%~63.34% (Table 3; Table 4).

1.4 Genetic structure analysis

Genetic structure analysis can quantify the number of ancestors of the samples studied and infer the genetic relationship of each sample. It is a genetic structure analysis method that is widely used at present, which is helpful to study the evolution process of research materials. In this study, admixture software (Alexander et al., 2009) was used to analyze the genetic structure of the developed SNP molecular markers of Forsythia suspensa germplasm resources. Based on the cross validation error rate of samples, the valley value of cross validation error rate is determined as the optimal grouping. The results of genetic structure analysis (Figure 2) showed that when K-value was 4, it was the best subgroup. Therefore, 39 Forsythia suspensa germplasm resources could be divided into 4 subgroups according to the developed SNP molecular markers (Table 5).

2 Discussion

Forsythia suspensa has rich germplasm resources and long cultivation history in China. It has high medicinal development value and wide application prospect. With the increasing market demand of Forsythia suspensa, the phenomenon of preemption in Forsythia suspensa habitats is serious, Forsythia suspensa is picked before maturity, resulting in the quality of Forsythia suspensa medicinal materials failing to meet the requirements of Pharmacopoeia. In addition, Forsythia suspensa resources are mainly wild. Due to different climate, soil and other ecological environment factors, the yield and quality of Forsythia suspensa are uneven. Therefore, the collection and evaluation of Forsythia suspensa germplasm resources and the cultivation of excellent germplasm have become the key problems to be solved for the sustainable development of Forsythia suspensa.

In recent years, molecular markers such as RAPD, SRAP and SSRs have been gradually applied to the evaluation and genetic analysis of Forsythia suspensa germplasm resources. Wu et al. (2016) used RAPD technology to study the genetic diversity of 14 Forsythia suspensa. The results showed that the genetic diversity of Forsythia suspensa was rich, and there was a significant correlation with the habitats of Forsythia suspensa. Sequence-related amplified polymorphism (SRAP) is a new molecular marker developed by Li et al. (2010) from the University of California from Brassica crops. He (2013) used SRAP molecular marker technology to analyze the genetic diversity of 26 Forsythia suspensa germplasm resources, from which 12 pairs of primer combinations were selected and 153 polymorphic bands were obtained. Shen et al. (2019) used EST-SST molecular marker technology to study the genetic diversity of 77 Forsythia suspensa germplasm resources from 12 habitats in Henan and Shanxi. The results showed that the genetic similarity of Forsythia suspensa was related to growth environment and altitude, but not to geographical distance.

With the development of the third generation DNA sequencing technology, single nucleotide polymorphism (SNP) molecular marker technology has been applied to germplasm resources evolution and genetic relationship identification, animal and plant genetic linkage map construction, QTL mapping and genetic diversity analysis for its advantages of high genetic stability, rich distribution, high polymorphism and easy detection. SLAF-seq is a high-throughput sequencing technology, which can develop SNP molecular markers with high density, good stability and low cost. The technology has been widely used in genetic diversity analysis of plants such as Theobroma cacao, Hevea brasiliensis, Citrus reticulata and Pinales (Zhou et al., 2018). Zhou et al. (2017) used SLAF-seq technology to analyze the genetic evolution of 300 Camellia oleifera germplasm resources, a total of 238 771 polymorphic SLAF tags were obtained, and 1 197 282 SNP markers were developed. Zhao et al. (2016) used SNP molecular markers to analyze Exocarpium Citri Grandis, and screened 21 SNP loci with good polymorphism, which provided effective SNP molecular markers for identification of Exocarpium Citri Grandis. Sun et al. (2013) used SNP and EST-SSR molecular markers to identify the genetic relationship of 363 litchi germplasm (Litchi chinensis Sonn.). The results showed that SNP molecular markers could be used to identify the parental origin of new litchi germplasm.

In this study, genetic diversity analysis and SNP molecular marker development of 39 Forsythia suspensa germplasm resources were carried out by SLAF-seq sequencing technology. The results of sequencing data showed that the reads information of 112.28 Mb was obtained in this study. Based on the obtained reads data, 535 357 SLAF tags were developed, including 262 297 polymorphic SLAFs, and a total of 1 809 741 SNP markers were obtained. These SNP molecular markers were used to analyze the genetic structure of Forsythia suspensa, and 39 Forsythia suspensa germplasm resources were divided into four groups, providing new information for the classification of genetic structure of Forsythia suspensa. The development of SNP molecular markers can provide data support for the investigation of agronomic traits, population identification and genetic analysis of Forsythia suspensa.

