Genetic diversity in <Emphasis Type="Italic">in vitro</Emphasis>-conserved germplasm of <Emphasis Type="Italic">Curcuma</Emphasis> L. as revealed by RAPD markers

A set of 30 accessions of five Curcuma species-C. latifolia, C. malabarica, C. manga and C. raktakanta and 13 morphotypes (identified on the basis of morphological markers) of C. longa conserved in the In Vitro Genebank at National Bureau of Plant Genetic Resources, New Delhi, were subjected to RAPD analysis. Of the 200 RAPD primers screened, 21 polymorphic primers were selected for further study. Mean genetic similarities based on Jaccard’s similarity coefficient ranged from 0.18 to 0.86 in accessions of cultivated species, i.e., C. longa and from 0.25 to 0.86 in wild species. The dendrogram derived from the RAPD data corroborated the morphological classification of the morphotypes. The efficiency of individual RAPD primers was also compared; primers OPC-20, OPO-06, OPC-01 and OPL-03 were adjudged highly informative in discriminating the germplasm of Curcuma.     

Genetic variability and differentiation of <Emphasis Type="Italic">Caragana microphylla</Emphasis> populations as revealed by RAPD markers

Genetic variability in random amplified polymorphic DNA (RAPD) was studied in 90 individuals of Caragana microphylla, an outcrossing perennial shrub species, from five natural populations sampled in Inner Mongolia steppe of China on a small scale. Nineteen selected primers were used to amplify DNA samples, and totally 225 bands were detected. The percentage of polymorphic bands within populations ranged form 58.22% to 63.56%, with an average of 60% at the population level and 71.11% at the species level, indicating relatively high genetic variations in C. microphylla species. Shannon’s information index (l) and Nei’s gene diversity (h) showed the similar trend with each other. According to the analysis of Nei’s gene diversity, the percentage of genetic variation among populations was 7.13%, indicating a low level of genetic differentiation among populations. There existed a strong gene flow (N _m = 3.26) among populations. Although AMOVA analysis also revealed most variation was within populations (Φ_ST = 4.1%), a significant proportion was observed among populations (P < 0.001) in the present study, suggesting genetic differentiation occurred among populations at a certain extent. Based on Mantel’s tests and the results of previous studies, the genetic structure pattern of C. microphylla accorded with the isolation-by-distance model on a very large scale, however, on a small scale, the significant genetic differentiation among populations might be enhanced by the micro-environmental divergence among the sampling sites, rather than by geographic factors. Analysis of the genetic variations of C. microphylla populations provided useful information for the adaptive strategy of Caragana species.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

Genetic differentiation of <Emphasis Type="Italic">Sorbus torminalis</Emphasis> in Eastern Europe as determined by microsatellite markers

The genetic variation in fourteen Sorbus torminalis (L.) Crantz. populations distributed over the eastern and south-eastern part of its range was studied using seven nuclear microsatellite loci. The differentiation level was relatively high (F _ST = 0.228), as expected for a species with a fragmented range. The distance-based approach to the analysis of differentiation patterns (neighbour-joining tree based on pairwise coefficients of differentiation) did not reveal a clear geographical structure. On the other hand, model-based Bayesian methods (BAPS and STRUCTURE) gave geographically continuous clusters of populations. The occurrence of populations deviating strongly from the general pattern is attributed to founder effect. In spite of a generally high differentiation, a significant isolation-by-distance pattern was found, which might be a consequence of postglacial migration and gene flow among descendants of different refugia.     

Genetic diversity of <Emphasis Type="Italic">Magnolia ashei</Emphasis> characterized by SSR markers

The Ashe magnolia (Magnolia ashei) is a deciduous small tree most noted for its large 1–2 foot long leaves and fragrant creamy white flowers. Although the species is adapted to and used in landscapes in many parts of the U.S., it is endemic only to Northwest Florida where it is limited to ten counties growing on undisturbed bluffs and ravine banks. The populations are highly fragmented and are threatened by degradation of habitat, leading the species to be listed as endangered in the state of Florida. SSR markers were developed to determine the genetic diversity of wild populations of M. ashei in order to guide long-term conservation strategies. 18 marker loci identified a total of 82 alleles that were used to characterize allelic diversity of M. ashei from 11 wild populations, 14 cultivated sources, five accessions of M. macrophylla, and three interspecific hybrids. Results indicated a higher than expected level of heterozygosity within populations, and a clear distinction between Eastern and Western populations; conservation efforts should therefore focus on maintaining these distinct groups in corresponding ex situ seed orchards to counteract pressures due to overcollection, pollution, and loss of habitat due to development. Clustering of individuals was similar using several analytical methods, indicating that despite relatively small sample sizes, our analysis is a valid reflection of the diversity among and relationships between these populations.     

