Neighbor-joining Research Articles

Applying a cytochrome c oxidase I barcode for Leishmania species typing.

Species delimitation has always been a challenge for taxonomists and for Leishmania studies there is no exception. Herein we attempt to display the usefulness of the mitochondrial gene Cytochrome Oxidase I-coI in classical and barcode-based approaches for Leishmania characterization. A total of 228 samples were analyzed, comprising 28 Leishmania related taxa, mainly from cultures of the Oswaldo Cruz Foundation`s Leishmania Collection. Primers were designed for amplification of coI; sequences were analyzed by distance-based indicators and both the Neighbor Joining and NeighborNet as species grouping techniques. Automatic Barcode Gap Discovery was applied to define species delimitation while for the character-based analysis a software for Barcoding with Logic formulas was employed. Final sequences of 486 bp with 238 parsimonious sites were aligned and edited. Robust groups were formed for most of the genus species, distinctive nucleotide positions in the barcode sequence were observed for 11 of them. A good agreement between the techniques applied and the original characterization was observed. Few species were not distinguished by coI: (i) L. (V.) peruviana, L. (V.) lindenbergi, and L. (V.) utingensis; (ii) L. (L.) venezuelensis and (iii) L. colombiensis and L. equatorensis with identical sequences. Some of these taxa have been, at one time or another, classified as controversial and, for most of them, a higher number of isolates should be studied to properly infer their taxonomic status. CoI represents a mitochondrial target that stands out as a taxonomically important asset with multiple advantages over other genes. This paper corresponds to the first report of coI analysis in Leishmania, a potentially advantageous target for the characterization of this parasite.

Sparse Neighbor Joining: rapid phylogenetic inference using a sparse distance matrix.

Phylogenetic reconstruction is a fundamental problem in computational biology. The Neighbor Joining (NJ) algorithm offers an efficient distance-based solution to this problem, which often serves as the foundation for more advanced statistical methods. Despite prior efforts to enhance the speed of NJ, the computation of the n 2 entries of the distance matrix, where n is the number of phylogenetic tree leaves, continues to pose a limitation in scaling NJ to larger datasets. In this work, we propose a new algorithm which does not require computing a dense distance matrix. Instead, it dynamically determines a sparse set of at most O(n log n) distance matrix entries to be computed in its basic version, and up to O(n log 2n) entries in an enhanced version. We show by experiments that this approach reduces the execution time of NJ for large datasets, with a trade-off in accuracy. Sparse Neighbor Joining is implemented in Python and freely available at https://github.com/kurtsemih/SNJ. Supplementary data are available at Bioinformatics online.

Using Multiplex Virtual PCR For Genetic Barcoding Of Tequatrovirus Bacteriophages

In this paper, we investigate the possibility of using the multiplex virtual PCR method to develop approaches to barcoding the genomes of T4-type bacteriophages. A comparison of the multiplex virtual PCR method and phylogenomic analysis based on alignment-free approach was performed. A dataset of Tequatrovirus genomes previously selected for pangenomic analysis was used for this study. The genomes used were preliminarily checked for their adequate annotation and, accordingly, sequencing accuracy. A study using the multiplex virtual PCR method was carried out for 67 genomes, barcodes were generated, and similarity parameters were determined. Based on the obtained data, a phylogenetic tree was constructed using the neighbor joining method. For the same set of Straboviridae bacteriophage genomes, a phylogenomic tree was constructed based on pairwise distance matriх between oligonucleotide 10-mer sets. Comparison of these two trees showed differences in branching between the control distantly related genomes from the genera Mosigvirus and Dhakavirus, and the branch of the genomes of bacteriophages of the genus Tequatrovirus. The authors of the work suggested that these differences may reflect the features of the multiplex virtual PCR method, which is more applicable for assessing the similarity of closely related genomes than within genomes with an identity degree of less than 75 %. It can be concluded that the multiplex virtual PCR method allows one to distinguish taxonomic groups of large bacteriophages, which include T4-type bacteriophages, at the genus level. Thus, using this method, within the genus of such bacteriophages one can more accurately compare the genomes of viruses with a length of about 170 kb. From a practical point of view, multiplex virtual PCR is quite applicable for including barcodes compiled on its basis in the bacteriophage passport for use in the practice of phage therapy.

First Report of Rhizopus arrhizusi causing rot of Dictyophora rubrovolvata in China.

