Paper mulberry (Broussonetia papyrifera (L.) L'Hér. ex Vent.), belonging to the family Moraceae, is a multifunctional tree of cultural importance in Asia that has been used for centuries in the manufacture of high-quality paper. Broussonetia papyrifera is native to southern and central China, Vietnam, Thailand, and Taiwan (Matthews, 1996; Chang et al., 2015), where it is common in secondary forests growing at moderate elevations. Broussonetia papyrifera was intentionally transported into the Pacific region by prehistoric voyagers for making barkcloth, a nonwoven textile, and several centuries ago to Japan as a high-quality fiber source (Whistler, 2009).
The genetic diversity of B. papyrifera has been studied using intersimple sequence repeat (ISSR) markers (Ho and Chang, 2006; Liao et al., 2014; González-Lorca et al., 2015), sequence-related amplified polymorphism (SRAP) markers (Liu et al., 2009), hypervariable chloroplast DNA (cpDNA) sequences, and internal transcribed spacer (ITS) sequences of ribosomal DNA (Chang et al., 2015). These molecular markers have been useful for characterizing the genetic diversity and population structure of this species within its native range. However, studies using ITS sequences and ISSR data (Seelenfreund et al., 2011; Gonzalez-Lorca et al., 2015) and a sex marker (Peñailillo et al., 2016) in B. papyrifera in the introduced range in the Pacific region have not provided the resolution to understand its dispersal across this geographic area where it has been propagated asexually (Peñailillo et al., 2016). Therefore, the development of microsatellite markers is crucial to genotype the species' genetic diversity in Japan and the Pacific region. Here, we present the isolation and characterization of 20 microsatellite markers that will provide information on the fine structure of B. papyrifera populations both in its native and introduced range.
METHODS AND RESULTS
To isolate microsatellites, genomic DNA from 10 samples of B. papyrifera collected in Taiwan, Japan, Chile, and several islands in Oceania was used (Appendix 1). These samples are deposited at the University of Chile, and one sample (BQUCH0152) has a voucher (SGO162505) at the herbarium of the National Museum of Natural History, Chile. Total DNA was extracted from young silica gel–dried leaves following the cetyltrimethylammonium bromide (CTAB) extraction protocol (Lodhi et al., 1994) and modified as described in Moncada et al. (2013). Approximately 1 cm2 of tissue was homogenized, mixed with extraction buffer (50 mM EDTA, 100 mM Tris-HCl, 0.3 M NaCl, 2.0% [w/v] CTAB, 0.5% [v/v] 2-mercaptoethanol [pH 8.0]), and incubated at 65°C for 25 min, followed by organic extraction. Total DNA was precipitated and stored at −20°C until analysis. Purified DNA was quantified by spectrophotometric absorbance (Nanodrop, ThermoFisher Scientific, Wilmington, Delaware, USA) and Picogreen (Synergy H1, Winooski, Vermont, USA) and its integrity verified by 0.8% agarose gel electrophoresis.
Ecogenics GmbH (Zurich, Switzerland) constructed an enriched library using magnetic streptavidin beads and biotin-labeled CT and GT repeat oligonucleotides to develop size-selected fragments of genomic DNA for enrichment of microsatellite sequences. The microsatellite-enriched library was analyzed with GS FLX Titanium chemistry on a Roche 454 platform (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). A total of 32,947 reads was found with an average length of 321 bp. Microsatellite simple sequence repeats (SSRs) with tri- or tetranucleotide motifs, repeated at least six times, and dinucleotides of at least 10 repeat units, were found in 10,024 reads. Primers were designed for 190 of these reads, and 36 were tested for the presence of polymorphisms on 10 samples from different geographic areas.
