Comparative Analysis of Begonia Plastid Genomes and Their Utility for Species-Level Phylogenetics

Recent, rapid radiations make species-level phylogenetics difficult to resolve. We used a multiplexed, high-throughput sequencing approach to identify informative genomic regions to resolve phylogenetic relationships at low taxonomic levels in Begonia from a survey of sixteen species. A long-range PCR method was used to generate draft plastid genomes to provide a strong phylogenetic backbone, identify fast evolving regions and provide informative molecular markers for species-level phylogenetic studies in Begonia.     

Development and characterization of microsatellite markers in sawih tree (Duabanga moluccana Blume) using ISSR-suppression PCR techniques

Duabanga moluccana (or locally known as sawih) is an indigenous fast growing tropical tree species that confers various advantages for the timber industry and for planted forests development. In this paper, we isolated and characterized 8 polymorphic microsatellite markers from the D. moluccana genome using ISSR-suppression PCR techniques. The number of alleles and PIC values ranged from 3 to 8 alleles per locus and from 0.488 to 0.792, respectively. Three microsatellite loci were deviated from Hardy-Weinberg equilibrium (P < 0.05). The transferability rate ranged from 24 to 100 % among the three indigenous tree species tested. This indicates that the newly developed microsatellite markers would be useful tools for population genetic studies on D. moluccana and other indigenous tree species.     

Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera)

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.     

Development and characterization of 24 chloroplast microsatellite markers for two species of <Emphasis Type="Italic">Eranthis</Emphasis> (Ranunculaceae)

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.     

Polymorphic microsatellite markers for the Socotran endemic herb Begonia socotrana

Six polymorphic microsatellite markers have been developed to examine population structure and outcrossing rates in the narrow‐range endemic Begonia socotrana. Only two of the markers amplify products in its recently discovered sister species B. samhaensis. All of the loci amplify in winter‐flowering Begonia hybrids derived from B. socotrana, revealing little polymorphism and demonstrating the narrow genetic base of the material used in their production.     

Development of microsatellite markers in Qualea grandiflora (Vochysiaceae) and transferability to congeneric species,typical trees of the Brazilian savanna

This study aimed to isolate and characterize microsatellite markers for Qualea grandiflora and to test their transferability to congeneric species Qualea multiflora and Qualea parviflora. These three species are widespread in the Cerrado, the largest, richest and probably the most threatened tropical savanna in the world. We characterized ten markers in 40 individuals belonging to two populations of Q. grandiflora and eight markers in 20 individuals belonging to one population of Q. multiflora and Q. parviflora. In Q. grandiflora, considering all 40 analyzed individuals, the number of alleles per locus ranged from eight to 21, and the average was 11.60. The mean number of alleles per locus was 8.8 and 7.3 in each population. The observed and expected heterozygosities (H_o and H_e) within populations varied from 0.235 to 0.944 and from 0.225 to 0.932, respectively. In Q. multiflora the number of alleles varied from two to 11 with an average of 5.75; the H_o ranged from 0.150 to 0.950, while H_e ranged from 0.191 to 0.817. In Q. parviflora, considering the seven polymorphic loci, the number of alleles ranged from two to 13, with an average of 7.5, while H_o ranged from 0.211 to 0.944, and H_e ranged from 0.193 to 0.906. The polymorphism level of the microsatellite markers here described enable them as powerful tools for future population genetic studies in these species, helping to answer ecological and evolutionary questions.     

Characterization of microsatellite DNA markers in a critically endangered species,Mekong giant catfish,Pangasianodon gigas

Microsatellite DNA markers for a critically endangered Mekong giant catfish (Pangasianodon gigas Roberts and Vidthayanon, 1991) were developed from fin clips collected from captive fish using (GT)₁₅ probe. The number of alleles per locus ranged from two to four. The expected heterozygosities ranged from 0.13 to 0.68. Also, these primers were successfully amplified in four closely related species, Pangasius bocourti, Pangasius conchophilus, Pangasius larnaudii and Pangasius sanitwongsei with the number of alleles per locus ranged from 1 to 13, 1 to 16, 1 to 12 and 1 to 4, respectively. These markers should prove to be very useful for the evaluation of genetic diversity for this species and other related Pangasius species.     