Dictyophora rubrovolvata, as an edible fungus with high medicinal value, is widely cultivated in several provinces in China (Hang et al. 2012). However, between December 2023 and March 2024, a rot disease occurred in the main production area in Fengxian District, Shanghai, China (N30°93', E121°49'). The disease incidence was 25% in the affected 1.33-ha growing area. High temperatures (>25℃) and poor ventilation provide favorable conditions for the spread of this disease. The disease mainly occurs at the stage of fruiting bodies formation of D. rubrovolvata. When the epidermis is damaged and broken, it becomes infested with mold, which then produces a layer of moldy rot with pus. The infected D. rubrovolvata tissues at the edge of the lesions were isolated, surface sterilized and cultured on potato dextrose agar (PDA) at 30 ℃ under dark conditions. Pure cultures were obtained by single-spore isolation. After 3 days, isolates were transferred to Czapek Yeast agar (CYA) (Samson et al, 2014). On CYA, the fungal colony consisted of white flocculent hyphae. Scanning electron microscopy analysis showed that the mycelium was white, and the internodes of the stolons formed characteristic pseudoroots, from which upwardly clustered erect, unbranched sporocarp peduncles expanded apically to form rounded sporocarp sacs, within which sporocarp spores were produced. (Hariprasath P, 2019). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS) and intergenic spacer (IGS) gene of the isolate were amplified by PCR (White et al. 1990; Liu XY. 2008). The resulting sequence was deposited in GenBank with accession PP951880 and PQ001670, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as DIC01. Comparative results showed that the sequences with accession numbers MT603964.1 and DQ990323.1 showed high homology of 99.15% and 98.96% to the ITS and IGS sequences of Rhizopus arrhizusi DIC01, respectively. Phylogenetic analysis with ITS and IGS genes of the isolated strain and 7 Rhizopus spp. strains were performed using MEGAX with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as R. arrhizusi. A spore suspension of the R. arrhizusi DIC01 (1 x107 conidia/mL) was inoculated back to healthy D. rubrovolvata. Five healthy fruit bodies of D. rubrovolvata were injected, and another five healthy morels were treated with potato dextrose broth (PDB) as controls. D. rubrovolvata was incubated at 25°C and 90% relative humidity without ventilation for 5 days. The pathogen successfully infected the D. rubrovolvata, which developed white moldy lesions similar to those of natural diseases. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as R. arrhizusi DIC01 based on its morphological characteristics and phylogenetic marker genes. R. arrhizusi has been reported to cause endothelial cell damage and mycelial invasion into blood vessels, leading to thrombosis and tissue necrosis. (Hariprasath P, 2019). To our knowledge, this is the first report of R. arrhizusi causing rot disease of D. rubrovolvata. This study confirmed that R. arrhizusi is the pathogenic fungus responsible for rotting disease in D. rubrovolvata farms in Fengxian, Shanghai.

First Report of Ralstonia pseudosolanacearum Causing Bacterial Wilt in Ginger in Peru.

Around 90% of Peru's ginger (Zingiber officinale) production is concentrated in the Junín region, due to the optimal agroecological conditions for its cultivation. In March 2024, fields with ginger plants (cultivar Criollo) in Junín region, provinces Chanchamayo and Satipo, specifically in the cities of Pichanaqui and Satipo respectively, exhibited approximately 40% of plants with severe symptoms of a disease characterized initially by plant yellowing and rapid progressing to necrosis. Affected rhizomes showed dark vascular bundles with milky white exudates upon cutting, while stems displayed vascular necrosis hindering water and nutrient transport, often resulting in plant death. Fifteen plants were sampled and diseased vascular tissues from rhizomes and stems were cultured on nutrient agar (NA) and incubated at 28°C. After 72 h, all isolations resulted in colonies with typical characteristics of Ralstonia solanacearum species complex (RSSC) were produced, with appearing fluid, irregularly round, and creamy white. Three isolates were selected for the identification steps (UNALM-RP01 to 03) were identified by PCR using primers 759/760 (Opina et al. 1997) confirming as RSSC with a 282 bp amplification product. Additionally, isolates were assigned to biovar 3 based on their ability to metabolize three acid-producing disaccharides (maltose, lactose, cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) (Hayward, 1964). Phylotype I was identified by multiplex PCR (primers Nmult) with a 114 bp amplification product (Fegan and Prior 2005). For the identification of the sequevars of the three isolates, DNA was extracted and PCR with primers ENDO-F/R (Ji et al. 2007) were performed to amplify and sequence the partial gene sequence of egl gene with 681 bp in length. The phylogeny by Neighbor joining with 10,000 bootstraps clustered the UNALM isolates along other sequevar 30 of R. pseudosolanacearum. The sequences were deposited in Genbank under accessions PQ213016, PQ213017 and PQ213018. For pathogenicity tests, bacterial colonies of isolate UNALM-RP01 were scraped from the culture media with a sterile needle and introduced into the stems of three 2-month-old ginger plants (cultivar Gigante). The plants subsequently exhibited yellowing seven days post-inoculation. Additionally, the rhizomes showed internal discoloration and bacterial exudation. Three plants were used as a control, which were pierced with a sterilized needle and showed no symptoms. All tested plants were kept in a greenhouse with controlled temperature (20-40 °C) The pathogen was successfully re-isolated from infected plants on NA medium, presenting typical colonies of RSSC and identified via PCR with primers 759/760, fulfilling Koch's postulates. This represents the first case in Peru of ginger plants infected with a Ralstonia species, specifically R. solanacearum phylotype I, corresponding to R. pseudosolanacearum. This species of RSSC and sequevar is known for causing disease in ginger. Its presence in Peru, however, may be the result of the pathogen's introduction, as its geographical origin is associated with Asia (Fegan and Prior 2005). To our knowledge, this is the first report of R. pseudosolanacearum causing ginger wilt disease in Peru. In 2024, an estimated average yield loss of 30% has been attributed to wilt disease in the Junín region, posing a significant threat to cultivation. Urgent and effective disease management strategies are essential to control and mitigate further losses.

