Development and characterization of microsatellite markers for endangered species Stipa pennata (Poaceae) and their usefulness in intraspecific delimitation

Abstract

Stipa pennata (Poaceae), has become a rare and endangered species in Central Europe due habitat loss and fragmentation. This species is characterized by high morphological variability, which has resulted in the description of numerous intraspecific taxa. The aim of present work is to develop microsatellite markers useful in population genetics studies as well as in intraspecific taxonomy of S. pennata s.l. We developed ten microsatellite markers using Illumina high-throughput. Polymorphism at each marker was evaluated using 4–15 individuals from four morphotypes of S. pennata s.l. Seven markers showed polymorphism while three were monomorphic. The number of alleles per locus ranged from 7 to 12, and the observed and expected heterozygosity varied from 0.000 to 1.000 and 0.000 to 0.8670, respectively. Our results confirm that three of four studied morphotypes are genetically distinct. The microsatellite markers developed here will be useful for evaluating levels of genetic diversity and differentiation, to study gene flow, population dynamics and in future conservation studies as well as for intraspecific delimitation of morphologically similar taxa within S. pennata s.l.

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter

“…DNA from fresh, young leaves was extracted using E.Z.N.A.® Plant DNA DS Kit (Omega) following manufacturer’s instructions and DNA concentration was measured with Thermo 2000 Nanodrop. Ten pairs of SSR primers (Eurofins, Table 2 ) were selected based on the previous study on a closely related grass species ( Stipa pennata ) [ 65 ] as there is lack of any type of nucleotide sequence information available on the species of our interest. Polymerase Chain Reaction (PCR) was performed in 20 µL reaction mixture containing 50 ng genomic DNA, 0.5 µM of each primer (forward and reverse), 200 µM dNTP (Thermo Scientific™), 2.5 units DreamTaq DNA Polymerase (Thermo Scientific™), 1X PCR buffer (10X buffer with 20 mM MgCl 2 ) and 1 µL Bovine Serum Albumin (BSA, 100 mg·mL –1 stock).…”

De novo assembly, annotation, marker discovery, and genetic diversity of the Stipa breviflora Griseb. (Poaceae) response to grazing

“…Using transcriptome sequencing, Ren et al [31] developed 21 microsatellite markers of S. breviflora and tested the polymorphism in six related species. Klichowska et al [32] mined 10 microsatellite markers of S. pennata using Illumina high-throughput. Transcriptome analysis has also been used to uncover the adaptive mechanisms of plants subjected to grazing.…”

“…breviflora and tested the polymorphism in six related species. Klichowska et al [ 32 ] mined 10 microsatellite markers of S . pennata using Illumina high-throughput.…”

The first draft genome of feather grasses using SMRT sequencing and its implications in molecular studies of Stipa

“…SSR markers are widely distributed across the genome and they are commonly applied in establishing genetic structure in Stipa . Previously, polymorphic microsatellite primers were reported in populations of S. purpurea (11 113 , 15 114 and 29 115 loci), S. pennata (7 loci 116 ), S. breviflora (21 loci 117 ) and S. glareosa (9 loci 118 ). In the present study, we identified 77,614 perfect SSR markers (Supplementary File 3) and 58 imperfect repeat motifs presented only in a single copy (Supplementary Table S3 ).…”