Molecular Systematics of Polygonum minus Huds. Based on ITS Sequences

Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.     

Phylogenetic Relationships between Four Salix L. Species Based on DArT Markers

The objectives of this study were to evaluate the usefulness of DArT markers in genotypic identification of willow species and describe genetic relationships between four willow species: Salix viminalis, S. purpurea, S. alba and S. triandra. The experimental plant material comprised 53 willow genotypes of these four species, which are popularly grown in Poland. DArT markers seem to identify Salix species with a high degree of accuracy. As a result, the examined species were divided into four distinct groups which corresponded to the four analyzed species. In our study, we observed that S. triandra was very different genetically from the other species, including S. alba which is generally classified into the same subgenus of Salix. The above corroborates the findings of other authors who relied on molecular methods to reveal that the classification of S. triandra to the subgenus Salix was erroneous. The Principal Coordinate Analysis (PCoA) and the neighbor-joining dendrogram also confirmed the clear division of the studied willow genotypes into four clusters corresponding to individual species. This confirmed the usefulness of DArT markers in taxonomic analyses and identification of willow species.     

Molecular Systematics of Genus Atractylodes (Compositae,Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.     

Efficiency of ITS Sequences for DNA Barcoding in Passiflora (Passifloraceae)

DNA barcoding is a technique for discriminating and identifying species using short, variable, and standardized DNA regions. Here, we tested for the first time the performance of plastid and nuclear regions as DNA barcodes in Passiflora. This genus is a largely variable, with more than 900 species of high ecological, commercial, and ornamental importance. We analyzed 1034 accessions of 222 species representing the four subgenera of Passiflora and evaluated the effectiveness of five plastid regions and three nuclear datasets currently employed as DNA barcodes in plants using barcoding gap, applied similarity-, and tree-based methods. The plastid regions were able to identify less than 45% of species, whereas the nuclear datasets were efficient for more than 50% using “best match” and “best close match” methods of TaxonDNA software. All subgenera presented higher interspecific pairwise distances and did not fully overlap with the intraspecific distance, and similarity-based methods showed better results than tree-based methods. The nuclear ribosomal internal transcribed spacer 1 (ITS1) region presented a higher discrimination power than the other datasets and also showed other desirable characteristics as a DNA barcode for this genus. Therefore, we suggest that this region should be used as a starting point to identify Passiflora species.     

Homogeneous Nature of Malaysian Marine Fish Epinephelus fuscoguttatus (Perciformes; Serranidae): Evidence Based on Molecular Markers,Morphology and Fourier Transform Infrared Analysis

Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance–Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish.     

Isolation and Characterization of 13 Microsatellite Loci from a Korean Endemic Species, Sophora koreensis (Fabaceae)

To evaluate the population genetics structure as a means of devising conservation strategies, we developed microsatellite primers for Sophora koreensis, a narrowly endemic and endangered species in Korea. Thirteen polymorphic microsatellite markers were developed in Korean populations of S. koreensis. Genetic diversity was analyzed in 40 individuals from two populations. The number of alleles per locus ranged from 4 to 14, with observed and expected heterozygosities ranging from 0.200 to 1.000 and from 0.189 to 0.864, respectively. The microsatellite markers described here are valuable tools for the population genetics research of S. koreensis. They can be used to obtain information for creating suitable management strategies to conserve this endemic and endangered species.     

New PCR Assays for the Identification of Fusarium verticillioides, Fusarium subglutinans, and Other Species of the Gibberella fujikuroi Complex

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.     

Molecular Identification of Fusarium Species in Gibberella fujikuroi Species Complex from Rice, Sugarcane and Maize from Peninsular Malaysia

The objective of this study was to identify Fusarium species in the Gibberella fujikuroi species complex from rice, sugarcane and maize as most of the Fusarium species in the species complex are found on the three crops. Isolates used were collected from the field and obtained from culture collection. The Fusarium isolates were initially sorted based on morphology and identifications confirmed based on the DNA sequence of the translation elongation factor 1-α (TEF-1α) gene. Based on the closest match of BLAST analysis, five species were recovered, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi and F. verticillioides. This is the first report regarding F. andiyazi from rice in Malaysia and Southeast Asia. The phylogenetic tree generated by using the neighbor joining method showed that isolates from the same species were grouped in the same clade. The present study indicated that Fusarium species in the G. fujikuroi species complex are widespread in rice, sugarcane and maize in Peninsular Malaysia. The findings also suggest that the use of morphological characters for identification of Fusarium species in the G. fujikuroi species complex from the three crops will lead to incorrect species designation.     