First Report of Bacterial Leaf Spot Disease on Ginger Caused by Enterobacter quasiroggenkampii in China.

In autumn 2023, an unknown leaf spot disease has occurred on ginger (Zingiber officinale Roscoe) in two fields of approximately 1800 m2 in Yongning District (22°49'N; 108°48'E), Nanning, China, with a incidence of 20-30%. The symptoms began as yellow spots on the leaves, expanding into elliptical to irregular lesions with yellow edges, the middle of the lesion turning grey-white in dry weather. Finally, multiple spots caused necrosis of the whole leaf. Twelve diseased leaves from six plants of two fields were collected, surface disinfected and ground. The ground samples were diluted and plated on nutrient agar (NA) medium at 28 °C for 48-72 h. The purified colonies appeared milky white and round, with smooth edges. Three isolates (GL1, GL2 and GL3) were selected for identification and pathogenic determination. They were gram negative, could utilize sorbitol, mannitol, inositol, raffinose, melibiose, disaccharides, and citrate; negative for methyl red, phenylalanine decarboxylase, hydrogen sulfide, urease; positive for voges-proskauer test and ornithine decarboxylase. These characteristics were consistent with Enterobacter genus (Wu et al., 2020). Genomic DNA was extracted from three isolates. The 16S rDNA region was amplified using 27F/1492R primers (Weisburg et al. 1991) and sequenced (accession no. PP837703-PP837705). Blastn analysis revealed that 16S rDNA sequences for GL1 was 99% identical (1373/1387 nt), GL2 96% (1364/1422 nt) and GL3 95% (1365/1435 nt) to Enterobacter quasiroggenkampii WCHECL1060 (NR_179166). To determine the species, the sequences of gyrB, rpoB and atpD genes were amplified using primers gyrB 01-F/gyrB 02-R, rpoB CM7/rpoB CM31b, and atpD 01-F/atpD 02-R, respectively (Lin et al. 2015; Zhu at al. 2010; Zhang et al. 2013). The GenBank accession numbers for the sequences were PP857680-PP857688. A multilocus phylogenetic tree was constructed with the concatenated sequence of 16S rDNA-gyrB-rpoB-atpD by using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA6 software. The three isolates clustered with E. quasiroggenkampii. Fifteen Darou ginger variety plants at the 4-5 leaf stage were tested for pathogenicity. Two to three leaves of each ginger plant were pricked with a syringe needle of 0.36mm in diameter or not and inoculated by spraying the bacterial suspension (108 CFU/mL), sterile water was used as a control. Five plants were inoculated with each isolate and the test was repeated three times. After 3-4 days of inoculation, all wounded leaves and about 10% of the unwounded leaves showed symptoms similar to those observed in the field. Control plants did not develop symptoms. Enterobacter quasiroggenkampii isolates were re-isolated from the inoculated leaves with symptoms, and their identity was confirmed by gyrB sequencing and colony morphology, completing Koch's postulates. Enterobacter quasiroggenkampii is a pathogen of humans that can cause nosocomial infections (Wu et al., 2020). In Guangxi, E. quasiroggenkampii was identified as one of the pathogens causing mulberry wilt (Jiao, 2022). To our knowledge, this is the first report of E. quasiroggenkampii causing bacterial leaf spot disease of ginger. The results of this study not only have practical significance for the control of ginger leaf spot, but also can provide excellent materials for the study of the differentiation and pathogenic mechanism of the genus Enterobacter, which has important academic value.