Studies on Middle American Begonia (Begoniaceae) I

Begonia xilitlensis, a new species inBegonia sectionGireoudia from Mexico is described, illustrated, and discussed, and the taxonomic and nomenclatural status of two others,Begonia glandulosa andB. stigmosa, is evaluated with a lectotype chosen for the former.     

Development of thirty-five novel polymorphic microsatellite markers in Pseudosciaena polyactis (Perciformes:Sciaenidae) and cross-species amplification in closely related species,Pseudosciaena crocea

Small yellow croaker, Pseudosciaena polyactis, is an economically important marine fishery species. In this study, we isolated 35 novel polymorphic microsatellite primers in P. polyactis by using the combined biotin capture method. The polymorphism of each locus was assessed in 30 individuals from Donggang, Northern Yellow Sea, China. A total of 519 alleles were detected and the number of alleles per locus ranged from 3 to 23. The PIC values of these 35 microsatellite loci ranged from 0.367 to 0.940. The observed (H_o) and expected heterozygosity (H_e) per locus ranged from 0.233 to 1.000 and from 0.438 to 0.943, respectively. Seven loci significantly deviated from Hardy–Weinberg equilibrium (HWE) after Bonferroni correction and no significant linkage disequilibrium (LD) was found between pairs of loci. In addition, cross-species amplification was performed in Pseudosciaena crocea, a closely related species of P. polyactis, to assess the applicability of these markers. These polymorphic microsatellites will provide useful tools for the study of genetic diversity and population structure of P. polyactis and P. crocea.     

Development of 12 microsatellite markers for Aconitum brachypodum (Ranunculaceae), a critically endangered and endemic medicinal plant

In this study, 12 microsatellite markers were developed from two microsatellite-enriched libraries (AG, AC) of Aconitum brachypodum, which were constructed using a FIASCO method. Polymorphism of each locus was assessed in 24 individuals of A. brachypodum. Number of alleles per locus (N_A) ranged from 2 to 4. The average allele number of the microsatellites was 2.58 per locus. The observed (H_O) and expected (H_E) heterozygosities ranged from 0.125 to 0.875 and 0.117 to 0.612, respectively. Polymorphic information content (PIC) ranged from 0.110 to 0.531. Among the 12 microsatellite markers, three deviated from Hardy–Weinberg equilibrium significantly. These markers will facilitate further studies on the population genetics of A. brachypodum.     

Microsatellite DNA markers for American shad (Alosa sapidissima) and cross‐species amplification within the family Clupeidae

Sixteen microsatellite loci were identified and characterized for American shad (Alosa sapidissima). The number of alleles per locus observed ranged from eight to 32 and averaged 15.4 alleles. Average observed heterozygosity was 81.1%. The markers were screened using four other species from the family Clupeidae. Amplification success among Alosa species was 79.2% with 81.6% polymorphism among those markers that amplified successfully. Amplification success was poor in Dorosoma (31.3%). Due to allelic diversity and estimates of heterozygosity, these markers can be useful in A. sapidissima for population level analyses, parentage assignment and broodstock management.     

Genetic differentiation and species cohesion in two widespread Central American Begonia species

