Species-specific Molecular Markers Research Articles

Complete chloroplast genome of Albizia kalkora (Fabaceae) in Korea and its taxonomic position

Albizia kalkora (Roxb.) Prain (Fabaceae) is a temperate and subtropical deciduous tree distributed in East and Southeast Asia and India. However, the taxonomic position of the Korean A. kalkora remains unclear. Here, we provide the complete chloroplast (cp) genome of the Korean A. kalkora to clarify its taxonomic position and phylogenetic relationship within the genus. The cp genome was assembled based on Illumina HiSeq reads and was 176,402 bp in length. The GC content of the species was 35.4%. The cp genome of A. kalkora revealed a typical quadripartite structure consisting of a pair of inverted repeats (39,716 bp) separated by the large single copy (91,862 bp) and small single copy (5,108 bp) regions. The phylogenetic trees constructed using the maximum likelihood and Bayesian inference methods based on 73 protein-coding genes revealed that A. kalkora is positioned within the Albizia clade, which is a sister to the Archidendron clade. Unexpectedly, the accession of the Korean A. kalkora was found to be a sister to Albizia julibrissin instead of an individual of the A. kalkora from China. Our complete cp genome data of A. kalkora are useful for identifying species by developing species-specific molecular markers, thereby providing a guideline for conservation.

Comparative Analysis of Six Chloroplast Genomes in Chenopodium and Its Related Genera (Amaranthaceae): New Insights into Phylogenetic Relationships and the Development of Species-Specific Molecular Markers.

Species within the genus Chenopodium hold significant research interest due to their nutritional richness and salt tolerance. However, the morphological similarities among closely related species and a dearth of genomic resources have impeded their comprehensive study and utilization. In the present research, we conduct the sequencing and assembly of chloroplast (cp) genomes from six Chenopodium and related species, five of which were sequenced for the first time. These genomes ranged in length from 151,850 to 152,215 base pairs, showcased typical quadripartite structures, and encoded 85 protein-coding genes (PCGs), 1 pseudogene, 37 tRNA genes, and 8 rRNA genes. Compared with the previously published sequences of related species, these cp genomes are relatively conservative, but there are also some interspecific differences, such as inversion and IR region contraction. We discerned 929 simple sequence repeats (SSRs) and a series of highly variable regions across 16 related species, predominantly situated in the intergenic spacer (IGS) region and introns. The phylogenetic evaluations revealed that Chenopodium is more closely related to genera such as Atriplex, Beta, Dysphania, and Oxybase than to other members of the Amaranthaceae family. These lineages shared a common ancestor approximately 60.80 million years ago, after which they diverged into distinct genera. Based on InDels and SNPs between species, we designed 12 pairs of primers for species identification, and experiments confirmed that they could completely distinguish 10 related species.

Isolation of a Novel Mosquitocidal Bacterium <i>Bacillus cereus</i> VCRC-641 from Fresh Water Fish <i>Clarias batrachus</i>

In this study, search for new mosquitocidal bacterium was attempted from the gut content of the fresh water fish Clarias batrachus (walking cat fish) which resulted in the isolation of a highly potential mosquitocidal bacterium. The isolate was identified as Bacillus cereus VCRC 641 by Bacillus species specific molecular markers ilvD, pur and pycA gene sequence analysis. Laboratory mosquito larval toxicity assay (bioassay) against mosquito larvae revealed that this new isolate was highly potent against Culex quinquefasciatus followed by Aedes aegypti and Anopheles stephensi. The lethal concentration values at LC50 for these three mosquito species were 0.001, 0.004 and 0.007mg/ L respectively. Therefore, in the present study, this is the first report that a highly potential mosquitocidal bacteria of B. cereus VCRC 641 was isolated from the gut content of Clarias batrachus and suggested that it can be used for mosquito vector control program.

RNA-seq analysis revealed considerable genetic diversity and enabled the development of specific KASP markers for Psathyrostachys huashanica.

Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat-P. huashanica addition lines. Four and eight SNP markers were detected in wheat-P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat-P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.

Cytogenetic characterization and molecular marker development for a wheat-T. boeoticum 4Ab (4B) disomic substitution line with stripe rust resistance.

