Abstract: Background and Objective: Koelreuteria as important native tree species, have multiple ornamental, ecological and medicinal values. However, research on the germplasm resources of Koelreuteria is relatively limited and relies primarily on the use of morphological differences to distinguish trees; thus the aim of this study was to develop effective EST-SSR markers to provide reference for the genetic diversity evaluation of the species. Methodology: In this study, 2871 microsatellite markers were assessed by mining a transcriptomic database of Koelreuteria paniculata Laxm. and 65,360 pairs of primers were designed. Based on the length of the products, the stability and permeability of primers and primer annealing temperatures, 96 pairs were screened and tested in a preliminary test. Thirteen pairs were selected for capillary electrophoresis analysis. The observed heterozygosity and expected heterozygosity were collected by GenAlEx software and the allelic polymorphism information content were calculated using CERVUS. Results: Ninety two pairs that gave clear bands in PCR amplification. For capillary electrophoresis analysis, in the microsatellite loci that showed polymorphisms, the number of alleles ranged from 2-5 per locus and the polymorphic information content ranged from 0.124-0.726, respectively. Additionally, based on capillary electrophoresis results, this study established unique fingerprints for four particular variants of K. paniculata Laxm. Conclusion: These polymorphic markers will be in a great favour of evaluating genetic biodiversity. The fingerprints would provide a foundation for variety protection.
INTRODUCTION
Koelreuteria is an important ornamental tree that has various economically important uses1. Plants in the genus contain a variety of compounds with great medicinal value, such as K. paniculata Laxm., which contains flavonoids2. However, research on the germplasm resources of Koelreuteria is relatively limited and relies primarily on the use of morphological differences to distinguish trees; this means it lacks clear molecular markers. Thus, this study used the universal molecular marker, simple sequence repeats (SSRs), to assess K. paniculata with a view to developing primers for helpful SSR markers. The study also used the SSRs to develop unique fingerprints for four special variants of K. paniculata Laxm., samples A2, A3, A4 and A5, which differ from ‘ordinary’ individuals in terms of leaf color (Fig. 1).
In recent years, many genetic markers have been developed, such as random amplified polymorphic DNA (RAPD), simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNPs). The SSRs occupy an important position in the field of genetic markers because of their high polymorphism, especially in plants3. The SSRs, also known as microsatellites, consist of short repeated DNA motifs. Compared with other genetic markers, SSRs also have the advantages of high abundance, a random distribution in the genome and co-dominant inheritance. As a result, SSRs play important roles in linkage mapping and DNA fingerprinting, genetic diversity analysis, identification of genetic relationships and functional gene tagging4.
However, the development of SSRs from the genome is relatively expensive and has limitations in terms of the research material, which should be genome-wide sequences. Alternatively, expressed sequence tag (EST)-SSRs, developed from expressed sequence regions, reduce the costs and are used widely in many fields.
Compared with genomic SSRs, EST-SSRs have a high level of transferability in both intraspecific and interspecific manners5,6. With these advantages, EST-SSRs have great value in genetic research and have been used in many plants, including angiosperms and gymnosperms, such as kenaf7, black pepper8, Amentotaxus5, pigeon pea9, grape10, white poplar11, eucalyptus6 and wheat12. In this study, EST-SSR markers are first developed in K. paniculata Laxm. and the study provide a primary situation about the distribution of SSR markers in the species expressed sequence.
The aim of study was the development of primers for EST-SSRs in K. paniculata Laxm. and the assessment of the utility of 92 primers pairs. Furthermore, this study obtained fingerprints of four special varieties and provided a foundation for variety protection.
MATERIALS AND METHODS
Plant materials: In total, 7 genotypes were used: Five K. paniculata Laxm. from Changzhi, Shanxi province, China (adult tree, collected on May 10, 2016) and two from Beijing, China (adult tree, collected on March 1, 2016). The detailed information about materials is shown in Table S1. All genotypes were collected and stored in -76°C. Part of the material was used for transcriptome sequencing and the rest was used for the development of primers.