Genotyping-by-sequencing shows high genetic diversity in Corylus avellana germplasm resistant to eastern filbert blight

European hazelnut (Corylus avellana) is an anemophilous, dichogamous, self-incompatible tree nut species. It is native to a large portion of Europe, Turkey, and the Caucasus region, across which a wealth of plant genetic resources is present. The objective of this study was to evaluate the genetic diversity of a core set of C. avellana representing the world’s germplasm using genotyping-by-sequencing derived single nucleotide polymorphism (SNP) markers and to classify novel eastern filbert blight (EFB) resistant or tolerant accessions. Two-hundred-twenty-two accessions underwent next-generation sequencing (NGS) to generate SNP markers. From this, 1,250 SNP markers were used to construct a neighbor-joining (NJ) dendrogram and perform a STRUCTURE and discriminant analysis of principal coordinates (DAPC) analyses. In the dendrogram, five major groups were established, which generally corresponded to geographic origins of the plant materials studied. In STRUCTURE, support was found for groupings at (K) = 3, (K) = 6, and (K) = 10 populations, with the greatest Δ-(K) value occurring at (K) = 10. Although the three different analyses indicated slightly different solutions, the overall results were generally consistent from the standpoint of identifying similar accession groupings. For many of the accessions, recorded origins tended to correspond with their genetic grouping, although there was also evidence of intermixing and likely movement of plant materials. Interestingly, in all three analyses, a vast majority of the new accessions from the Republic of Georgia formed their own distinct group, highlighting this geographic region as a unique pool of C. avellana genetic resources. Overall, EFB resistant/tolerant accessions were placed across a wide range of genetic backgrounds. Thus, our results indicate EFB resistance/tolerance is present across a wide spectrum of C. avellana genetic resources, with the Georgian accessions representing a new and relatively unique germplasm pool that can be incorporated into hazelnut breeding programs.

Population genetic differentiation and structure of rare plant Anemone shikokiana based on genotyping-by-sequencing (GBS)

BackgroundAnemone shikokiana (Makino) Makino is a perennial herb of the genus Anemone in the family Ranunculaceae. Endemic to the Shandong Peninsula in China and Shikoku Island in Japan, it is a rare and endangered plant with a narrow, disjunct distribution. It is threatened with extinction and is in urgent need of conservation. Evaluating the genetic diversity of species, revealing the population genetic structure and gene flow, and inferring the population history are of great importance for species conservation, especially for rare and endangered plants.ResultsIn our study, 73 samples from eight wild populations in China were sequenced by Super-GBS, yielding a total of 40.59 G clean reads and 52,231 SNPs. Based on the obtained SNP data set, we evaluated the population genetic diversity, genetic structure, and gene flow of A. shikokiana. A low level of genetic diversity was found (He = 0.1925, Ho = 0.1422). The neighbor-joining (NJ) tree, principal component analysis and ADMIXTURE analysis suggested that these 73 A. shikokiana could be considered as two groups. Pairwise genetic differentiation coefficients (Fst) indicated that genetic differentiation was lower between adjacent populations and higher between geographically separated populations. The gene flow between Kunyu Mountain and Lao Mountain was very low. However, neither of the two regions showed evidence of Isolation by Distance.ConclusionsHere, we revealed the population genetic structure and gene flow of A. shikokiana from the Shandong Peninsula, China. This research provides valuable genetic resources for A. shikokiana and contributes to the scientific and effective conservation of the species.