Triticum boeoticum (2n = 2x = 14, AbAb) is an important relative of wheat. This species tolerates many different types of environmental stresses, including drought, salt, and pathogenic infection, and is lower in dietary fiber and higher in antioxidants, protein (15-18%), lipids, and trace elements than common wheat. However, the gene transfer rate from this species to common wheat is low, and few species-specific molecular markers are available. In this study, the wheat-T. boeoticum substitution line Z1889, derived from a cross between the common wheat cultivar Crocus and Triticum boeoticum line G52, was identified using multicolor fluorescence in situ hybridization (mc-FISH), multicolor genomic in situ hybridization (mc-GISH) and a 55K single nucleotide polymorphism (SNP) array. Z1889 was revealed to be a 4Ab (4B) substitution line with a high degree of resistance to stripe rust pathogen strains prevalent in China. In addition, 22 4Ab chromosome-specific molecular markers and 11 T. boeoticum genome-specific molecular markers were developed from 1145 4Ab chromosome-specific fragments by comparing the sequences generated by specific-length amplified fragment sequencing (SLAF-seq), with an efficiency of up to 55.0%. Furthermore, the specificity of these markers was verified in four species containing the Ab genome. These markers can not only be used for the detection of the 4Ab chromosome but also provide a basis for molecular marker-assisted selection-based breeding in wheat.

Genome survey sequencing and mining of genome-wide microsatellite markers in yellow-billed babbler (Turdoides affinis)

Turdoides affinis is a species of group dwelling old world passerine of family Leiothrichidae. Unavailability of genome-wide sequence and species-specific molecular markers have hindered comprehensive understanding of cooperative breeding behaviour in T. affinis. Therefore, we generated genome-wide microsatellite markers through whole genome short read sequencing of T. affinis. A total of 68.8 gigabytes of paired-end raw data were sequenced containing 195,067,054 reads. Total sequenced reads spanned a coverage of 17X with genome size of 1.18 Gb. A large number of microsatellite markers (265,297) were mined in the T. affinis genome using Krait, and 50 most informative markers were identified and validated further. In-silico PCR results validated 47 markers. Of these 47 markers, five were randomly selected and validated in-vitro in twelve individuals of T. affinis. Genotyping data on these five loci estimated observed heterozygosity (H0) and expected heterozygosity (He) ratios between 0.333 – 0.833 and 0.851–0.906, respectively. Effective allele size ranged from 6.698 to 10.667, inbreeding coefficient of the population ranged from 0.080 to 0.631 and null allele frequency was calculated at 0.055 to 0.303. Polymorphic information content of all the five loci varied between 0.850 and 0.906. Probabilities of exclusion and identity across 5 loci was estimated to be 0.95 and 0.0036, respectively. All the loci showed significant adherence to Hardy-Weinberg equilibrium. The microsatellite markers reported in this study will facilitate future population genetics studies on T. affinis and other congeneric species.

The complete chloroplast genome of two Firmiana species and comparative analysis with other related species.

Firmiana is a small genus within the subfamily Sterculioideae of the Malvaceae. There are nine Firmiana species distributed in South and South-west China, most of which are endangered. Due to the shortage of plastid genomes data, the phylogenetic relationships and the evolutionary history of this genus remain unclear. Therefore, the complete chloroplast genomes of F. calcarean and F. hainanensis were sequenced using high-throughput sequencing and then compared with the chloroplast genomes of other reported Firmiana species. The genome size of F. calcarean and F. hainanensis is 161,263 and 160,031bp long, respectively, containing a total of 131 genes (including 85 protein coding genes, 37 tRNAs, 8 rRNAs, and one pseudogene). Comparative analysis revealed that the genome structure, GC content, gene content and order, as well as the RNA editing sites within the chloroplast genomes of F. calcarean and F. hainanensis were similar to previously reported Firmiana species. ML phylogenetic analysis revealed that F. danxiaensis, F. hainanensis, F. calcarean, F. simplex, and F. major form a sister group to F. colorata, F. pulcherrima, and F. kwangsiensis. The SSRs, long repeats, and 21 highly divergent regions (Pi > 0.01) identified in this study might provide potential DNA markers for further population genetics and phylogenetic studies of Firmiana. Our findings can help design new species-specific molecular markers and the general framework to further explore the evolutionary history of Firmiana and to address their conservation challenges.

Comparative Analyses of Complete Chloroplast Genomes and Karyotypes of Allotetraploid Iris koreana and Its Putative Diploid Parental Species (Iris Series Chinenses, Iridaceae).

