Development and characterization of genomic SSR markers for Tamarix chinensis (Tamaricaceae)

Premise of the Study We developed a set of microsatellite markers to study the population genetic structure, mating system, and interspecific hybridization of Tamarix chinensis (Tamaricaceae), an alkali‐ and salt‐tolerant shrub endemic to China, Korea, and Japan. Methods and Results Using Illumina sequencing, we developed 10 polymorphic and 11 monomorphic microsatellite primers. High levels of polymorphism were detected in four T. chinensis populations. Allele numbers ranged from two to 11, and the levels of observed and expected heterozygosity ranged from 0.182 to 0.846 and from 0.165 to 0.794, respectively. The polymorphism information content values ranged from 0.201 to 0.803. Cross‐species amplification showed two to 15 alleles per locus in 24 individuals from one natural population of the congener T. ramosissima, and the levels of observed and expected heterozygosity ranged from 0.042 to 0.864 and from 0.041 to 0.892, respectively. Conclusions These markers should be useful for exploring the population genetic structure, mating system, and gene flow of T. chinensis.

HiSeq 2500 high-throughput sequencing system (Illumina, San Diego, California, USA) to generate 125-bp paired-end reads by a commercial company (Novogene Co. Ltd., Beijing, China). Raw data were cleaned up by trimming the adapters and low-quality reads with a custom script by the company who did the sequencing, and by filtering reads with read depth of <10 and >400 using CD-HIT-EST (Li and Godzik, 2006) to avoid false positives.
Assembly of paired reads was performed using Velvet version 1.1.06 (Zerbino and Birney, 2008). Contigs with a length of less than 125 bp were deleted. Raw sequences were deposited to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA accession PRJNA492209). A total of 818,142 contigs were generated with an average length of 323 bp and an N50 length of 474 bp. The MISA perl script (Thiel et al., 2003) was used to search for SSRs with mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs with a minimum repeat number of 14, eight, six, five, four, and four, respectively. Compound microsatellites were defined as having two or more motifs separated by an interval of ≤100 bp. A total of 31,140 SSRs were identified using the MISA perl script for 28,454 contigs. There were 2567 contigs that contained more than one SSR, and there were 2027 compound SSRs. Di-and trinucleotide motifs were the most abundant, comprising 41.53% and 41.24%, respectively. SSR primers were designed with Primer3 version 1.1.4 (Rozen and Skaletsky, 1999) with the following qualifications: primer length range from 18 to 22 bp and annealing temperature 55-60°C. Twenty-four primers were synthesized by a commercial company (GenScript, Nanjing, China).
In total, 58 individuals of T. chinensis from four natural populations and 24 individuals of T. ramosissima from one population were sampled (Appendix 1). We collected no more than 20 individuals for each T. chinensis population because the sampled T. chinensis individuals were scattered along riverbanks, often 50 m apart, and some individuals were damaged. Genomic DNA was extracted with the DNeasy kit (QIAGEN) and tested on agarose gel.
As an initial primer test, two individuals were randomly chosen from each of the four studied T. chinensis populations (Appendix 1) to run PCR using the 24 primer pairs. The PCR had a 10-μL volume and contained: 1 μL of 1× buffer (Tiangen, Beijing, China), 1 μL of MgCl 2 (20 mM), 1 μL of dNTPs (2.5 mM each), 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.2 μL of Taq polymerase (5 U/μL) (TaKaRa Bio Inc., Kyoto, Japan), 0.5 μL of DNA (50 ng/μL), and 5.9 μL of ddH 2 O. The PCR profile was as follows: 4 min denaturation at 94°C; followed by 30 cycles of 30 s denaturation at 94°C, 30 s annealing step at 55°C, and 30 s elongation at 72°C; and a final extension step at 72°C for 3 min. PCR products were tested by polyacrylamide gel electrophoresis (PAGE) as an initial polymorphism test. The verified polymorphic primers (altogether 10 polymorphic SSRs, Table 1) were synthesized by GenScript (Nanjing, China) with forward primers labeled with 6-FAM. The fluorescent-labeled primers were used to run PCR in 58 T. chinensis individuals and 24 T. ramosissima individuals using the reaction volume and PCR profile as described above. PCR products combined with deionized formamide, using GeneScan 500 LIZ (Thermo Fisher Scientific) as a size standard, were run on a 3730 Genetic Analyzer (Thermo Fisher Scientific), and alleles were assigned to bins in GeneMarker version 2.6.7 (SoftGenetics, State College, Pennsylvania, USA). The levels of observed (H o ) and expected heterozygosity (H e ) and number of alleles were calculated in GenAlEx 6.502 (Peakall and Smouse, 2005). The GENEPOP allele .txt file was exported from GenAlEx for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium analysis in GENEPOP (version 1.2) (Raymond and Rousset, 1995). The polymorphism information content (PIC) value of each locus was calculated in CERVUS version 3.0.3 (Kalinowski et al., 2007), according to the following formula: PIC = 1 − ∑ n i=1 f 2 i , in which f i is the frequency of the ith allele and n is the allele number. Significance levels were tested with sequential Bonferroni corrections (Rice, 1989).
In total, 21 (GenBank accession no.: MG856343-MG856363) out of 24 primers amplified the target sequences. Of these, 11 were monomorphic and 10 were polymorphic in the PAGE test (Table 1). The 10 polymorphic primers showed high polymorphism in both species (Table 2). For T. chinensis, the allele number per locus per population varied from two to 11, with the highest average allele number (6.2) for the Kenli (KL) population, followed by Fangshan (FS) (5. The PIC values ranged from 0.040 to 0.882. HWE testing revealed no loci (for KL), one locus (for LJ and FS), and two loci (for HK) for T. chinensis; for the single population of T. ramosissima one locus deviated from equilibrium significantly (P < 0.01; Table 2). No significant linkage disequilibrium was detected between markers after Bonferroni correction for both species.

CONCLUSIONS
This is the first report of genomic microsatellites for T. chinensis. The 10 polymorphic markers showed comparatively high genetic variation, transferability to congeneric species, little or no deviation from HWE, and were in linkage equilibrium. These properties make them especially useful for genetic analysis of population genetic structure, mating system, and gene flow in T. chinensis and its congeners.