Begonia is one of the ten largest plant genera, with over 1500 species. This high species richness may in part be explained by weak species cohesion, which has allowed speciation by divergence in allopatry. In this study, we investigate species cohesion in the widespread Central American Begonia heracleifolia and Begonia nelumbiifolia, by genotyping populations at microsatellite loci. We then test for post-zygotic reproductive barriers using experimental crosses, and assess whether sterility barriers are related to intraspecific changes in genome size, indicating major genome restructuring between isolated populations. Strong population substructure was found for B. heracleifolia (F_ST=0.364, F′_ST=0.506) and B. nelumbiifolia (F_ST=0.277, F′_ST=0.439), and Bayesian admixture analysis supports the division of most populations into discrete genetic clusters. Moderate levels of inferred selfing (B. heracleifolia s=0.40, B. nelumbiifolia s=0.62) and dispersal limitation are likely to have contributed to significant genetic differentiation (B. heracleifolia Jost''s D=0.274; B. nelumbiifolia D=0.294). Interpopulation crosses involving a divergent B. heracleifolia population with a genome size ∼10% larger than the species mean had a ∼20% reduction in pollen viability compared with other outcrosses, supporting reproductive isolation being polymorphic within the species. The population genetic data suggest that Begonia populations are only weakly connected by gene flow, allowing reproductive barriers to accumulate between the most isolated populations. This supports allopatric divergence in situ being the precursor of speciation in Begonia, and may also be a common speciation mechanism in other tropical herbaceous plant groups.     

Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae)

The Pilosocereus aurisetus group contains eight cactus species restricted to xeric habitats in eastern and central Brazil that have an archipelago-like distribution. In this study, 5–11 microsatellite markers previously designed for Pilosocereus machrisii were evaluated for cross-amplification and polymorphisms in ten populations from six species of the P. aurisetus group. The genotypic information was subsequently used to investigate the genetic relationships between the individuals, populations, and species analyzed. Only the Pmac101 locus failed to amplify in all of the six analyzed species, resulting in an 88?% success rate. The number of alleles per polymorphic locus ranged from 2 to 12, and the most successfully amplified loci showed at least one population with a larger number of alleles than were reported in the source species. The population relationships revealed clear genetic clustering in a neighbor-joining tree that was partially incongruent with the taxonomic limits between the P. aurisetus and P. machrisii species, a fact which parallels the problematic taxonomy of the P. aurisetus group. A Bayesian clustering analysis of the individual genotypes confirmed the observed taxonomic incongruence. These microsatellite markers provide a valuable resource for facilitating large-scale genetic studies on population structures, systematics and evolutionary history in this group.     

Development and characterization of microsatellite markers for rice leaffolder, Cnaphalocrocis medinalis (Guenée) and cross-species amplification in other Pyralididae

Rice leaffolder, Cnaphalocrocis medinalis (Guenée), is a destructive and widespread pest on rice. In this study, 20 microsatellite markers were isolated and characterized from C. medinalis partial genomic libraries using the method of fast isolation by AFLP of sequence containing repeats. Of these markers, 18 markers displayed polymorphisms. Polymorphisms were evaluated in 48 individuals from two natural populations. The number of alleles per locus ranged from 2 to 15, and the expected and observed heterozygosities ranged from 0.324 to 0.934 and from 0.304 to 0.917, respectively. Cross-species amplification was also performed to test the transferability of the 20 microsatellite markers and a moderate level of cross amplication was observed across the three species of Pyralididae (26.67 %). These microsatellite loci would facilitate the future study on population genetics and molecular genetics of rice leaffolder and would also be useful for study in Chilo suppressalis, Scirpophaga incertulas and Pyrausta nubilalis.     

Development, characterization, and inheritance of 113 novel EST-SSR markers in the Pacific oyster (Crassostrea gigas)

A total of 113 novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the Pacific oyster (Crassotrea gigas). Polymorphisms of these markers were evaluated in a wild population of 30 individuals. The number of alleles per locus ranged from 2 to 27 with an average of 6.3, and the observed and expected heterozygosities varied from 0 to 0.9667 and from 0.0333 to 0.9701, respectively. Mendelian segregations were tested for 24 of the markers that were polymorphic in one family produced by single-pair mating. Null alleles were discovered at four loci. Nine tests of segregation ratios revealed significant departures from expected Mendelian ratios. As a useful addition to the collection of the microsatellites that are now available for C. gigas, these EST-SSR markers will help the advance in investigation of QTL mapping and genetic diversity in this species.     