Polymorphisms in these 36 loci were assessed using the procedure described by Schuelke (2000). A universal 18-bp fluorophore-labeled M13 tail (5′-TGTAAAACGACGGCCAGT-3′) was incorporated into the PCR products during the first PCR cycles. In subsequent cycles, these products function as templates for the fluorophore-labeled universal M13 primer to produce fluorescent PCR products. PCR reactions were performed in a reaction volume of 10 µL containing 2–10 ng of genomic DNA, 1× buffer with 15 mM MgCl2, 200 µM dNTP mix, 0.04 µM forward primer, 0.16 µM of the reverse and the M13 primer, and 0.05 µL of HotStarTaq DNA polymerase (QIAGEN, Hilden, Germany). PCR amplifications were conducted under the following conditions: an initial denaturation of 15 min at 95°C; 30 cycles at 95°C for 30 s, at an annealing temperature specific for each primer for 45 s, and at 72°C for 45 s; followed by eight cycles at 95°C for 30 s, at 53–55°C for 45 s, and at 72°C for 45 s; and a final extension step at 72°C for 30 min.
Characteristics of 20 polymorphic microsatellite loci developed in Broussonetia papyrifera.
The amplified products were analyzed on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA) with GeneScan 500 ROX Size Standard (Applied Biosystems). Genotypes were determined using GeneMapper version 3.2 (Applied Biosystems) with default settings. Due to the M13 tail attached to each forward primer, 18 bp were subtracted from the experimentally determined amplicons to obtain the length of actual alleles.
Twenty primer pairs were successfully amplified, with the expected sizes and banding patterns displaying clear polymorphisms in B. papyrifera (Table 1). Polymorphisms were evaluated in samples of B. papyrifera from its native range (three Asian populations, n = 70). Table 2 shows the number of alleles per marker, observed and expected heterozygosity (Ho and He), polymorphism information content (PIC), coefficient of inbreeding (FIS), null allele frequency (r), and Hardy–Weinberg equilibrium (HWE) of the analyzed samples from three populations. Ho, He, and FIS were estimated using Arlequin 126.96.36.199 (Excoffier and Lischer, 2010). CERVUS 3.0.7 was used to calculate PIC (Kalinowski et al., 2007). Null allele frequency was calculated using MICRO-CHECKER (van Oosterhout et al., 2004) and HWE with GenAlEx 6.502 (Peakall and Smouse, 2006, 2012). The total number of alleles ranged from four to 35 with a mean of 19.2 (Table 1). Ho and He ranged from 0.038 to 0.846 and from 0.191 to 0.935 with averages of 0.495 and 0.722, respectively. PIC values ranged from 0.169 to 0.911 with averages of 0.678 and FIS values ranged from −0.024 to 0.895 with an average of 0.329. Null allele frequency values ranged from −0.037 to 0.396 (Table 2). No significant deviation of HWE in terms of heterozygosity deficiency was detected for four markers (Bropap02214, Bropap02359, Bropap23758, Bropap26773) in the three populations (Table 2). Across the three populations (in Guangdong and Yunnan in southern China and Taiwan), 384 alleles were scored. Samples were from nonadjunct individuals because B. papyrifera is a widely distributed and common species in East Asia.
Genetic properties of the 20 newly developed polymorphic microsatellites of Broussonetia papyrifera.a
Transferability of the 20 microsatellite markers developed in Broussonetia papyrifera across three Moraceae and one Rosaceae species.
The reported genetic diversity for B. papyrifera represents only part of the diversity found in Asia. The transferability of these markers was tested in three additional Moraceae and one Rosaceae species (Table 3, Appendix 2). Some of the developed markers exhibited limited interspecific transferability. Six markers showed transferability to Ficus carica L. Eight markers exhibited no transferability to any of the tested species (Table 3).
We identified and characterized 20 highly polymorphic and informative microsatellite markers for B. papyrifera, presenting an average of 19.2 alleles per marker. Some of the markers show limited transferability to other Moraceae and one Rosaceae species. The described genetic diversity represents a subset of the genetic diversity in the native range. These microsatellite markers may be able to serve as useful tools to analyze genetic diversity, population genetic structure, and gene flow of B. papyrifera in its native range, to trace its worldwide dispersal history, and to help in germplasm conservation.
The study received financial support from the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (grants 1080061 and 1120175 to A.S.) from Chile, and from the Biodiversity Research Center, Academia Sinica, Taiwan (to K.F.C.). The authors thank Dr. K.-Y. Ho (National Chiayi University, Taiwan) for providing some of the Taiwanese samples, P. Matthews (National Museum of Ethnology, Osaka, Japan) for providing samples of B. papyrifera from Japan, and M. Floor for valuable assistance.