Sequence Markers Research Articles

Phylogeny and taxonomy of the genera of Erysiphaceae, part 5: Erysiphe (the “Microsphaera lineage” part 1)

ABSTRACT In this contribution, we offer the fifth installment of a series focusing on the phylogeny and taxonomy of powdery mildews. This paper is the second segment evaluating the genus Erysiphe. The first treatment of Erysiphe focused on phylogenetically basal species in the “Uncinula lineage.” This research presents a phylogenetic-taxonomic assessment of species that form the group previously referred to as the “Microsphaera lineage.” Given the size of the group, we split the treatment of this lineage of Erysiphe species into two parts based on their phylogenetic placement. Phylogenetic trees based on ITS+28S data are supplemented by sequences of additional markers (CAM, GADPH, GS, RPB2, and TUB). Included in the analysis of the Microsphaera lineage is the “Erysiphe aquilegiae complex” (group, clade, cluster), which encompasses sequences obtained from an assemblage of Erysiphe species with insufficient resolution in rDNA analyses. Attempts have been made to resolve this group at the species level by applying a multilocus approach. A detailed discussion of the “Erysiphe aquilegiae complex” is provided. Sequences are provided for the first time for several species, particularly North American species, such as Erysiphe aggregata, E. erineophila, E. parnassiae, and E. semitosta. Ex-type sequences for Microsphaera benzoin and M. magnusii have been retrieved. Alphitomorpha penicillata, Microsphaera vanbruntiana, and M. symphoricarpi are epitypified with ex-epitype sequences. The new species Erysiphe alnicola, E. deutziana, E. cornigena, E. lentaginis, and E. sambucina are described, the new combinations E. lauracearum, E. passiflorae, and E. sambucicola are introduced, and the new name E. santali is proposed.

Phylogenetic analysis of a region of mitochondrial cox-1 as a DNA barcode marker sequence of Gazella subgutturosa (Goitered gazelle) in Mongolia

Gazella subgutturosa, a vulnerable species, is threatened by illegal hunting for meat and sport. The mitochondrial cytochrome c oxidase subunit 1 gene (cox-1) is used as a DNA marker to distinguish mammalian species for the investigation of illegal hunting. In this study, we sequenced a part of the cox-1 gene (709 bp) of six Mongolian G. subgutturosa individuals. Our DNA sequences were clustered in a clade of Gazella which is distinct from other clades of mammalian species in the phylogenetic tree. Our findings suggest that DNA sequences can be useful in the investigation of illegal hunting.

Using Next Generation Sequencing of Flow Cytometry CD Markers and Machine Learning As a Replacement to Flow Cytometry Analysis for the Diagnosis of Hematologic Neoplasms