Species delimitations in the Campomanesia xanthocarpa group (Myrtaceae): insights from molecular markers and taxonomy

Different views on recognising taxa associated with the Campomanesia xanthocarpa group (Myrtaceae) demonstrate the difficulties in clearly delimiting species. Studies using Inter Simple Sequence Repeats (ISSR) molecular markers were carried out on 201 individuals from 13 populations of C. xanthocarpa Mart. ex O.Berg, C. adamantium (Cambess.) O.Berg, C. costata M.Ibrahim & Landrum and C. littoralis D.Legrand in an attempt to improve understanding of species boundaries between these species. SplitsTree, analysis of molecular variance (AMOVA), principal component analysis (PCA), Neighbour-Joining (NJ) dendrogram and STRUCTURE showed inconsistencies between morphological and genetic data in these taxa. Therefore C. adamantium and C. xanthocarpa are treated as distinct taxa in this study, as are C. costata, C. littoralis and C. rhombea O.Berg that were previously considered part of C. xanthocarpa. Structured populations in C. adamantium were not congruent with taxonomic data or poorly supported in the data analysed. These were maintained as a single polymorphic species and new integrative approaches are necessary to improve understanding of taxon boundaries. We present a taxonomic treatment based on these decisions. This study contributes to the systematic treatment of Campomanesia and encourages specific delimitation studies to resolve remaining taxonomic issues within the genus.

First report of basal stem rot on sugarcane (var. Badila) caused by Sclerotium rolfsii in China.

Badila (Saccharum officinarum) is one of the important chewing cane in south China. During the year 2019-2020, as much as 60.2%-87.5% of sugarcane plants stem showed red rot developments were observed in the fields of Yongning District, Nanning city, Guangxi province. Symptomatic plants showed red rot at basal stem nodes and sheath, when the disease serious, the epidermis and aerial roots decomposed and exfoliated, then formed sclerotiums, the upper stem also occurred the symptom. Infected plant tissues were dissected into small pieces with 0.1 × 0.1cm in size and surface sterilized in 0.1% HCl2 for 2 min, followed by 75% ethanol for 30 s, rinsed three times with sterile distilled water. Then the tissues were placed onto potato dextrose agar (PDA) plates and incubated at 25 °C for 3 days. Numerous white globoid sclerotia were formed on PDA after 5 days of growth. The sclerotia (2 to 3 mm in diameter) were white at first and then gradually turned dark brown. Aerial mycelia usually formed many narrow hyphal strands 4 to 9 μm wide. Five uniform isolates were obtained from diseased sugarcane plants. Pathogenicity of representative strain W1 was confirmed by inoculating 120-day-old Badila plants grown in field. Five plants were inoculated with colonized agar discs (6mm in diameter) by applying toothpick tips to the lower part of the stem. Five non-inoculated plants served as control. The inoculated and non-inoculated plants were sprayed sterile water then incubated with plastic film for maintained high moisture. All the plants were placed inside of a growth chamber at 26 ± 2°C with a 14-h photoperiod and 80% relative humidity. All inoculated plants showed red rot at stem and sheath after 2 weeks, whereas the control plants were symptomless. By the third week, mycelium and sclerotia developed on the crown on the inoculated plants. The fungus was re-isolated from the artificially inoculated plants. To confirm the species-level identification, partial of the ribosomal DNA internal transcribed spacer (ITS), mitocondrial small subunit (SSU), and nuclear ribosomal large subunit (LSU) regions of representative strain W1 were amplified and sequenced using the primers pairs ITS1/ITS4 (White et al. 1990), ITS-Fu-F /ITS-Fu-R and SRLSU1//SRLSU2 (Kumar et al., 2016), respectively. The resulting ITS, SSU and LSU sequences were deposited in GenBank (GenBank accession no. MW620994, MW617878, and MW617872) and shared 99.42%, 100% and 100% sequence identity with Athelia rolfsii isolate (JN017199, OM319631, and MT225781). Phylogenetic analysis conducted with neighbor-joining (NJ) method using MEGA6.0 revealed that the isolate share a common clade with reference sequence of A. rolfsii in GenBank Data Library. Based on morphological and molecular characteristics, the fungus was identified as A. rolfsii (anamorph: Sclerotium rolfsii) (Paul et al. 2017; Paparu et al. 2020). Although S. rolfsii has been reported causing sugarcane sett rot in Australia (Bhuiyan et al., 2019) and seedlings of sugarcane in Indian (Gopi et al., 2023), as we know, this is the first report of sugarcane basal stem rot disease caused by this fungus in China. This study will be helpful for the prevention and control sugarcane basal stem rot in the future.