Purification and Structural Identification of Polysaccharides from Bamboo Shoots (Dendrocalamus latiflorus)

Three kinds of polysaccharides, namely, BSP1A, BSP2A, and BSP3B, were isolated from raw bamboo shoot (Dendrocalamus latiflorus) after purification and classification by DEAE cellulose-52 (ion-exchange chromatography) and Sephadex G-50. The molecular weights of BSP1A, BSP2A, and BSP3B were 10.2, 17.0 and 20.0 kDa, respectively, which were measured through GPC (gel performance chromtatography) methods. BSP1A contained arabinose, glucose, and galactose in a molar ratio of 1.0:40.6:8.7. BSP2A and BSP3B contained arabinose, xylose, glucose, and galactose in molar ratios of 6.6:1.0:5.2:10.4 and 8.5:1.0:5.1:11.1, respectively. The existence of the O-glycopeptide bond in BSP1A, BSP2A, and BSP3B was demonstrated by β-elimination reaction. FTIR spectra of the three polysaccharides showed that both BSP2A and BSP3B contained β-d-pyranose sugar rings. However, BSP1A exhibited both β-d-pyranose and α-d-pyranose sugar rings. Congo red test indicated that BSP1A and BSP2A displayed triple helix structures, but BSP3B did not. NMR spectroscopy revealed that BSP1A may exhibit a β-1,6-Glucan pyran type as the main link, and few 1,6-glycosidic galactose pyranose and arabinose bonds were connected; BSP2A mainly demonstrated →5)β-Ara(1→and→3)β-Gal(1→connection. Furthermore, BSP3B mainly presented →3)β-Glu(1→and→3)β-Gal(1→connection and may also contain few other glycosidic bonds.     

Association Study Identifying a New Susceptibility Gene (AUTS2) for Schizophrenia

Schizophrenia (SCZ) is a severe and debilitating mental disorder, and the specific genetic factors that underlie the risk for SCZ remain elusive. The autism susceptibility candidate 2 (AUTS2) gene has been reported to be associated with autism, suicide, alcohol consumption, and heroin dependence. We hypothesized that AUTS2 might be associated with SCZ. In the present study, three polymorphisms (rs6943555, rs7459368, and rs9886351) in the AUTS2 gene were genotyped in 410 patients with SCZ and 435 controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forced PCR-RFLP methods. We detected an association between SCZ and the rs6943555 genotype distribution (odds ratio (OR) = 1.363, 95% confidence interval (CI): 0.848–2.191, p = 0.001). The association remained significant after adjusting for gender, and a significant effect (p = 0.001) was observed among the females. In the present study, rs6943555 was determined to be associated with female SCZ. Our results confirm previous reports which have suggested that rs6943555 might elucidate the pathogenesis of schizophrenia and play an important role in its etiology.     

Assessing Molecular Signature for Some Potential Date (Phoenix dactylifera L.) Cultivars from Saudi Arabia, Based on Chloroplast DNA Sequences rpoB and psbA-trnH

Phoenix dactylifera L. (date palm), being economically very important, is widely cultivated in the Middle East and North Africa, having about 400 different cultivars. Assessment of date cultivars under trading and farming is a widely accepted problem owing to lack of a unique molecular signature for specific date cultivars. In the present study, eight different cultivars of dates viz., Khodry, Khalas, Ruthana, Sukkari, Sefri, Segae, Ajwa and Hilali were sequenced for rpoB and psbA-trnH genes and analyzed using bioinformatics tools to establish a cultivar-specific molecular signature. The combined aligned data matrix was of 1147 characters, of which invariable and variable sites were found to be 958 and 173, respectively. The analysis clearly reveals three major groups of these cultivars: (i) Khodary, Sefri, Ajwa, Ruthana and Hilali (58% BS); (ii) Sukkari and Khalas (64% BS); and (iii) Segae. The economically most important cultivar Ajwa showed similarity with Khodary and Sefri (67% BS).The sequences of the date cultivars generated in the present study showed bootstrap values between 38% and 70% so these sequences could be carefully used as molecular signature for potential date cultivars under trading and selection of genuine cultivars at the seedling stage for farming.     

Population Genetics of the Endemic Hawaiian Species Chrysodracon hawaiiensis and Chrysodracon auwahiensis (Asparagaceae): Insights from RAPD and ISSR Variation

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.     

Genetic Diversity and Differentiation of Dendrocalamus membranaceus (Poaceae: Bambusoideae), a Declining Bamboo Species in Yunnan, China, as Based on Inter-Simple Sequence Repeat (ISSR) Analysis

Dendrocalamus membranaceus Munro is a woody bamboo with a high economic and ecological value that often occurs as natural stands, such as in the large-scale forested areas of China's Yunnan Province. Due to its overexploitation, the habitat of D. membranaceus in Yunnan has been dramatically reduced, and the quality of the stands has declined. As a preliminary analysis in considering the effective protection for these germplasm resources, we assessed the genetic diversity of 12 natural populations in Yunnan, using inter-simple sequence repeat (ISSR) markers. From 10 ISSR primers, we generated 155 bands, of which 153 were polymorphic (98.71%). Compared with other species in the genus, this species demonstrated a greater genetic diversity (S = 0.349) and lower genetic differentiation (G(ST) = 0.252). Our analysis of molecular variance revealed that the genetic differentiation among the populations is significant. A large proportion of the genetic variation (78.95%) resides among the individuals within populations, whereas only 21.05% are found among populations. Mantel tests indicated no significant correlation between genetic and geographic distances among the populations. Given the low sexual reproducibility and characteristics of monocarpic plants, we recommend implementing in situ conservation measures for all of the D. membranaceus populations in Yunnan and collecting sufficient samples for ex situ conservation. Furthermore, the conservation area should be extended to its main natural habitats, the Lancang-Mekong River Valley.     