Development of EST-SSR primers: The study screened for potential SSRs having at least five repeated SSR motifs from the transcriptome of K. paniculata Laxm. Based on the data, 65,360 pairs of primers were designed for 2871 SSR loci using QDD (ver. 3.1)13.
| Fig. 1(a-e): | (a) Photo of sample A1, the ‘ordinary’ golden rain tree and ( b-e) Four specific varieties of K. paniculata Laxm. (A2-A5) |
Based on the length of the products, stability and permeability of the primers and primer annealing temperature (which affects testing methods and results), 96 primer pairs were screened and tested. The information about the primers is shown in Table S2.
DNA extraction and PCR amplification: Genomic DNA samples were extracted from young leaves with a modified CTAB method4.
| Table S1: | Material sources |
| Table S2: | Primers used for SSR analysis of Koelreuteria |
The PCR amplification was performed using a Thermal Cycler (Bio-Rad T-100TM series, USA) and a reaction mix consisting of 25 μL 2×PCR reaction mixture (New England Biolabs, Ipswich, M A, USA), 2 μL genomic DNA (10-20 ng), 1 μL each of forward and reverse primer (10 μM) and 21 μL ddH2O in a final volume of 50 μL. The PCR conditions were divided into two steps. First, initial denaturation at 94°C for 3 min, three cycles of 94°C for 30 sec, 50-60°C (Tm+1, Tm+0.5 and Tm, where Tm is the annealing temperature of the primers) for 30 sec, 72°C for 40 sec. Then 35 cycles of 94°C for 30 sec, 50-60°C for 30 sec, 72°C for 40 sec and a final extension at 72°C for 7 min. The PCR products were separated by polyacrylamide gel electrophoresis and agarose gel electrophoresis and they were observed using white and UV light, respectively.
Capillary electrophoresis: The primers were selected that showed stable amplification and redesigned the forward primers. Three primers were used: A 5’ M13-tailed forward primer (primer A), a reverse primer (primer B) and a fluorescently labeled M13 primer (primer C). The reaction mix was as follows: 12.5 μL 2×PCR reaction mixture (New England Biolabs, Ipswich, MA, USA), 2 μL genomic DNA and primer A, 0.5 μL primer B, 5 μL primer C and 3 μL ddH2O in a final volume of 25 μL. The PCR conditions were the same as already discussed in this study.
Statistical analysis: The raw data was analyzed using the GeneMarker software (ver. 2.2.0) and the observed heterozygosity (Ho) and expected heterozygosity (He), the allelic Polymorphism Information Content (PIC) were calculated using GenAlEx (ver. 6.5)14 and CERVUS (ver. 3.0)15, respectively.
RESULTS
Characterization of EST-SSRs in K. paniculata Laxm.: In total, 2871 unigenes having putative EST-SSRs were discovered when we screened the transcriptome of K. paniculata Laxm. Among them, dinucleotide repeats, which constituted the largest group of repeated motifs, accounted for more than half of the total EST-SSR content (64.51%), followed by trinucleotide (34.38%), tetranucleotide (0.90%) and pentanucleotide (0.21%) repeats (Fig. 2). Dinucleotides and trinucleotides formed most of EST-SSRs, with the remaining repeats constituting only 1.11% of EST-SSRs.
The number of EST-SSR repeats ranged widely, from 5-12, in dinucleotide repeats in K. paniculata Laxm. and the frequencies of EST-SSR repeats decreased with increased motif length. Additionally, AG/TC accounted for the highest proportion (67.71%) of dinucleotide repeats, followed by AT/TA (21.00%), AC/TG (10.21%) and CG/GC (1.08%; Fig. 3a). Among trinucleotide repeats, AAG/TTC was the most abundant (25.43%), followed by ACC/TGG (16.21%), ATC/TAG (14.79%), AGG/TCC (11.14%) and AAC/TTG (10.13%; Fig. 3b).
Polymorphism analysis and fingerprint construction: Through the SSR amplification of samples from K. paniculata Laxm., 92 pairs of primers gave clear bands on PCR amplification; these accounted for 95.83% of the total primers tested, demonstrating the utility of our primers (Fig. S1, S2). Of the microsatellite loci that had clear amplification results, we selected 13 pairs for the construction of fingerprints with capillary electrophoresis (Fig. 4). In these 13 loci, the number of alleles ranged from 1-5 per locus and 10 pairs were polymorphic among individuals, whereas 3 pairs were monomorphic.
