Abstract
Premise of the Study Aconitum reclinatum is the only representative species of Aconitum subg. Lycoctonum in North America, with restricted ranges and endangered populations. Polymorphic microsatellite markers were developed for A. reclinatum for further investigation of genetic diversity and population structure.Methods and ResultsUsing Illumina HiSeq technology, we sequenced a genomic library for identification of simple sequence repeat markers. A total of 12 polymorphic primer pairs were developed and tested on 66 individuals from four populations in North America. The number of alleles ranged from one to seven per locus with an average of 3.48. Levels of observed and expected heterozygosity varied from 0 to 1.000 and 0 to 0.736, respectively, at population level. Three primer pairs were successfully amplified in three of four closely related species.ConclusionsThe microsatellites isolated in this study will be useful in further research on the genetic diversity and conservation genetics of A. reclinatum populations in North America.
Highlights
PREMISE OF THE STUDY: Aconitum reclinatum is the only representative species of Aconitum subg
The microsatellites isolated in this study will be useful in further research on the genetic diversity and conservation genetics of A. reclinatum populations in North America
The largest existing populations of A. reclinatum are known to be located in North Carolina in the eastern United States (Hardin, 1964; Brink, 1982)
Summary
Tender leaves were collected from A. reclinatum and dried instantly in silica gel. A total of 66 individuals and four populations were sampled. Total genomic DNA was extracted from the dried leaves of A. reclinatum using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). We used one individual of A. reclinatum collected in Three Top Mountain, North Carolina, USA, to construct a genomic DNA. Using MISA (Thiel et al, 2003), a total of 197,407 simple sequence repeat (SSR) loci were detected from the 2,464,714 scaffolds. To screen the SSR primers, we conducted PCR amplification using one individual from each of the four populations. The PCR products of 17 primer pairs produced the expected size (Table 1). Sequencing Analysis 5.2 (Applied Biosystems, Carlsbad, California, USA) was used to measure the size of the PCR products in the ABI 3730 DNA sequencer (Applied Biosystems). Among a total of 17 primer pairs, five were monomorphic and 12 produced polymorphic sites. The number of alleles per locus (A), observed and expected heterozygosity (Ho and He), polymorphism information content (PIC), coefficient of inbreeding (FIS), null allele frequency (r), and Hardy–Weinberg equilibrium
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