Analysis of genetic diversity and environmental associations of wild citron (Citrus medica L.) in northeast India

Citron (Citrus medica L.) is a major citrus fruit crop known for its medicinal, nutritional, and aromatic uses. Native to northeast India, Myanmar, and south China, citron is cultivated mostly in the Mediterranean and South Asian countries. The truly wild populations of citron are found in the hills and valleys of northeast India. The extent of genetic diversity in wild citron in relation to environmental variables has not been documented. In the present study, we analyzed genetic diversity and environmental association of wild populations of C. medica in northeast India using 15 polymorphic nuclear SSR (simple sequence repeat) markers and 21 environmental variables. SSR profiling revealed a moderate genetic diversity (Ho = 0.40; He = 0.41; I = 0.70). Neighbor-joining (NJ) and principal coordinate analysis (PCoA) grouped 16 natural populations into two genetic clusters. Bayesian structure results showed two genetic populations with a high admixture of individuals. Analysis of molecular variance (AMOVA) revealed low genetic differentiation (Fst = 0.14; P < 0.001), indicative of high gene flow (Nm = 1.53) in citron. The isolation by distance (IBD) analysis showed a weak correlation, indicating that geographic distance was not a determining factor for genetic distance in C. medica populations. The higher metrics of outcrossing rate (tm > 0.9) revealed outbreeding nature of the species. The genetic diversity of citron showed a positive correlation with precipitation of driest month and precipitation of coldest quarter, but negative with mean diurnal range and isothermality. Overall, the results of the present study could benefit the conservation and management of citron resources and their natural habitat in northeast India. Genetically highly diverse populations identified in the present study can be used for conservation and utilization of citron genetic resources.

First Report of 'Candidatus Phytoplasma ziziphi'-Related Strains Infecting Peony (Paeonia suffruticosa Andr.) Showing Leaf Yellows Symptoms in China.

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5'-ATGGCTGAAATATTTTCAAGAG-3') and JWB-tuforfR1 (5'-TTATTCTATGATTTTAATAACAG-3') followed by JWB-tuforfF2 (5'-ATGTAAACGTAGGAACTATTGG-3') and JWB-tuforfR2 (5'- TCCGATAGTTCTTCCACCTTCAC-3'). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with 'Candidatus Phytoplasma ziziphi' (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with 'Ca. P. ziziphi' (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of 'Ca. P. ziziphi' with 68% bootstrap value. Although there are several reports available on 'Ca. P. solani' infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of 'Ca. P. ziziphi'-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Machine learning can be as good as maximum likelihood when reconstructing phylogenetic trees and determining the best evolutionary model on four taxon alignments

Phylogenetic tree reconstruction with molecular data is important in many fields of life science research. The gold standard in this discipline is the phylogenetic tree reconstruction based on the Maximum Likelihood method. In this study, we present neural networks to predict the best model of sequence evolution and the correct topology for four sequence alignments of nucleotide or amino acid sequence data. We trained neural networks with different architectures using simulated alignments for a wide range of evolutionary models, model parameters and branch lengths. By comparing the accuracy of model and topology prediction of the trained neural networks with Maximum Likelihood and Neighbour Joining methods, we show that for quartet trees, the neural network classifier outperforms the Neighbour Joining method and is in most cases as good as the Maximum Likelihood method to infer the best model of sequence evolution and the best tree topology. These results are consistent for nucleotide and amino acid sequence data. We also show that our method is superior for model selection than previously published methods based on convolutionary networks. Furthermore, we found that neural network classifiers are much faster than the IQ-TREE implementation of the Maximum Likelihood method. Our results show that neural networks could become a true competitor for the Maximum Likelihood method in phylogenetic reconstructions.

Molecular identification and genetic variation of forensically important fly species (Order: Diptera) in Thailand using DNA barcoding

Forensic entomology plays a crucial role in criminal investigations by providing vital insights into minimum postmortem interval (PMImin) and corpse relocation by identifying insect species that colonize in decomposing remains. This study aimed to identify and analyze the genetic variation of forensically significant fly species in Thailand, using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I COI gene. A total of 3,220 fly specimens were collected from 18 provinces across six regions of Thailand from October 2017 to September 2022. These specimens were classified by morphological identification into 21 species among three Dipteran families: Calliphoridae, Muscidae, and Sarcophagidae, with Chrysomya megacephala Diptera: Calliphoridae being the most abundant species. DNA barcoding confirmed the morphological identifications with 100 % accuracy, showing low intraspecific K2P distances0.0 to 1.1 %) and significant interspecific K2P distances 2.5 % to 17.2 %. A Neighbour-Joining (NJ) analysis was conducted to assess the molecular identification capabilities of the barcoding region. This analysis successfully recovered nearly all species as distinct monophyletic groups. The species groupings obtained were generally consistent with both morphological and molecular identifications. These findings underscore the effectiveness of DNA barcoding for precise species identification and contribute to a comprehensive database of forensically important flies in Thailand, thus facilitating improved forensic investigations and biodiversity studies.

