Taxillus nigrans (Hance) Danser (Loranthaceae) is a mistletoe species that is found attached to many canopy tree species in low mountains, hills, and river basins in subtropical areas of southwestern China at elevations of 300–1300 m. Flowering can occur throughout the year, and the fruiting period is mainly in November. The entire plant of this species can be used as raw material for Chinese traditional medicine (Jiang, 1998). However, because the range of the species has undergone rapid expansion mediated by birds in the urban area of Chengdu (Sichuan Province, China), it forms large groves on garden tree species and is sometimes harmful to its host trees, so that individuals of this species are often removed by gardeners. To date, apart from some basic taxonomic data on the species (Gong et al., 2004) and genome studies on other species of Taxillus Tiegh. (Rist et al., 2011; Wei et al., 2017), nearly all published research has focused on aspects relating to its medicinal value, for example, the extraction and identification of medicinal components and the optimization of extraction methods (Li et al., 2006, 2009; Zhang et al., 2016; Zhao et al., 2016). There is little information on the genetic diversity and population structure of the species. We are also interested in developing genetic approaches for identification of individuals and assignment testing, which will help in understanding how this species expands its distribution and jumps from host to host in urban areas as well as in the field.

Simple sequence repeat (SSR) markers, also known as microsatellites or short tandem repeats, are highly polymorphic and are therefore useful as molecular markers in population genetic studies (Zhang et al., 2012; Jiang et al., 2015). Transcriptome sequencing has proven to be a powerful and cost-effective tool that has greatly accelerated the process of discovering molecular markers, including single nucleotide polymorphisms (SNPs) and SSRs (Ashrafi et al., 2012; Qi et al., 2016). In this study, we sequenced and assembled the transcriptome of T. nigrans and developed a set of expressed sequence tag (EST)–SSR markers for population genetic studies of T. nigrans. We also tested the transferability of these markers in herbarium samples of T. delavayi (Tiegh.) Danser and five individuals of Scurrula parasitica L. (collected from the field), another Loranthaceae parasite that co-occurs with T. nigrans.

METHODS AND RESULTS

Approximately 10 µg (400 ng/µL) of total RNA was extracted from fresh leaf material of one individual of T. nigrans using TRIzol Reagent (Invitrogen, Carlsbad, California, USA). Subsequently, mRNA was isolated using magnetic oligo (dT) beads (Illumina, San Diego, California, USA); it was then fragmented into short fragments using the Ambion RNA Fragmentation Kit (Ambion, Austin, Texas, USA) according to the manufacturer's protocols. First-strand cDNA synthesis was performed using reverse transcriptase (Invitrogen) with random primers, and second-strand cDNA was synthesized by RNase H and DNA Polymerase I (Invitrogen). Finally, the transcriptome was sequenced on an Illumina HiSeq 2000 system at Novogene (Beijing, China). Prior to the assembly, a stringent filtering process of raw sequencing reads was conducted. The number of low-quality (Q ≤ 3) bases in a single read was restricted to less than 50%, and paired reads were discarded if the number of unknown nucleotide bases in either of the paired reads exceeded 3% following the sequencing company's protocol (Novogene). After removing the adapter sequences and ambiguous reads, the clean reads obtained were de novo assembled using Trinity (release 2013-02-25; Grabherr et al., 2011) with default settings. The final assembly was composed of 299,147 unigenes and had an N50 size of 1056 bp. Raw transcriptome read data were deposited in the National Center for Biotechnology Information (NCBI) Short Read Archive (accession no. SRP105083).

Table 1.

Characteristics of 19 polymorphic microsatellite loci developed for Taxillus nigrans.

Table 2.

Genetic properties of 17 newly developed polymorphic microsatellite loci in three populations of Taxillus nigrans. Loci exhibiting null alleles are not included.^a

SSRs were detected using the Perl script MISA (Thiel et al., 2003) with a motif size of one to six nucleotides and thresholds of eight, four, four, three, three, and three repeat units for mono-, di-, tri-, tetra-, penta-, and hexanucleotide SSRs, respectively. We selected 83,954 microsatellite loci and used the primer design software package Primer3 version 2.3.6 (Untergasser et al., 2012) to design primer sets. Following random browsing across the output files of these primer sets, 40 markers were selected based on length (19–20 bp), GC content (40–65%) of the primers, and annealing temperatures (59–61°C) of the primer sets. Nineteen of the 40 tested markers were selected based on PCR success rate and degree of polymorphism (difference in band length), and these were used to genotype individual Taxillus plants (Table 1).

Table 3.

Fragment sizes detected in cross-amplification tests of the 19 newly developed microsatellite markers in Taxillus delavayi and Scurrula parasitica.^a

Genomic DNA was extracted from the silica-dried leaves of 160 individuals from three populations of T. nigrans, two individuals of T. delavayi, and five individuals of S. parasitica (Appendix 1) using the cetyltrimethylammonium bromide (CTAB) method of Doyle and Doyle (1987). PCR reactions were performed in 25-µL volumes containing 12.5 µL 2× PCR buffer, 300.0 µM each dNTP, 0.3 µM each primer, 1.25 unit Taq DNA polymerase (Vazyme Biotech, Nanjing, China), and ca. 50 ng of genomic DNA. The cycling conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 45 s, 55°C for 30 s, and 72°C for 45 s; the reactions were completed by a final elongation step at 72°C for 10 min. The PCR products were checked on 1% agarose gels to confirm PCR success and then sent to TsingKe (Chengdu, China) for microsatellite genotyping. Primer pairs were synthesized with the forward primer of each pair 5′ end-labeled with either 6-FAM, TAMRA, or HEX (Applied Biosystems, Foster City, California, USA), and amplicons were analyzed on an ABI PRISM 3100 genetic analyzer. The microsatellite genotype at each locus for each individual was determined using GeneMarker (SoftGenetics, State College, Pennsylvania, USA). Allele sizes at each locus were then scored and checked for possible genotyping errors, such as stuttering, large allele dropouts, or null alleles, using CERVUS (Dakin and Avise, 2004). In total, null alleles (null allele frequency [r] > 0.4) were detected at two loci (Table 1). These loci were eliminated, and the remaining 17 microsatellite loci were subjected to further analyses (Table 2).

These 17 microsatellite loci were highly polymorphic, with two to 21 alleles per locus. We used GenAlEx version 6 (Peakall and Smouse, 2006) to calculate the number of alleles and the observed and expected heterozygosity at each locus (Table 2). When using GIMLET version 1.3.3 (Valière, 2002), a minimum of two loci and six loci are needed to estimate, respectively, the unbiased probability that a genotype is shared by two individuals (P_ID) in a population, and the probability that a genotype is shared by two siblings (P_ID(sib)).

In the cross-species transferability test, eight of the 19 loci were successfully genotyped in two individuals of T. delavayi taken from herbarium specimens (Table 3). In contrast, all polymorphic loci were successfully amplified in S. parasitica (Table 3). The difference in success between T. delavayi and S. parasitica may have been due to a higher proportion of degraded DNA from T. delavayi herbarium specimens.

CONCLUSIONS

We developed and amplified a set of polymorphic EST-SSR markers for T. nigrans. These new SSR markers will serve as a basis for studies assessing the genetic diversity and population structure of T. nigrans. Our research will be useful for conservation genetic, ecological, and evolutionary studies of the genus Taxillus, a group of plant species of importance in Chinese traditional medicine. We plan to use these markers to explain the rapid demographic expansion and host specificity of T. nigrans in urban areas in southwestern China.