| Fig. 2: | Frequency distributions of various K. paniculata Laxm. EST-SSRs with differing numbers of repeats |
| Fig. 3(a-b): | No. of various (a) Dinucleotide and (b) Trinucleotide repeat motifs in K. paniculata Laxm. |
| Fig. 4(a-c): | Capillary electrophoresis results with primers, (a) 17 (b) 48 and (c) 69 for all samples (B1, B2, A1, A2, A3, A4 and A5) |
| Fig. S1: | Polyacrylamide gel electrophoresis results for all primers |
| Mixed DNA was used in the amplification | |
| Fig. S2: | Agarose gel electrophoresis results for some primers |
| The numbers indicate different primers and the letters are the numbers of the samples | |
The 10 pairs were used to analyze polymorphism information content and for fingerprint construction. The PIC value of the 10 pairs ranged from 0.124-0.726. The Ho and He levels were in the ranges of 0.143-1.000 and 0.133-0.765, respectively (Table 1). Based on the capillary electrophoresis results, the study established fingerprints for each sample (Table 2).
DISCUSSION
This is the first study about EST-SSR markers development in K. paniculata Laxm. and the information about EST-SSRs distribution in the species was obtained, which was similar to that reported for the rubber tree16. As an important biotechnology, the study about SSRs in the species could play an important role in future research and similar reports have been used in many species17, espically in agricultural18,19, commercial20,21 and horticultural22 crops. However, the lacking sequence limit the development of SSR markers in most species. The advent of next-generation sequencing technology provides resources for the development of potential SSR markers, especially non-sequenced species. For example, EST-SSRs was studied in Amentotaxus species to obtain amplifiable EST-SSR markers in the interspecies5 using transcriptome; EST-SSRs developed from transcriptome can do a great favour in variety identification as well as identification of parent-offspring relationships for some species like caprifig23 and tea24, Dutta et al.9 and Li et al.7 also made a research prograss about the distribution of EST-SSRs in pigeonpea and kenaf using next generation squencing, respectively.
Koelreuteria paniculata Laxm., a unique native Chinese tree, has great value in both landscaping and pharmacy1. However, in the current study, it is insufficient about the research in molecular markers for the species.
| Table 1: | Characteristics of 13 microsatellite loci developed in K. paniculata Laxm. |
| Na = No. of different alleles, Ho: Observed heterozygosity = no. of hets/N, He: Expected heterozygosity = 1-Sum pi2, PIC: Polymorphic information content | |
| Table 2: | Fingerprints of seven samples of K. paniculata Laxm. |
| 1: Presence of polymorphic alleles in the samples, 0: Absence of polymorphic alleles in the samples | |
The markers obtained in the study have been proved to be valuable and effective. Ninety two primers were proved to have effective amplification in the species and 10 of 13 primers are with polymorphism. These primers all can be used for future genomic studies in K. paniculata Laxm. For example, they can be used in genomic diversity analyses as has been reported in the white poplar11, Jatropha curcas L.25, the chickpea26 and wheat27. They will also be of value in genetic mapping, including linkage mapping and comparative mapping as has been described in wheat and rice28, red raspberry29, Populusnigra L.30 and pepper31. Moreover, the cross-species/genera transferability of EST-SSRs can also be used in research on phylogenetic relationships32,33. The EST-SSR markers, which have the advantages of SSR markers are also more widely applicable. For some non-model plants, especially those with large genomes, it is more effective to develop EST-SSRs from the transcriptome. Indeed, it may be cheaper and more reliable.
CONCLUSION AND FUTURE RECOMMENDATIONS
In this study, effective EST-SSR markers were developed and 10 pairs of primers were confirmed to be polymorphic. The construction of fingerprints for four special varieties in the species provided protection for variety promotion. The study about EST-SSRs in this tree has great potential utility in future studies about the species. Abundant and highly transferable EST-SSR markers would do a great favour for future breeding of K. paniculata Laxm..
SIGNIFICANCE STATEMENTS
This study developed new EST-SSR markers in Koelreuteria paniculata Laxm. at the first time and provides information about the distribution of EST-SSRs in the species. This study will help researchers to estimate genetic diversity and population structure within this species and can be benificial for the development of a breeding strategy for Koelreuteria paniculata Laxm.
ACKNOWLEDGMENTS
We gratefully acknowledge the infrastructure support of the National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University. This study was supported by "Graduate Training and Development Program of Beijing Municipal Commission of Education"(BLCXY201512).