Development of EST‐derived microsatellite markers in the aquatic macrophyte Ranunculus bungei (Ranunculaceae)

Abstract

Premise of the study:Microsatellite or simple sequence repeat (SSR) markers were developed to investigate the influence of ecological factors on gene flow and spatial genetic structuring of the submerged plant Ranunculus bungei (Ranunculaceae), which is regarded as an important species for understanding how plants adapt to an aquatic environment.Methods and Results:Twenty-two microsatellite loci were identified from an expressed sequence tag (EST) library. The number of alleles per locus ranged from one to five, and the expected heterozygosity varied from 0.0 to 0.5 in four Chinese populations of R. bungei. Fourteen loci were polymorphic and significantly deviated from Hardy–Weinberg equilibrium. All of the loci were found to be amplifiable in two other species of Ranunculus section Batrachium, and cross-amplification in six riparian and aquatic species of Ranunculaceae was also partially successful.Conclusions:These novel EST-SSR markers will be useful for ecological and evolutionary studies of R. bungei as well as related species.

Influence of environmental factors on the genetic variation of the aquatic macrophyte Ranunculus subrigidus on the Qinghai-Tibetan Plateau

“…Genomic DNA was extracted using the Novel DNA Plant Kit (Kangwei Biotech, Beijing, China). Thirteen self-developed EST-SSRs and four feasible SSRs isolated in Ranunculus species were used in R. subrigidus [39]. All PCR amplifications were performed in 20 μL reaction mixtures containing 1.5 μL genomic DNA (~ 30 ng/μL), 0.5 μL of each primer (10 μM), and 10 μL 1x master PCR Mix (Tiangen Biotech, Beijing, China).…”

“…All PCR amplifications were performed in 20 μL reaction mixtures containing 1.5 μL genomic DNA (~ 30 ng/μL), 0.5 μL of each primer (10 μM), and 10 μL 1x master PCR Mix (Tiangen Biotech, Beijing, China). The PCR conditions comprised an 5 min initial denaturation step at 95 °C; followed by 35 cycles of 30 s at 95 °C, 30 s at suggested annealing temperature according to Wu et al [39], 1 min at 72 °C; and a final extension at 72 °C for 7 min. PCR products were analyzed on the ABI 3730XL analyzer (Applied Biosystems, Foster City, CA, USA).…”

“…Cook and Ranunculus trichophyllus Chaix) based on our field identifications and herbarium studies. The species differ in flower characteristics, life cycle and genetics [35, 39]. In present study, our aim was estimating the extent and spatial pattern of genetic variation of R. subrigidus .…”

Extensive Sympatry and Frequent Hybridization of Ecologically Divergent Aquatic Plants on the Qinghai-Tibetan Plateau

“…Total genomic DNA was extracted using the DNAsecure Plant Kit (Tiangen Biotech, Beijing, China). Microsatellite markers (7 loci for Stuckenia, Nies and Reusch, 2004 ; Wu et al, 2020 ; 17 loci for Ranunculus, Wu et al, 2017 ; Wu et al, 2019 ) and one cpDNA fragment ( rpl32-trnL ) were chosen to identify kinships. The primers “rpL32-F” and “trnL (UAG) ” ( Shaw et al, 2007 ) were used to amplify and sequence the region.…”