3 Materials and Methods

3.1 Test materials

39 Forsythia suspensa (Thunb.) Vahl germplasm resources were collected from different counties and cities in Shaanxi Province, Shanxi Province, Henan Province and Hebei Province (Table 6). The healthy and mature leaves of the above materials were collected and frozen in liquid nitrogen at the same time.

3.2 Acquisition of Forsythia suspensa DNA

Plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd. DP305) was used to extract DNA from 39 Forsythia suspensa germplasm resources. The DNA concentration was detected by NanoDrop2000 spectrophotometer, and the extraction quality of DNA was detected by electrophoresis. Ensure that the extracted genomic DNA of Forsythia suspensa meets the requirements of database construction.

3.3 Construction of SLAF library

The Olea europaea genome was selected as the reference genome in this study because the genome of Forsythia suspensa has not been published. The genome size of Olea europaea was 1.14 Gb, and the GC content was 35.40% (https://www.ncbi.nlm.nih.gov/genome/?term=Olea+europaea). Based on the genome size and GC content, the electronic enzyme digestion prediction of Olea europaea was carried out. The suitable restriction enzyme was selected to carry out the enzyme digestion experiment on Forsythia suspensa. The 3' end of the enzyme section was treated with A, and the Dual-index sequencing joint was connected. PCR amplification and electrophoresis gel cutting were carried out to recover the target fragment (Kozich et al., 2013). The SLAF sequencing of Forsythia suspensa was carried out on the Illumina platform.

3.4 Evaluation of sequencing data

In this study, the Dual-index method was used for statistical analysis of the sequencing data of Forsythia suspensa. The data information of 39 Forsythia suspensa samples was obtained by sequencing, and the sequencing data joints were filtered. The data volume and quality of the sequencing data were analyzed, including the number of reads, Q30 and GC content. To analyze the quality of SLAF library construction, Nipponbare (rice) was selected as the control in this study, and the same enzyme was used for double enzyme digestion to construct the library. Nipponbare (rice) genome data reference was from http://rapdb.dna.affrc.go.jp.

3.5 SNP loci development

The sequencing data in this study were from the enzyme section of the same endonuclease, and the data with integrity less than 0.2 and depth less than 2 were filtered out. Then, according to the principle of sequence similarity, 39 Forsythia suspensa samples were clustered in this study. The data clustered together were defined as the same SLAF tags. If a SLAF tag had differences in sequences between different Forsythia suspensa samples, it could be considered as a polymorphic SLAF tag. The development of SNP loci is based on the SLAF tag, using the alignment software BWA (Li and Durbin, 2009) to compare the sequencing data with the reference genome, using GATK (McKenna et al., 2010) and SAM tools (Li et al., 2009a) method to detect SNP. Based on sequence consistency, the detected polymorphism SNP information is considered to be the most reliable SNP data set, and the SNP screening standard is secondary genotype frequency of (MAF)>0.05, integrity>0.8.

3.6 Analysis of genetic structure

In this study, admixture software (Alexander et al., 2009) was used to carry out the analysis of genetic population structure for the developed SNP molecular markers of Forsythia suspensa. Cluster analysis was carried out according to the cluster number of Forsythia suspensa samples (assuming that K value was 1~15), and the clustering results were verified by cross validation. According to the verification results, the valley value of cross validation error rate was determined as the optimal cluster number of Forsythia suspensa samples.

Authors’ contributions

JT was the experimental designers and executor of this study. JT and WCX completed the data analysis and the first draft of the paper. TW, XXL, LRK and WSQ participated in some parts of the experiments. LLD was the designer and director of the project, guiding experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the National Program on Key Research and Development Project of China (2017YFC1701702; 2017YFC1700702), Innovation Team Project of Traditional Chinese Medicine in the Technology system of Modern Agricultural Industry in Hebei Province (HBCT2018060201), Innovative Engineering Project of Hebei Academy of Agricultural and Forestry Sciences (2019-2-3) and Science and Technology Support Project in Hebei Province (19226345D).

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