Transcriptome-wide mining, characterization, and development of microsatellite markers in Lychnis kiusiana (Caryophyllaceae).

BACKGROUND: Lychnis kiusiana Makino is an endangered perennial herb native to wetland areas in Korea and Japan. Despite its conservational and evolutionary significance, population genetic resources are lacking for this species. Next-generation sequencing has been accepted as a rapid and cost-effective solution for the identification of microsatellite markers in nonmodel plants. RESULTS: Using Illumina HiSeq 2000 sequencing technology, we assembled 67,498,600 reads into 91,900 contigs and identified 11,403 microsatellite repeat motifs in 9563 contigs. A total of 4510 microsatellite-containing transcripts had Gene Ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 124 pathways with significant scores. Many microsatellites in the L. kiusiana leaf transcriptome were linked to genes involved in the plant response to light intensity, salt stress, temperature stimulus, and nutrient and water deprivation. A total of 12,486 single-nucleotide polymorphisms (SNPs) were identified on transcripts harboring microsatellites. The analysis of nucleotide substitution rates for 2389 unigenes indicated that 39 genes were under strong positive selection. The primers of 6911 microsatellites were designed, and 40 of 50 selected primer pairs were consistently and successfully amplified from 51 individuals. Twenty-five of these were polymorphic, and the average number of alleles per SSR locus was 6.96, with a range from 2 to 15. The observed and expected heterozygosities ranged from 0.137 to 0.902 and 0.131 to 0.827, respectively, and locus-specific FIS estimates ranged from - 0.116 to 0.290. Eleven of the 25 primer pairs were successfully amplified in three additional species of Lychnis: 56% in L. wilfordii, 64% in L. cognata and 80% in L. fulgens. CONCLUSIONS: The transcriptomic SSR markers of Lychnis kiusiana provide a valuable resource for understanding the population genetics, evolutionary history, and effective conservation management of this species. Furthermore, the identified microsatellite loci linked to the annotated genes should be useful for developing functional markers of L. kiusiana. The developed markers represent a potentially valuable source of transcriptomic SSR markers for population genetic analyses with moderate levels of cross-taxon portability.

Phylogeny and chromosomal variations in East Asian Carex, Siderostictae group (Cyperaceae), based on DNA sequences and cytological data.

Carex (Cyperaceae) is one of the largest genera of the flowering plants, and comprises more than 2,000 species. In Carex, section Siderostictae with broader leaves distributed in East Asia is thought to be an ancestral group. We aimed to clarify the phylogenetic relationships and chromosomal variations within the section Siderostictae, and to examine the relationship of broad-leaved species of the sections Hemiscaposae and Surculosae from East Asia, inferred from DNA sequences and cytological data. Our results indicate that a monophyletic Siderostictae clade, including the sections Hemiscaposae, Siderostictae and Surculosae, as the earliest diverging group in the tribe Cariceae. Low chromosome numbers, 2n = 12 or 24, with large sizes were observed in these three sections. Our results suggest that the genus Carex might have originated or relictly restricted in the East Asia. Geographical distributions of diploid species are restricted in narrower areas, while those of tetraploid species are wider in East Asia. It is concluded that chromosomal variations in Siderostictae clade may have been caused by polyploidization and that tetraploid species may have been able to exploit their habitats by polyploidization.

Phylogeography of the Japanese common sedge, Carex conica complex (Cyperaceae), based on chloroplast DNA sequence data and chromosomal variation.

PREMISE OF THE STUDY: Genetic and chromosomal variations in plants are often reflected in the geographical distribution patterns. Therefore, identifying such phylogeographical patterns on population is important for understanding the process of plant diversification and speciation, and analyzing both molecular and cytological aspects is necessary. • METHODS: We investigated the phylogeographic pattern and genetic diversity of the widespread Japanese sedge, Carex conica complex, based on chloroplast DNA haplotypes and chromosomal variations. • KEY RESULTS: Sixteen distinct haplotypes were detected from 258 individuals in the C. conica complex inferred from the cpDNA sequences of intergenic spacer (IGS) between atpB and rbcL, IGS between trnT and trnL, trnL intron, and IGS between trnL and trnF. Most haplotypes showed distinct geographical structures. Phylogenetic analyses revealed two major clades, clades I and II, among 16 haplotypes of the complex. The haplotypes of clade I were mainly found in eastern Japan, while most haplotypes of clade II were found in western Japan. Four intraspecific aneuploids of 2n = 32, 34, 36, and 38, with forming normal bivalents in meiotic divisions, were also found in the complex. The geographical distribution of these intraspecific aneuploids corresponded well with those of the haplotypes. • CONCLUSIONS: Our results suggest that the genetic diversity and chromosomal variations in the C. conica complex may have originated from contractions and expansions of geographical ranges affected by Quaternary climatic oscillations.

Nepalensinols D-G, new resveratrol oligomers from Kobresia nepalensis (Cyperaceae) as potent inhibitors of DNA topoisomerase II.

Four new resveratrol oligomers, nepalensinols D-G, were isolated from the stem of Kobresia nepalensis (Cyperaceae). The structures were determined by detailed NMR spectral analysis. The compounds were assessed for their inhibitory activity against human topoisomerase II, a potential target of anti-tumor agents. These stilbenoids showed potent inhibitory activity against human topoisomerase II with IC50 values of 5-15 microM.

Stilbenoids of Kobresia nepalensis (Cyperaceae) exhibiting DNA topoisomerase II inhibition.

Resveratrol oligomers, nepalensinol A, B and C, were isolated from the stem of Kobresia nepalensis (Cyperaceae). The structures were established on the basis of chemical properties and spectroscopic evidence including 2D NMR spectroscopic analysis. Nepalensinol A, B and C showed a potent inhibitory effect on topoisomerase II -- stronger than etoposide (VP-16), a topoisomerase II inhibitor used as an anti-cancer drug. Nepalensinol B, in particular, exhibited the most potent activity with an IC(50) of 0.02 microg/ml.

Molecular phylogeny of Japanese Eleocharis (Cyperaceae) based on ITS sequence data, and chromosomal evolution.

ITS sequence data were used to estimate the phylogeny of 24 Japanese Eleocharis species and to make karyomorphological observations on 19 of these taxa. Two major clades were identified in Japanese Eleocharis molecular phylogenetic trees: (1) one including all species of section Limnochloa, and (2) another comprising two sections, Pauciflorae and Eleocharis. Phylogenetic analysis including both Japanese and North American species also shows strong support for monophyly of the Mutatae/ Limnochloa clade. The width of the spikelets in the species Mutatae/ Limnochloa is the same as that of the culms, indicating that the relative widths of spikelets and culms are useful characteristics for classification. Two major clades were supported by karyomorphological data. All taxa of section Limnochloa had very small chromosomes, while sections Pauciflorae and Eleocharis had large chromosomes. The basic chromosome number of sections Eleocharis and Pauciflorae is thought to be x=5. Chromosomal evolution in the genus Eleocharis with diffuse centromeric chromosomes may be caused by both aneuploidization and polyploidization. Our data suggest that a 3-bp insertion near the 3' end of the 5.8S gene is useful for intrageneric delimitations of the genus Eleocharis.