Introduction: Flow cytometry performs multi-parameter analysis of cells and analyzes surface and intracellular markers for accurate phenotypic characterization of a cell population. Flow cytometry is used extensively in the diagnosis and classification of various hematologic neoplasms. However, analysis of the generated data is time consuming and remains subjective, requiring special skill and experience. Furthermore, some diagnostic classes, such as myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS), are difficult to diagnose using flow cytometry. The RNA levels of the CD markers used in flow cytometry can be reliably quantified using next generation sequencing (NGS). However, when all cells are jointly sequenced, studying subpopulation of cells is lost, which hinders accurate diagnosis. However, machine learning algorithms are capable of multi-marker normalizing and compensate for the loss of subclonal analysis. To validate this assumption, we explored the potential of using the RNA levels of 30 CD markers along with a machine learning algorithm in the differential diagnosis between various types of hematologic neoplasms. Methods: RNA was extracted from fresh bone marrow and peripheral blood samples from 172 acute myeloid leukemia (AML), 369 normal control, 68 MPN, 218 MDS, 93 acute lymphoblastic leukemia (ALL), 74 chronic lymphocytic leukemia (CLL), 38 mantle cell lymphoma, and 83 multiple myeloma cases. The samples were consecutive and collected without selection. RNA sequencing was performed using a targeted hybrid capture panel that included CD1A, CD2, CD3D, CD3E, CD3G, CD4, CD5, CD7, CD8A, CD8B, CD10, CD14, CD19, CD20, CD22, CD33, CD34, CD38, CD40, CD44, CD47, CD68, CD70, CD74, CD79A, CD79B, CD81, CD138, CD200, and CD274 genes. Salmon v1.4.0 software was used for expression quantification (TPM). Machine learning algorithm (random forest) was used for classifying diseases. Two thirds of samples were used for training the random forest algorithm and one third was used for testing. Results: While frequently a diagnosis can be made by simply inspecting the RNA levels of various CD markers, machine learning is needed when the fraction of the neoplastic cells is low. Using machine learning (random forest), diagnosis of most hematologic neoplasms was achieved with high sensitivity and specificity in the testing set. Area under the curve (AUC) was at 0.972 (95% CI: 0.950-0.994) for AML vs. normal, 0.936 (95% CI: 0.898-0.974) for normal vs. MM, 0.965 (95% CI: 0.909-1.00) for mantle vs. CLL, 0.962 (95% CI: 0.907-1.00) for CLL vs. ALL, 0.935 (95% CI: 0.866-1.00) for CLL vs. normal, and 0.964 (95% CI: 0.927-1.00) for AML vs. ALL. Diseases that are difficult to diagnose by routine flow cytometry were diagnosed by RNA expression and machine learning at acceptable accuracy. For example, AUC was at 0.761 (95% CI: 0.689-0.834) for MDS vs. normal, 0.831 (95% CI: 0.762-0.901) for MDS vs. AML, 0.888 (95% CI: 0.822-0.954) for MDS vs. MPN, and 0.785 (95% CI: 0.698-0.872) for MPN vs. normal. Conclusions: This data demonstrates that NGS quantification of RNA from 30 CD markers when combined with machine learning is adequate for reliable diagnosis of various types of hematologic neoplasms. This approach can provide valuable information to distinguish between MPN, MDS, and normal bone marrow that flow cytometry cannot provide. Furthermore, this technology can be automated and less susceptible to human error and practically can be used as a replacement to routine flow cytometry analysis.

DNA Markers, Pathogenicity Test, and Multilocus Sequence Analysis to Differentiate and Characterize Cereal-Specific Xanthomonas translucens Strains.

Xanthomonas translucens contains a group of bacterial pathogens that are closely related and have been divided into several pathovars based on their host range. X. translucens pv. undulosa (Xtu) and X. translucens pv. translucens (Xtt) are two important pathovars that cause bacterial leaf streak disease on wheat and barley, respectively. In this study, DNA markers were developed to differentiate Xtu and Xtt and were then used to characterize a collection of X. translucens strains with diverse origins, followed by confirmation and characterization with pathogenicity tests and multilocus sequence analysis/typing (MLSA/MLST). We first developed cleaved amplified polymorphic sequence markers based on the single-nucleotide polymorphisms within a cereal pathovar-specific DNA sequence. In addition, two Xtt-specific markers, designated Xtt-XopM and Xtt-SP1, were developed from comparative genomics among the sequenced Xtt/Xtu genomes. Using the developed markers, a collection of X. translucens strains were successfully identified as Xtu or Xtt. Pathogenicity tests on wheat and barley plants and MLSA of four housekeeping genes validated the pathovar assignation of those strains. Furthermore, MLSA revealed distinct subclades within both Xtu and Xtt groups. Seven and three sequence types were identified from MLST for Xtu and Xtt strains, respectively. The establishment of efficient Xtt/Xtu differentiation methods and characterization of those strains will be useful in studying disease epidemiology and host-pathogen interactions and breeding programs when screening for sources of resistance for these two important bacterial pathogens.

Investigation of phylogenetic relationships within Saxifraga diversifolia complex (Saxifragaceae) based on restriction-site associated DNA sequence markers.