Nuclear Microsatellite Primers for the Endangered Relict Fir,Abies pinsapo (Pinaceae) and Cross-Amplification in Related Mediterranean Species

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.     

A Simple Strategy for Development of Single Nucleotide Polymorphisms from Non-Model Species and Its Application in Panax

Single nucleotide polymorphisms (SNPs) are widely employed in the studies of population genetics, molecular breeding and conservation genetics. In this study, we explored a simple route to develop SNPs from non-model species based on screening the library of single copy nuclear genes (SCNGs). Through application of this strategy in Panax, we identified 160 and 171 SNPs from P. quinquefolium and P. ginseng, respectively. Our results demonstrated that both P. ginseng and P. quinquefolium possessed a high level of nucleotide diversity. The number of haplotype per locus ranged from 1 to 12 for P. ginseng and from 1 to 9 for P. quinquefolium, respectively. The nucleotide diversity of total sites (π_T) varied between 0.000 and 0.023 for P. ginseng and 0.000 and 0.035 for P. quinquefolium, respectively. These findings suggested that this approach is well suited for SNP discovery in non-model organisms and is easily employed in standard genetics laboratory studies.     

Molecular Method for Sex Identification of Half-Smooth Tongue Sole (Cynoglossus semilaevis) Using a Novel Sex-Linked Microsatellite Marker

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most important flatfish species for aquaculture in China. To produce a monosex population, we attempted to develop a marker-assisted sex control technique in this sexually size dimorphic fish. In this study, we identified a co-dominant sex-linked marker (i.e., CyseSLM) by screening genomic microsatellites and further developed a novel molecular method for sex identification in the tongue sole. CyseSLM has a sequence similarity of 73%–75% with stickleback, medaka, Fugu and Tetraodon. At this locus, two alleles (i.e., A244 and A234) were amplified from 119 tongue sole individuals with primer pairs CyseSLM-F1 and CyseSLM-R. Allele A244 was present in all individuals, while allele A234 (female-associated allele, FAA) was mostly present in females with exceptions in four male individuals. Compared with the sequence of A244, A234 has a 10-bp deletion and 28 SNPs. A specific primer (CyseSLM-F2) was then designed based on the A234 sequence, which amplified a 204 bp fragment in all females and four males with primer CyseSLM-R. A time-efficient multiplex PCR program was developed using primers CyseSLM-F2, CyseSLM-R and the newly designed primer CyseSLM-F3. The multiplex PCR products with co-dominant pattern could be detected by agarose gel electrophoresis, which accurately identified the genetic sex of the tongue sole. Therefore, we have developed a rapid and reliable method for sex identification in tongue sole with a newly identified sex-linked microsatellite marker.     

Genetic Diversity and Population Structure: Implications for Conservation of Wild Soybean (Glycine soja Sieb. et Zucc) Based on Nuclear and Chloroplast Microsatellite Variation

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.     

Comparison of the Phenolic Content and Antioxidant Activities of Apocynum venetum L. (Luo-Bu-Ma) and Two of Its Alternative Species

The leaves of Apocynum venetum L. (AV), a native Chinese plant, have been used as folk medicine in China and Japan. This study evaluated the content of the active antioxidant component and antioxidant activities of AV, and its two alternative species, Poacynum pictum (Schrenk) Baill. (PP) and Poacynum hendersonii (Hook.f.) Woodson (PH). The total phenolic and total flavonoid contents were determined. In addition, the quantitative analysis of two major flavonoid compounds (hyperoside and isoquercitrin) was carried out by HPLC. The antioxidant activities were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity method, the reducing power test and the chelating ability of ferrous ions. The highest total phenolic and flavonoid contents were observed in the AV methanolic extract, followed by the PP and PH methanolic extracts. HPLC analysis indicated that isoquercitrin was one of the major components in all three species, however, hyperoside was only detected in AV at high levels. All the antioxidant assays we performed demonstrated that the AV extract was markedly superior to those of the other two species.     

Identification of a Male-Specific Amplified Fragment Length Polymorphism (AFLP) and a Sequence Characterized Amplified Region (SCAR) Marker in Eucommia ulmoides Oliv

Eucommia ulmoides Oliv. is a dioecious species. Currently, there is no method to identify the sex during the juvenile stage that lasts a relatively long time. This study aimed to develop a sex specific Amplified Fragment Length Polymorphism (AFLP) marker for Eucommia ulmoides Oliv. Of a total of 64 AFLP primer combinations screened, primer combination E-ACA/M-CTT produced a 350 bp male-specific marker. This 350 bp AFLP marker was converted into a 247 bp Sequence Characterized Amplified Region (SCAR) marker. Results suggest that the SCAR marker can be utilized for early sexual identification in Eucommia ulmoides Oliv., and it will greatly facilitate future breeding programs.