The Iris series Chinenses in Korea comprises four species (I. minutoaurea, I. odaesanensis, I. koreana, and I. rossii), and the group includes some endangered species, owing to their high ornamental, economic, and conservation values. Among them, the putative allotetraploid, Iris koreana (2n = 4x = 50), is hypothesized to have originated from the hybridization of the diploids I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28) based on morphological characters, chromosome numbers, and genome size additivity. Despite extensive morphological and molecular phylogenetical studies on the genus Iris, little is known about Korean irises in terms of their complete chloroplast (cp) genomes and molecular cytogenetics that involve rDNA loci evolution based on fluorescence in situ hybridization (FISH). This study reports comparative analyses of the karyotypes of the three Iris species (I. koreana, I. odaesanensis, and I. minutoaurea), with an emphasis on the 5S and 35S rDNA loci number and localization using FISH together with the genome size and chromosome number. Moreover, the cp genomes of the same individuals were sequenced and assembled for comparative analysis. The rDNA loci numbers, which were localized consistently at the same position in all species, and the chromosome numbers and genome size values of tetraploid Iris koreana (four 5S and 35S loci; 2n = 50; 1C = 7.35 pg) were additively compared to its putative diploid progenitors, I. minutoaurea (two 5S and 35S loci; 2n = 22; 1C = 3.71 pg) and I. odaesanensis (two 5S and 35S loci; 2n = 28; 1C = 3.68 pg). The chloroplast genomes were 152,259–155,145 bp in length, and exhibited a conserved quadripartite structure. The Iris cp genomes were highly conserved and similar to other Iridaceae cp genomes. Nucleotide diversity analysis indicated that all three species had similar levels of genetic variation, but the cp genomes of I. koreana and I. minutoaurea were more similar to each other than to I. odaesanensis. Positive selection was inferred for psbK and ycf2 genes of the three Iris species. Phylogenetic analyses consistently recovered I. odaesanensis as a sister to a clade containing I. koreana and I. minutoaurea. Although the phylogenetic relationship, rDNA loci number, and localization, together with the genome size and chromosome number of the three species, allowed for the inference of I. minutoaurea as a putative maternal taxon and I. odaesanensis as a paternal taxon, further analyses involving species-specific molecular cytogenetic markers and genomic in situ hybridization are required to interpret the mechanisms involved in the origin of the chromosomal variation in Iris series Chinenses. This study contributes towards the genomic and chromosomal evolution of the genus Iris.

Efficient Anchoring of Erianthus arundinaceus Chromatin Introgressed into Sugarcane by Specific Molecular Markers

Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.

Phylogeny of the order Phoenicopteriformes and population genetics of the Caribbean flamingo (Phoenicopterus ruber: Aves)

AbstractFlamingos are highly mobile waterbirds that can be found in tropical and subtropical regions worldwide. The large distribution range of flamingos, the inaccessibility of most of their breeding sites and the lack of species-specific molecular markers, such as microsatellites, have hampered population genetics studies of these majestic birds. Here, we developed a library of microsatellite loci primers using high-throughput sequencing technology that could be used for further genetic studies on Phoenicopteriformes. Microsatellite and mitochondrial markers were employed for the genetic characterization of individuals of all flamingo species and their hybrids, and to conduct a population genetics study of the Caribbean flamingo. The phylogeny of Phoenicopteriformes was confirmed with six species grouped in two major clades that diverged approximately 13 Mya. This suggests the existence of two genera of flamingos, not three, as is currently accepted. The analysis of the genetic structure of the Caribbean flamingo shows that all Cuban demes constitute a single population isolated from the Bonairean colony. This supports suggestions of the existence of limited connectivity between northern and southern Caribbean colonies. The small Galápagos colony was confirmed as an isolated population with low genetic diversity; thus, it should be considered as threatened.