Subsect. Hirculoideae Engl. & Irmsch., belonging to Saxifraga sect. Ciliatae Haw., has high species richness. It can be divided into S. diversifolia, S. pseudohirculus, and S. sinomontana complexes based on morphological characteristics. The species with prominent leaf veins on the posterior leaf edge were placed in the S. diversifolia complex, which is mainly distributed on the eastern and southern margins of the Qinghai-Tibetan Plateau. In this study, 53 samples, representing 15 of the 33 described species in the S. diversifolia complex, were sequenced using the Restriction-site Associated DNA Sequence (RAD-seq) technique. A total of 111,938 high-quality SNP loci were screened to investigate the phylogenetic relationships within the S. diversifolia complex. The result of the neighbor-joining (NJ) tree shows that the S. diversifolia complex is a paraphyletic group. Despite of some inconsistencies as revealed by genetic structural analysis, clustering results of representative species reconstructed by both NJ and principal component analysis analyses support previous biogeographic and morphological evidences. In addition, long-distance gene flow events for 11 taxa were detected in the S. diversifolia complex, respectively from S. implicans 1 to S. implicans 2, S. diversifolia and S. maxionggouensis, and from S. maxionggouensis to S. nigroglandulifera. These findings may improve our comprehension of the phylogeny, classification, and evolution of the S. diversifolia complex.

Molecular Mapping of Putative Genomic Regions Controlling Fruit and Seed Morphology of Watermelon.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F2 mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77-38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

Cyathus tongxinianus sp. nov. (Basidiomycota) from China

A new species of bird’s nest fungus, Cyathus tongxinianus, is proposed based on a combination of morphological and molecular evidence. Cyathus tongxinianus is characterized by the cupulate to obconical basidiomata with the smooth outer and inner surface of the peridium, a dimitic hyphal system of peridium walls with generative hyphae having clamp connections, the presence of the fimbriate mouth, brown black to dark grey peridioles, a monomitic hyphal system bearing the generative hyphae with clamp connections, the presence of crystals, and sub-globose, thick-walled basidiospores (8.5–19.5 × 7.5–11). Sequences of ITS and nLSU rRNA markers of the studied samples were generated, and the phylogenetic analyses were performed with maximum likelihood, maximum parsimony and bayesian inference methods on two datasets (ITS+nLSU and ITS). The analyses showed that the new species clustered into the genus Cyathus within the family Nidulariaceae based on the ITS+nLSU phylogenetic tree. The phylogenetic analyses based on molecular data from ITS sequences showed that C. tongxinianus is sister to C. annulatus, and groups with C. renweii.

Mapping of prehaustorial resistance against wheat leaf rust in einkorn (Triticum monococcum), a progenitor of wheat

Wheat leaf rust (Puccinia triticina) is one of the most significant fungal diseases of wheat, causing substantial yield losses worldwide. Infestation is currently being reduced by fungicide treatments and mostly vertical resistance. However, these measures often break down when the fungal virulence pattern changes, resulting in a breakdown of vertical resistances. In contrast, the prehaustorial resistance (phr) that occurs in the einkorn–wheat leaf rust interaction is race-independent, characterized by an early defense response of plants during the prehaustorial phase of infestation. Einkorn (Triticum monococcum) is closely related to Triticum urartu as a progenitor of wheat and generally shows a high level of resistance against leaf rust of wheat. Hence, einkorn can serve as a valuable source to improve the level of resistance to the pathogen in future wheat lines. In particular, einkorn accession PI272560 is known to exhibit a hypersensitive prehaustorial effector triggered immune reaction, preventing the infection of P. triticina. Remarkably, this effector-triggered immune reaction turned out to be atypical as it is non-race-specific (horizontal). To genetically dissect the prehaustorial resistance (phr) in PI272560, a biparental F2 population of 182 plants was established after crossing PI272560 with the susceptible T. boeoticum accession 36554. Three genetic maps comprising 2,465 DArT-seq markers were constructed, and a major QTL was detected on chromosome 5A. To locate underlying candidate genes, marker sequences flanking the respective QTL were aligned to the T. urartu reference genome and transcriptome data available from the parental accessions were used. Within the QTL interval of approximately 16.13 million base pairs, the expression of genes under inoculated and non-inoculated conditions was analyzed via a massive analysis of cDNA (MACE). Remarkably, a single gene located 3.4 Mbp from the peak marker within the major QTL was upregulated (20- to 95-fold) after the inoculation in the resistant accession in comparison to the susceptible T. boeoticum accession. This gene belongs to a berberine bridge enzyme-like protein that is suspected to interact on the plant surface with glycoside hydrolases (GH) secreted by the fungus and to induce a hypersensitive defense reaction in the plant after fungal infections.

