Ranunculus bungei Steud. (section Batrachium DC., Ranunculaceae) is a perennial submerged plant that can proliferate vegetatively through rhizomes or sexually via selfing or outcrossing (Cook, 1966). Ranunculus bungei is widely distributed in heterogeneous environments within the temperate and alpine regions of China and is significant for studies of the adaptation to aquatic habitats in angiosperms (Chen et al., 2015). In recent years, investigations have been carried out to examine genetic variation and population structure in R. bungei with intersimple sequence repeat (ISSR) markers or chloroplast noncoding spacers (Wang et al., 2010; Chen et al., 2014), but these markers are less powerful in studies on reproductive system, hybridization, patterns of gene flow, and fine-scale population structure. The development of suitable markers can provide a better understanding of the evolutionary progress and the underlying ecological factors of R. bungei and its related species.

Microsatellite or simple sequence repeat (SSR) markers are molecular markers with many desirable genetic attributes (e.g., codominant inheritance and hypervariability), which have been used to reveal genetic patterns in a wide variety of species (Kalia et al., 2011). For clonal plants, estimation of genetic variation is often biased with markers of low discriminatory ability (Arnaud-Haond et al., 2005); therefore, genetic studies on aquatic macrophytes, which are characterized by limited sexual proliferation (Barrett et al., 1993), should be assessed using appropriate polymorphic markers. Although a large number of SSR loci for Ranunculus L. species have been identified (e.g., Noel et al., 2005; Matter et al., 2012), we found that cross-species amplification was rarely successful in R. bungei based on preliminary experiments. Therefore, we developed 22 novel EST-SSR markers from R. bungei for use in investigations of population and landscape genetics of this widely distributed submerged species.

METHODS AND RESULTS

A mixture of tissues from roots, stems, and leaves was used for the transcriptome sequencing of R. bungei, conducted by Chen et al. (2015) using the Illumina HiSeq 2000 sequencer (Illumina, San Diego, California, USA). A total of 5,312,841 clean reads of R. bungei deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (accession no. SRR1822529; Chen et al., 2015) were used for de novo transcriptome assembly using Trinity version 2.1.0 with default parameters (Grabherr et al., 2011). The longest sequence was chosen for transcripts with several isoforms, as identified with a perl script (available at  https://github.com/jinweiwu/perl/blob/master/extract). The MIcroSAtellite identification tool (MISA) Perl script (Thiel et al., 2003) was then used to screen for microsatellite motifs from total unigenes, and the minimum number of each type of repeat was set to six. MISA recovered a total of 9903 SSR motifs for R. bungei, and 50 unigenes were randomly chosen for the EST-SSR development.

PCR primer design for the targeted unigenes was performed with Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, California, USA). An initial evaluation of the primers was facilitated in 20 individuals randomly selected from four Chinese populations of R. bungei (Appendix 1), and 22 primer pairs showing unique products ranging from 100–500 bp were individually labeled with the fluorescent dyes 6-FAM or HEX (Table 1). Characterization of the EST-SSR loci was estimated in the four populations of R. bungei, with 22, 20, 15, and 16 individuals, respectively (Appendix 1). Genomic DNA was extracted from the freeze-dried leaves of R. bungei individuals using the DNAsecure Plant Kit (Tiangen Biotech, Beijing, China). PCR amplifications were performed in 20-µL reaction mixtures containing 1.5 µL of genomic DNA (∼30 ng/µL), 0.5 µL of each primer (10 µM), and 10 µL 2× Master PCR Mix (Tiangen Biotech). Microsatellites were amplified under the following PCR conditions: a 5-min initial denaturation step at 95°C; followed by 30–35 cycles of 30 s at 95°C, 30 s at 50–58°C (Table 1), and 1 min at 72°C; and a final extension at 72°C for 7 min. PCR products differed in fluorescent label or length (>80 bp) and were multiplexed and analyzed on the ABI 3730XL sequencer (Applied Biosystems, Foster City, California, USA) with GeneScan 500 LIZ Size Standard (Applied Biosystems). Microsatellite genotyping was performed using GeneMarker version 1.5 software (SoftGenetics, State College, Pennsylvania, USA). The number of alleles, observed and expected heterozygosities, and deviations from Hardy–Weinberg equilibrium (HWE) at each locus were estimated using GenAlEx 6.5 (Peakall and Smouse, 2012). Linkage disequilibrium of locus pairs was tested using Arlequin version 3.5.1.3 (Excoffier et al., 2005).

Table 1.

Characteristics of 22 EST-SSR markers developed in Ranunculus bungei.

Cross-species amplification was conducted in two other species of Ranunculus section Batrachium (R. aquatilis L. var. eradicatus Laest. [n = 11] and R. trichophyllus Chaix ex Vill. [n = 14]), as well as six riparian and aquatic species of Ranunculaceae (R. cheirophyllus Hayata [n = 5], R. natans C. A. Mey. [n = 8], Halerpestes tricuspis (Maxim.) Hand.-Mazz. [n = 12], H. ruthenica (Jacq.) Ovcz. [n = 6], Caltha palustris L. [n = 3], and C. natans Pall. [n = 5]) (Appendix 1).

The characteristics of 22 EST-SSR loci are presented in Table 1. Fourteen loci were polymorphic, two of which were fixed for different alleles in multiple populations (Table 2). The loci BatrB6, BatrB9, and BatrB12 showed the highest number of alleles (five), and eight loci were monomorphic among all individuals (Table 2). The expected and observed heterozygosity ranged from 0.0 to 0.5 and 0.0 to 1.0 per locus, and all polymorphic loci showed significant deviation from HWE (Table 2). Significant linkage disequilibrium (P < 0.05) was observed among seven locus pairs in four R. bungei populations (BatrB5 and BatrB6, BatrB6 and BatrB10, BatrB6 and BatrB12, BatrB9 and BatrB15, BatrB10 and BatrB13, BatrB10 and BatrB15, and BatrB13 and BatrB15). The deviation from HWE and significant linkage disequilibrium could be explained by the small population size, inbreeding, and clonal reproduction in R. bungei. All of the loci were amplified successfully in two Ranunculus species from section Batrachium, and four loci could be amplified successfully in all eight species (Table 3). The allele size ranges were similar among Ranunculus section Batrachium species, but greatly differed among the taxa from different genera in loci BatrB1, BatrB9, BatrB11–13, BatrB17, and BatrB21.

Table 2.

Results of initial primer screening in four populations of Ranunculus bungei.^a

Table 3.

Cross-amplification of 22 EST-SSR markers developed in Ranunculus bungei across eight other species of Ranunculaceae. The number of alleles in populations of each species is presented for the loci that could be successfully amplified.^a

CONCLUSIONS

Fourteen polymorphic and eight monomorphic microsatellite loci were developed in R. bungei. The polymorphism observed for the SSRs in R. bungei is moderate when compared with other aquatic plants (Nies and Reusch, 2004; Wu et al., 2013). Cross-species amplification also indicates that these markers may be widely used in related Ranunculaceae species. We conclude that the EST-SSRs described here will facilitate ecological and evolutionary studies of R. bungei as well as related species.