Characterization of 11 novel polymorphic microsatellite loci in the threatened Korean loach,Iksookimia koreensis,isolated using a next-generation sequencing method

To investigate the genetic diversity and population structure of Iksookimia koreensis, we characterized 11 novel polymorphic microsatellite markers developed using next-generation sequencing. The number of alleles per locus ranged from 4 to 10 (average = 6.26). Observed and expected heterozygosities ranged from 0.333 to 0.866 and from 0.375 to 0.866, respectively. No loci showed significant deviation from Hardy–Weinberg equilibrium (HWE). These loci were also used successfully to study the genetic diversity of a closely related species, Iksookimia longicorpa. Four of the 11 loci amplified in the two species showed different allele frequency and distribution, indicating deep genetic divergence between I. koreensis and I. longicorpa. The newly developed microsatellite markers reported here will provide a useful tool for examining gene flow, population genetic structure, and genetic diversity of these species.     

Cytokinin Activity in Begonia and Bryophyllum

Cytokinin activity has been obtained in ethanol extracts of Begonia and Bryophyllum plants. Begonia x cheimantha Everett yielded 30 to 300 μg kinetin equivalents per kg of fresh leaves in the tobacco callus bioassay. Short day conditions appeared to increase the extractable cytokinin content in the tissue. Purification by fractionation on Dowex 50 H⁺ columns followed by organic solvent extraction, silver precipitation, and repeated paper chromatography yielded an apparently homogeneous product which accounted for most of the activity in the Begonia extracts. It was indistinguishable from zeatin in chromatograms developed with six solvent systems. Other cytokinin active fractions were also obtained from both Begonia and Bryophyllum. Crystalline picrate preparations of active products were insufficient for identification by mass spectrometry.     

Exploring genetic variability within lentil (Lens culinaris Medik.) and across related legumes using a newly developed set of microsatellite markers

Lentil (Lens culinaris Medik.) is an economically important grain legume, yet the genetic and genomic resources remain largely uncharacterized and unexploited in this crop. Microsatellites have become markers of choice for crop improvement applications. Hence, simple sequence repeat (SSR) markers were developed for lentil through the construction of genomic library enriched for GA/CT motifs. As a result 122 functional SSR primer pairs were developed from 151 microsatellite loci and validated in L. culinaris cv. Precoz. Thirty three SSR markers were utilized for the analysis of genetic relationships between cultivated and wild species of Lens and related legumes. A total of 123 alleles were amplified at 33 loci ranging from 2–5 alleles with an average of 3.73 alleles per locus. Polymorphic information content (PIC) for all the loci ranged from 0.13 to 0.99 with an average of 0.66 per locus. Varied levels of cross genera transferability were obtained ranging from 69.70 % across Pisum sativum to 12.12 % across Vigna radiata. The UPGMA based dendrogram was able to establish the uniqueness of each genotype and grouped them into two major clusters clearly resolving the genetic relationships within lentil and related species. The new set of SSR markers reported here were efficient and highly polymorphic and would add to the existing repertoire of lentil SSR markers to be utilized in molecular breeding. Moreover, the improved knowledge about intra- and inter-specific genetic relationships would facilitate germplasm utilization for lentil improvement.     

Isolation and characterization of microsatellite loci for the analysis of genetic diversity in Whitmania pigra

The objective of this study was to isolate microsatellite loci to analyze the genetic diversity of Whitmania pigra. Four new microsatellite markers of W. pigra were developed from an enriched library and ten from a modified SAMPL assay. A total of 127 alleles were detected, with an average of 9.1 alleles per locus. The expected heterozygosity (He) of each microsatellite locus varied from 0.451 to 0.857, with an average of 0.688. The polymorphism information content (PIC) of each microsatellite locus ranged from 0.361 to 0.838, with an average of 0.640. Analysis of molecular variance showed that the main variation component existed within the populations (81.64%) rather than among the populations (18.36%). Phylogenetic tree for 15 populations of Hirudo using the NJ method by MEGA 5.1 software were divided into two major clusters. These microsatellite markers will contribute to research on the individual identification, genetic diversity, population structure, genome mapping and conservation biology of Hirudo.