A Molecular Marker to Identify Spodoptera frugiperda (JE Smith) DNA in Predators’ Gut Content

Simple SummaryThis work aimed to build a molecular marker to detect Spodoptera frugiperda DNA in predators’ gut content. The molecular marker developed is highly specific, and it was able to detect S. frugiperda DNA in the gut content of ladybug and earwig predators in field conditions. Our results confirm that generalist predators feed on S. frugiperda in maize fields, and they must be considered in IPM programs for S. frugiperda suppression.Spodoptera frugiperda is a serious pest of maize and other crops worldwide. The integration of control tactics is recommended for S. frugiperda suppression because reports of insecticide and Btplant-resistance are frequent. Biological control agents would be an alternative to improve S. frugiperda control in agricultural areas. We constructed a species-specific molecular marker to detect S. frugiperda DNA in predators’ gut content and estimated the predation rates of ladybugs and earwigs on S. frugiperda in maize crops. Predators were sampled in Pirassununga, São Paulo state, Brazil, in 2020 and 2021. Using the species-specific molecular marker in laboratory conditions, we estimated the half-life time to detect S. frugiperda DNA in the gut contents of Hippodamia convergens as 6.16 h and Doru luteipes as 25.72 h. The weekly predation rate of S. frugiperda by predators in maize crop varied from 0 to 42.1% by ladybugs and from 0 to 9.2% by D. luteipes. Predation events on S. frugiperda by predators were more frequent during the maize reproductive stage. Our results confirmed that predators might contribute to S. frugiperda suppression in maize fields. However, further studies of prey–predator interactions and agricultural landscapes are essential for a better understanding of predator dynamics in crops.

Genome-Wide Survey and Development of the First Microsatellite Markers Database (AnCorDB) in Anemone coronaria L.

Anemone coronaria L. (2n = 2x = 16) is a perennial, allogamous, highly heterozygous plant marketed as a cut flower or in gardens. Due to its large genome size, limited efforts have been made in order to develop species-specific molecular markers. We obtained the first draft genome of the species by Illumina sequencing an androgenetic haploid plant of the commercial line “MISTRAL® Magenta”. The genome assembly was obtained by applying the MEGAHIT pipeline and consisted of 2 × 106 scaffolds. The SciRoKo SSR (Simple Sequence Repeats)-search module identified 401.822 perfect and 188.987 imperfect microsatellites motifs. Following, we developed a user-friendly “Anemone coronaria Microsatellite DataBase” (AnCorDB), which incorporates the Primer3 script, making it possible to design couples of primers for downstream application of the identified SSR markers. Eight genotypes belonging to eight cultivars were used to validate 62 SSRs and a subset of markers was applied for fingerprinting each cultivar, as well as to assess their intra-cultivar variability. The newly developed microsatellite markers will find application in Breeding Rights disputes, developing genetic maps, marker assisted breeding (MAS) strategies, as well as phylogenetic studies.

Utilization of an In Silico 16S Ribosomal RNA Gene-Based Restriction Enzyme Approach for Identification of Lactic Acid Bacteria

In silico 16S ribosomal RNA gene-based restriction enzyme approach is a computer-simulated polymerase chain reaction-restriction fragment length polymorphism analysis method, namely customizable in silico sequence evaluation for restriction sites. The purpose of this study was to identify lactic acid bacteria using restriction enzyme analysis of 16S gene (16S ribosomal RNA polymerase chain reaction-restriction fragment length polymorphism). An in silico investigation was conducted to detect species-specific molecular markers in different lactic acid bacteria strains. We selected six out of 20 16S ribosomal RNA gene sequences from probiotic-potential lactic acid bacteria strains. Initially, the corresponding 16S ribosomal RNA gene sequences were selected from the database aligned using the Clustal Omega program and their evolutionary relationships were determined. The results indicated that the studied lactic acid bacteria strains belonged to 6 distinct clades except for the genus of Lactobacillus. Furthermore, the in silico polymerase chain reactionrestriction fragment length polymorphism was conducted for further taxonomic classification. Different restriction enzymes were used to recognize sites present on the lactic acid bacteria 16S ribosomal RNA genes. Customizable in silico sequence evaluation for restriction sites output comprised a predicted agarose gel as virtual polymerase chain reaction-restriction fragment length polymorphism patterns, for proper selection of the appropriate enzyme (s) to be used for polymerase chain reaction-restriction fragment length polymorphism marker generation. Our study demonstrated that ApyPI, CchIII and NheI produced distinct restriction patterns as those of Ku324937_C species. The most abundant species-specific markers were seen for Ku324928_C (10 markers), followed by Ku324908_C (8 markers) of order Hemiptera, then Ku324909_C (5 markers), Ku324898_C (4 markers) and finally, Ku324937_C (3 markers). The results demonstrated that customizable in silico sequence evaluation for restriction sites is a powerful tool to detect species-specific markers to provide better understanding of organisms found in raw and/or fermented milk.

