Development of EST-SSR markers in the relict tree Davidia involucrata (Davidiaceae) using transcriptome sequencing.

Davidia involucrata, reputed to be a "living fossil" in the plant kingdom, is a relict tree endemic to China. Extant natural populations are diminishing due to anthropogenic disturbance. In order to understand its ability to survive in a range of climatic conditions and to design conservation strategies for this endangered species, we developed genic simple sequence repeats (SSRs) from mRNA transcripts. In total, 142,950 contigs were assembled. Of these, 30,411 genic SSR loci were discovered and 12,208 primer pairs were designed. Dinucleotides were the most common (77.31%) followed by trinucleotides (16.44%). Thirteen randomly selected primers were synthesized and validated using 24 individuals of D. involucrata. The markers displayed high polymorphism with the number of alleles per locus ranging from 3 to 12 and the observed and expected heterozygosities ranging from 0.083 to 1.0 and 0.102 to 0.69, respectively. This large expressed sequence tag dataset and the novel SSR markers will be key tools in comparative studies that may reveal the adaptive evolution, population structure, and resolve the genetic diversity in this endangered species.

Development of expressed sequence tag-based microsatellite markers for the critically endangered Isoëtes sinensis (Isoetaceae) based on transcriptome analysis.

Isoëtes sinensis is a critically endangered quillwort. To facilitate studies on the conservation genetics of this species, we developed expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of 50,063 unigenes were predicted by transcriptome sequencing, 5294 (10.6%) of which significantly matched 3011 Gene Ontology annotations and 2363 were assigned to Kyoto Encyclopedia of Genes and Genomes metabolic pathways. Most of these (2297) were involved in metabolism. A total of 1982 SSR motifs were identified, with trinucleotides being the dominant repeat motif, and 1438 (72.6%) SSR primers were designed. Eighteen randomly selected primer pairs were used to genotype 24 I. sinensis accessions, which confirmed the suitability of these novel markers for molecular studies of I. sinensis. The heterozygosity index value ranged between 0.0799 and 0.9106, while the Shannon-Wiener diversity index value ranged between 0.1732 and 2.5589. The EST-SSRs reported in this study are linked to genic sequences, and are therefore ideal for investigating the evolutionary history of I. sinensis. These markers, together with the large EST dataset generated in this study, will greatly facilitate conservation genetic studies of I. sinensis.

Development of microsatellite markers in Hagenia abyssinica (Bruce) J.F. Gmel, an endangered tropical tree of eastern Africa, using next-generation sequencing.

Hagenia abyssinica (Bruce) J.F. Gmel is an endangered tree species endemic to the high mountains of tropical Africa. We used Illumina paired-end technology to sequence its nuclear genome, aiming at creating the first genomic data library and developing the first set of genomic microsatellites. Seventeen microsatellite markers were validated in 24 individuals. The average number of alleles per locus was 7.6, while the observed and expected heterozygosities ranged from 0.000 to 0.958 and from 0.354 to 0.883, respectively. These polymorphic markers will be used as tools for further molecular studies to facilitate formulation of appropriate conservation strategies for this species.

Development of microsatellite markers in the tetraploid fern Ceratopteris thalictroides (Parkeriaceae) using RAD tag sequencing.

To understand the genetic variability of the tetraploid fern Ceratopteris thalictroides (Parkeriaceae), we described 30 polymorphic microsatellite markers obtained using the restriction site-associated DNA (RAD) tag sequencing technique. A total of 26 individuals were genotyped for each marker. The number of alleles per locus ranged from 4 to 10, and the expected heterozygosity and the Shannon-Wiener index ranged from 0.264 to 0.852 and 0.676 to 2.032, respectively. Because these 30 microsatellite markers exhibit high degrees of genetic variation, they will be useful tools for studying the adaptive genetic variation and sustainable conservation of C. thalictroides.

Genetic variation in wild populations of the tuber crop Amorphophallus konjac (Araceae) in central China as revealed by AFLP markers.

Amorphophallus konjac is an economically important crop. In order to provide baseline information for sustainable development and conservation of the wild plant resources of A. konjac, we studied the genetic diversity and population structure of this species using amplified fragment length polymorphism (AFLP) molecular markers. We sampled 139 individuals from 10 wild populations of A. konjac in central China. Using five AFLP primer combinations, we scored a total of 270 DNA fragments, most of which were polymorphic (98.2%). Percentage of polymorphic loci, Nei's genetic diversity index, and Shannon's information index showed high levels of genetic variation within A. konjac populations. Analysis of molecular variance indicated that most of the variance (68%) resided within populations. The coefficient of genetic differentiation between populations was 0.348 and the estimated gene flow was 0.469, indicating that there was limited gene flow among the populations. Unweighted pair group method with arithmetic mean cluster analysis and principal coordinates analysis indicated that geographically close populations were more likely to cluster together. The Mantel test revealed a significant correlation between geographic and genetic distances (R2 = 0.2521, P < 0.05). The special insect-pollination system of A. konjac and the complex geography of central China are likely to have contributed to the current pattern of genetic variation of this species. In the present study, we provide several suggestions on the future protection of the wild plant genetic resources of A. konjac.