Genetic diversity, population structure, and phylogenetic relationships of a widespread East Asia herb, Cryptotaenia japonica Hassk. (Apiaceae) based on genomic SNP data generated by dd-RAD sequencing.

Single-nucleotide polymorphisms (SNPs) represent the most prevalent form of genomic polymorphism and are extensively used in population genetics research. Using dd-RAD sequencing, a high-throughput sequencing method, we investigated the genome-level diversity, population structure, and phylogenetic relationships among three morphological forms of the widely distributed taxon Cryptotaenia japonica Hassk., which is native to East Asia. Our study aimed to assess the species status of C. japonica according to its genetic structure and genetic diversity patterns among 66 naturally distributed populations, comprising 26C. japonica f. japonica, 36C. japonica f. dissecta (Y. Yabe) Hara and 4C. japonica f. pinnatisecta S. L. Liou accessions. Based on genomic SNP data generated by dd-RAD sequencing, we conducted genetic diversity, principal component, neighbor-joining (NJ) phylogenetic, admixture clustering, and population differentiation analyses. The findings revealed the following: (1) 5,39,946 unlinked, high-quality SNPs, with mean π, H O, H E and F IS values of 0.062, 0.066, 0.043 and -0.014, respectively, were generated; (2) population divergence was unaffected by isolation through distance; (3) six main distinct regions corresponding to geographic locations and exhibiting various levels of genetic diversity were identified; (4) pairwise F ST analysis showed significant (P < 0.05) population differentiation in 0%-14% of populations among the six regions after sequential Bonferroni correction; and (5) three migration events (historical gene flow) indicated east‒west directionality. Moreover, contemporary gene flow analysis using Jost's D, Nei's G ST, and Nm values highlighted the middle latitude area of East Asia as a significant contributor to genetic structuring in C. japonica. Overall, our study elucidates the relatively low genetic differentiation and population structure of C. japonica across East Asia, further enhancing our understanding of plant lineage diversification in the Sino-Japanese Floristic Region.

Optimally Ordered Orthogonal Neighbor Joining Trees for Hierarchical Cluster Analysis.

We propose to use optimally ordered orthogonal neighbor-joining (O 3 NJ) trees as a new way to visually explore cluster structures and outliers in multi-dimensional data. Neighbor-joining (NJ) trees are widely used in biology, and their visual representation is similar to that of dendrograms. The core difference to dendrograms, however, is that NJ trees correctly encode distances between data points, resulting in trees with varying edge lengths. We optimize NJ trees for their use in visual analysis in two ways. First, we propose to use a novel leaf sorting algorithm that helps users to better interpret adjacencies and proximities within such a tree. Second, we provide a new method to visually distill the cluster tree from an ordered NJ tree. Numerical evaluation and three case studies illustrate the benefits of this approach for exploring multi-dimensional data in areas such as biology or image analysis.

Application effectiveness of "Youxin-1" in genetic diversity and structure analysis of local chickens.

China's local chicken breeds are rich in resources, and have formed different germplasm characteristics in the process of long-term selection and evolution. Scientific assessment of population genetic diversity and identification of inter-breed genetic structure are of great value to the protection and innovative utilization of local chicken breed resource. In order to evaluate the application effectiveness of 23K SNP chip "Youxin-1" in the analysis of genetic diversity and genetic structure of local chickens, we used RADseq to identify genomic genetic variation of 21 local chicken breeds and developed 23K chip "Youxin-1". The genetic statistics of each variety were calculated based on two sets of SNP data, and correlation, fitting and phylogenetic analysis were carried out to evaluate the application effectiveness of the chip. The results showed that the observed heterozygosity (Ho), polymorphism information content (PIC), inbred coefficient (FROH) and genetic differentiation coefficient (Fst) calculated based on the two SNP data sets were basically consistent in the 21 local chicken breeds. The genetic diversity of Langya chicken (LA), Piao chicken (PJ) and Wenchang chicken (WC) was relatively rich. The genetic diversity of Bian chickens (BJ), Langshan chickens (LS), Gushi chickens (GS), Dongxiang blue-eggshell chickens (DX) and Beijing fatty chickens (BY) was relatively poor, and the correlation coefficients of Ho, PIC, FROH and average Fst in the two groups were 0.794, 0.901, 0.926 and 0.984, respectively, all reaching extremely significant levels (P<0.01) with a high degree of fit (P<0.001) and R2 were 0.644, 0.827, 0.916 and 0.927. For the two sets of SNP data, the evolutionary tree constructed by neighbor-joining (NJ) method and maximum likelihood (ML) method was reasonable, and the 21 local chicken breeds were generally divided into six categories, which was consistent with the formation history and geographical distribution of the varieties. The 23K chip also realized reasonable clustering of the five new varieties without individual deviation. There are some differences in the estimation of genetic statistics using SNP with different densities, and data standardization is needed. 23K chip has good efficacy in the analysis of genetic diversity and structure of local chickens.