Genetic variation of <Emphasis Type="Italic">Picea jezoensis</Emphasis> populations in South Korea revealed by chloroplast,mitochondrial and nuclear DNA markers

Genetic variation associated with Picea jezoensis populations of South Korea was investigated using chloroplast (cp), mitochondrial (mt) and nuclear DNA markers. In South Korea, P. jezoensis is distributed across a very restricted area, being found on the summits of three mountains: Mts. Jiri, Dokyu and Gyebang. Examination of five region restriction enzyme combinations for mtDNA and four for cpDNA revealed haplotypes endemic to South Korea. The Gyebang population, the most northerly and most isolated, was genetically distinct from the other populations. Nuclear microsatellite markers indicated, overall, a low level of genetic diversity (H _e = 0.406) in South Korea; this could be attributed to genetic drift and/or founder effects associated with historical events. The Wilcoxon sign-rank test did not indicate a recent bottleneck in any of the populations irrespective of the model considered (infinite allele model, two-phased model of mutation, and stepwise mutation model). Microsatellite markers also demonstrated that the Gyebang population was distinct from the others. The results of this study could be used as the basis for conservation guidelines for the management of this species in South Korea.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Genetic diversity and population structure of two important medicinal plant species <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and <Emphasis Type="Italic">Schisandra sphenanthera</Emphasis> revealed by nuclear microsatellites

Seven polymorphic and transferable nuclear microsatellites were used to investigate the population structure of genetic diversity of Schisandra chinensis and Schisandra sphenanthera for facilitating their conservation and sustainable utilization. High levels of gene diversity were revealed in these two medicinal species, the majority of genetic diversity was harbored within populations, and population structure was might due to restricted gene flow among populations. Isolation by distance was close to significance in S. chinensis but not in S. sphenanthera. In S. chinensis, null alleles were identified as a cause for excess of homozygotes at loci G24 and WGA60, but inbreeding might also be partly responsible for the positive F _IS values in this species. In contrast, null allele frequencies were high at all the seven loci in S. sphenanthera and resulted in overestimation of fixation index. The strategy for ex situ conservation of these two medicinal species is discussed based on the genetic results.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Genetic variability of the banded murex (<Emphasis Type="Italic">Hexaplex trunculus</Emphasis>) revealed by <Emphasis Type="Italic">ND2</Emphasis> and <Emphasis Type="Italic">ITS2</Emphasis> sequences

The gastropod Hexaplex trunculus is widely distributed in a relatively large range of habitats, but has no dispersal stage. We investigated its genetic structure across its distribution range, from Mediterranean Sea to adjacent Atlantic coasts, by sequencing mitochondrial DNA portions of the NADH dehydrogenase gene ND2 (420 pb) and the internal transcribed spacer ITS2 (450 pb). Our results suggested a significant genetic variability of ND2 (π = 0.009 and Hd = 0.629) and low variability of the ITS2 sequences. A strong phylogeographic break, separated the Aegean populations from those of Western/Eastern Mediterranean and the Atlantic ones, was founded. The tow lineages may have been separated by vicariance events due to the Peloponnese break that separates the Aegean populations from other populations and was maintained until now by the quasi-circular anticyclonic front associated to the straits of Cretan Arc of the Peloponnesian Peninsula. Tunisian coasts appear particularly diverse since the two divergent lineages co-occured. These results may have management consequences since H. trunculus is a high commercial value harvested species.     

Genetic diversity and population structure of Chinese <Emphasis Type="Italic">Lentinula edodes</Emphasis> revealed by InDel and SSR markers

Genetic diversity and population structure of 88 Chinese Lentinula edodes strains belonging to four geographic populations were inferred from 68 Insertion-Deletion (InDel) and two simple sequence repeat (SSR) markers. The overall values of Shannon’s information index and gene diversity were 0.836 and 0.435, respectively, demonstrating a high genetic diversity in Chinese L. edodes strains. Among the four geographic populations, the Central China population displayed a lower genetic diversity. Multiple analyses resolved two unambiguous genetic groups that corresponded to two regions from which the samples were collected—one was a high-altitude region (region 1) and the other was a low-altitude region (region 2). Results from analysis of molecular variance suggested that the majority of genetic variation was contained within populations (74.8 %). Although there was a strong genetic differentiation between populations (F _ST ?=?0.252), the variability of ITS sequences from representative strains of the two regions (<3 %) could not support the existence of cryptic species. Pairwise F _ST values and Nei’s genetic distances showed that there were relatively lower genetic differentiations and genetic distances between populations from the same region. Geographic distribution could play a vital role in the formation of the observed population structure. Mycelium growth rate and precocity of L. edodes strains displayed significant differences between the two regions. Strains from region 2 grew faster and fructified earlier, which could be a result of adaptation to local environmental factors. To the best of our knowledge, this was the first study on the genetic structure and differentiation between populations, as well as the relationship between genetic structure and phenotypic traits in L. edodes.     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Genetic diversity of <Emphasis Type="Italic">avenin-like b</Emphasis> genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss

Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A–M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.