Rapid Detection of Red Rot Disease Pathogens (Pythium chondricola and P. porphyrae) in Pyropia yezoensis (Rhodophyta) with PCR-RFLP.

Red rot disease is one of the best-known algal diseases infecting red algae Pyropia species. This disease decreases the quality and quantity of Pyropia aquaculture products in Korea, Japan, and China. Recently we found that Pythium chondricola (Oomycetes) infects blades of Pyropia yezoensis. Therefore, two Pythium species (P. chondricola and P. porphyrae) have been reported as red rot disease pathogens. In this study, we developed a species-specific molecular marker for distinguishing between the two red rot disease pathogens. Using a polymerase chain reaction restriction fragment length polymorphism method based on the mitochondrial cytochrome c oxidase subunit 2 (cox2) and nuclear ribosomal RNA large subunit regions, we classified these two Pythium species without a sequencing step. This new method had high specificity and efficiency for detecting red rot disease pathogens at the species level for both of the cultured and field samples. Therefore, the molecular markers developed in this study are effective for long-term monitoring of the infection and distribution pattern of each Pythium species in Pyropia aquaculture farms. Moreover, molecular monitoring can provide useful information for predicting infection and preventing mass mortality of Pyropia species by red rot disease.

The Development of New Species-Specific Molecular Markers Based on 5S rDNA in Elaeagnus L. Species.

The Elaeagnus L. species are trees and bushes that mainly grow in temperate zones of Western Europe; Minor, Central, and Southeast Asia; the Far East; and North America. Some species are used as fruit or ornamental plants and have economic value. Problems with the identification of species in the Elaeagnus genus by molecular genetical methods arise in the study of populations, systematics, breeding, and other areas of plant science and practice. Recently, the polymorphism of 5S ribosomal DNA non-transcribed spacers (5S rDNA NTSs) in Elaeagnaceae Adans. has been described. The results were used in our study as a basis for development of new species-specific molecular markers for some members of the Elaeagnus genus. The author’s method was applied for finding regions that were potentially applicable for species-specific primer design. As a result, some species-specific molecular markers were developed for Elaeagnus angustifolia L., E. commutata Bernh., E. pungens Thunb., and E. multiflora Thunb. These markers were tested in a range of samples and showed the presence of amplified fragments in lanes of the marked species only. Samples of other species showed no amplifications. Thus, the developed markers may be useful for the species identification of the studied Elaeagnus plants in botanical, dendrological, and genetic research (especially in a leafless period of year), as well as in breeding and hybridization experiments.

Occasional long-distance dispersal may not prevent inbreeding in a threatened butterfly

BackgroundTo set up successful conservation measures, detailed knowledge on the dispersal and colonization capacities of the focal species and connectivity between populations is of high relevance. We developed species-specific nuclear microsatellite molecular markers for the grayling (Hipparchia semele), a butterfly endemic to Europe and of growing conservation concern in North-West Europe, and report on its population genetics, in a fragmented, anthropogenic landscape in Belgium. Our study included samples from 23 different locations nested in two regions and additional historical samples from two locations. We assessed contemporary, long-distance dispersal based on genetic assignment tests and investigated the effect of habitat loss and fragmentation on the population genetic structure and genetic variation using data of nine microsatellite loci.ResultsDetected dispersal events covered remarkably long distances, which were up to ten times larger than previously reported colonisation distances, with the longest movement recorded in this study even exceeding 100 km. However, observed frequencies of long-distance dispersal were low. Our results point to the consequences of the strong population decline of the last decades, with evidence of inbreeding for several of the recently sampled populations and low estimates of effective population sizes (Ne) (ranging from 20 to 54 individuals).ConclusionsOur study shows low frequencies of long-distance dispersal, which is unable to prevent inbreeding in most of the local populations. We discuss the significance for species conservation including future translocation events and discuss appropriate conservation strategies to maintain viable grayling (meta) populations in highly fragmented, anthropogenic landscapes.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing

BackgroundPlasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources.ResultsA population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable.ConclusionsThis two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

The response of rice varieties to Meloidogyne graminicola from the Mekong Delta, Vietnam