Genetic Diversity and Population Structure of Dülmen Wild, Liebenthal and Polish Konik Horses in Comparison with Przewalski, Sorraia, German Draught and Riding Horses.

The objective of the present study was to analyze the genetic diversity, individual-based assessment of population structure, and admixture in the Dülmen wild horse population in comparison to warmblood, coldblood, and primitive horse populations. The Dülmen wild horse is kept as a unique horse population in the Merfelder Bruch near Dülmen in Westphalia, Germany, and since 1856 has been managed by the Dukes of Croÿ. The Dülmen wild horse population is exposed to the natural conditions of the Merfelder Bruch all year round without human interventions for feeding and veterinary care. In the present study, genetic diversity was estimated for 101 Dülmen wild horses using multilocus genotypic information from a set of 29 autosomal microsatellites and compared with 587 horses from 17 different horse populations. Dülmen wild horses maintained a high degree of genetic diversity, with an average observed heterozygosity of 0.68, a mean number of 6.17 alleles, and heterozygote deficit of -0.035. Pairwise genetic distances (FST, Nei's standard, and Cavalli-Sforza distances) were closest to German coldblood breeds, Polish Konik, and Icelandic horses and most divergent from Sorraia and Przewalski's horses. Neighbor joining dendrogram and PCA plots showed a clear distinction of Dülmen wild horses from other populations, particularly from Przewalski horses. Posterior Bayesian analysis confirmed clear differentiation from other horse populations without an admixture pattern and a high membership index (0.92). It was possible to distinguish Dülmen wild horses from Dülmen and Polish Konik horses. In conclusion, Dülmen wild horses show a notable separation from other German horse breeds and primitive horse populations and may serve as a resource to study evolution of equine domestication.

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

Insights into the genomic homogeneity of Moroccan indigenous sheep breeds though the lens of runs of homozygosity

Numerous studies have indicated that Morocco’s indigenous sheep breeds are genetically homogenous, posing a risk to their survival in the challenging harsh climate conditions where they predominantly inhabit. To understand the genetic behind genetic homogeneity through the lens of runs of homozygosity (ROH), we analyzed the whole genome sequences of five indigenous sheep breeds (Beni Guil, Ouled Djellal, D’man, Sardi, Timahdite and Admixed).The results from principal component, admixture, Fst, and neighbour joining tree analyses consistently showed a homogenous genetic structure. This structure was characterized by an average length of 1.83 Mb for runs of homozygosity (ROH) segments, with a limited number of long ROH segments (24–48 Mb and > 48 Mb). The most common ROH segments were those ranging from 1–6 Mb. The most significant regions of homozygosity (ROH Islands) were mostly observed in two chromosomes, namely Chr1 and Chr5. Specifically, ROH Islands were exclusively discovered in the Ouled Djellal breed on Chr1, whereas Chr5 exhibited ROH Islands in all breeds. The analysis of ROH Island and iHS technique was employed to detect signatures of selection on Chr1 and Chr5. The results indicate that Chr5 had a high level of homogeneity, with the same genes being discovered across all breeds. In contrast, Chr1 displays some genetic variances between breeds. Genes identified on Chr5 included SLC39A1, IL23A, CAST, IL5, IL13, and IL4 which are responsible for immune response while genes identified on Chr1 include SOD1, SLAMF9, RTP4, CLDN1, and PRKAA2. ROH segment profile and effective population sizes patterns suggests that the genetic uniformity of studied breeds is the outcome of events that transpired between 250 and 300 generations ago. This research not only contributes to the understanding of ROH distribution across breeds but helps design and implement native sheep breeding and conservation strategies in Morocco. Future research, incorporating a broader sample size and utilizing the pangenome for reference, is recommended to further elucidate these breeds’ genomic landscapes and adaptive mechanisms.