Summary The rice root-knot nematode, Meloidogyne graminicola, is a serious pathogen of rice production worldwide. In Vietnam, scientific information on the rice root-knot nematode is limited. In this circumstance, our study aimed to investigate the characteristics of M. graminicola from rice fields in southern Vietnam and to evaluate the reaction of 20 rice varieties to this soil-borne pathogen in a glasshouse experiment. Ten populations of root-knot nematode collected from rice fields at different geographical regions in the Mekong Delta were identified as M. graminicola based on the perineal configuration of females and species-specific molecular markers. The morphological, morphometric and molecular characteristics of ten nematode isolates were defined, and the variations observed between these isolates were not significantly different. The pathogenicity of these isolates was examined in the susceptible Indica rice, Oryza sativa ‘IR64’, with a reproduction factor ranging from 18.1 to 37.8. Of the 20 Indica rice varieties tested in response to the nematode population MG-PN08, the ‘Tetep’ variety showed moderate resistance to the nematode (reproductive factor = 11.7; galling index = 2.4), while others were ranked at levels ranging from moderately susceptible to highly susceptible. The life cycle of M. graminicola in ‘Tetep’ root is 28 days compared to 21 in ‘IR64’. ‘Tetep’-nematode interaction at the histological and molecular levels needs further investigation to develop new rice varieties resistant to rice root-knot nematode.

Antibacterial activity of crude protein and development of species-specific molecular marker for fungus Ophiocordyceps sobolifera

The medicinal mushroom Ophiocordyceps sobolifera has been used in traditional Chinese medicine; however, the proven medical benefits of the crude protein are limited. Therefore, in this study, forty-two isolates of O. sobolifera were identified using their internal transcribed spacer (ITS) sequences. Then, the antibacterial activities of the crude proteins were evaluated against strains of methicillin-resistant and -susceptible Staphylococcus aureus and Bacillus cereus using agar well diffusion technique. The results revealed that the crude protein had a good inhibitory effect against S. aureus and B. cereus. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the crude proteins ranged from 0.156–10.00 mg/mL and 0.156–10.00 mg/mL, respectively. Among them, the Cod-NB1302 strain had the broadest inhibitory activity against the tested bacterial pathogens with MIC values of 0.625–1.25 mg/mL and MBC values of 0.625–1.25 mg/mL. Then, the species-specific molecular marker derived from random amplified polymorphic DNA (RAPD) was developed for the identification of O. sobolifera. The results indicated that the OPT13 arbitrary primer was able to generate two species-specific DNA fragments from O. sobolifera. Specific fragments were then cloned, sequenced, and used to design the species-specific primers. Only one species-specific primer set, the SpsoL_f01/SpsoL_r01 primer pair, produced a prominent amplicon of 118 bps in O. sobolifera under stringent conditions without non-specific detection after being applied to other species, with the exception of O. longissima. Moreover, the detection limit of this primer was 5.84 ng of genomic DNA, and this primer set could be applied in all strains of O. sobolifera.

Cytochrome C Oxidase Subunit I and II (COI and COII) Sequences Support Recognition of Papilio chikae Igarashi and Papilio hermeli Nuyda as Distinct Species

Papilio chikae and P. hermeli are two endemic swallowtail butterflies in the Philippines found primarily in the Cordillera region and Mindoro Island, respectively. Their species status is still subject to debate due to opposing interpretations of their genitalia and morphological traits. This study aims to delineate these taxa through the use of mitochondrial cytochrome C oxidase subunit I(COI) gene marker, with additional information on their cytochrome C oxidase subunit II(COII) gene. The COI nucleotide sequences from our Philippine P. chikae samples were surprisingly identical to P. hermeliEF514465.1 on the GenBank, whereas our P. hermelisamples showed a 100% similarity with P. hermeliEF514464.1.Based on our phylogenetic analysis, P. chikae and P. hermeli exhibit distinct COI barcodes supported by a high bootstrap value (85%). The P. hermeli voucher specimen with GenBank Acc. No. EF514465.1 clustered with our P. chikae samples, separate from the cluster containing our P. hermelispecimens. Nonetheless, the 3.1–3.4% genetic distance observed in the COI region supported by the formation of two separate clusters suggests species status for these taxa. Moreover, species-specific molecular markers obtained from the COII region displayed a 2.4% distance between these two taxa. These molecular markers could therefore be utilized to distinguish P. chikae from P. hermeli. Nevertheless, a thorough examination of their morphology, behavior, and ecology is still imperative to firmly establish their taxonomic status.