Species-specific coral microbiome assemblages support host bleaching resistance during an extreme marine heatwave

Scleractinian assemblages are threatened by marine heat waves with coral survivorship depending on host genetics and microbiome composition. We documented an extreme marine heat wave in Fiji and the response of corals in two thermally stressed reef flats. Through high-throughput amplicon sequencing of 16S and ITS rDNA phylogenetic markers, we assessed coral microbiomes (Symbiodiniaceae, prokaryotes, fungi, and Apicomplexa) of paired bleached and unbleached colonies of four common coral species representative of dominant genera in the South Pacific. While all coral species exhibited one or more pathways to bleaching resistance, harboring assemblages composed primarily of thermally tolerant photosymbionts did not always result in host bleaching resistance. Montipora and Pocillopora species, which associate with diverse Symbiodiniaceae and vertically transmit their photosymbionts, fared better than Acropora, which acquire their photosymbionts from the environment, and Porites, which associate with a narrow photosymbiont assemblage. Prokaryotic and fungal beta diversity did not differ between bleached and unbleached conspecifics, however, the relative abundance of the fungus Malassezia globosa was significantly greater in unbleached colonies of Montipora digitata. Each coral species harbored distinct assemblages of Symbiodiniaceae, prokaryotes, and Apicomplexa, but not fungi, reiterating the importance of host genetics in structuring components of its microbiome. Terrestrial fungal and prokaryotic taxa were detected at low abundance across coral microbiomes, indicating that allochthonous microbial inputs occur, but that coral microbiomes remain dominated by marine microbial taxa. Our study offers valuable insights into the microbiome assemblages associated with coral tolerance to extreme water temperatures.

Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus

BackgroundIxodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ).MethodsA multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis.ResultsSequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected.ConclusionsWe developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.Graphical abstract

<i>In Silico</i> Approach for DNA Barcoding using Phylogenetic Analysis of <i>Coelogyne</i> spp. based on the <i>mat</i>K, <i>rpo</i>C1, <i>rbc</i>L and nrDNA Markers

In silico biology is considered as an effective and applicable approach to initiate various research, such as biodiversity taxonomical conservation. Phylogenetic analysis using in silico taxonomy method for orchid species can provide data on genetic diversity and evolutionary relationships. One particular method that can be used to evaluate specific targets of gene loci in the taxonomic study is DNA barcoding. This research was conducted to determine the specific target locus gene using matK, rbcL, rpoC1, and nrDNA markers for DNA barcoding of the Coelogyne genus with in silico approach using phylogenetic analysis. All marker sequences were collected from the NCBI website and analysed using several softwares and methods, namely Clustal X for sample sequence alignment and MEGA 11 for phylogenetic tree construction and analysis. The results showed that the gene locus in Coelogyne recommended was the nrDNA gene locus. Phylogenetic analysis revealed that the use of the nrDNA gene locus was able to separate 17 Coelogyne species with two outgroup species, namely Cymbidium and Vanilla, then followed with ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) while the other gene loci, namely maturase K (matK) and polymerase beta' subunit (rpoC1) provided a visual phylogenetic tree in which the two outgroup species entered into the same clade as the Coelogyne species. Thus, the results of this study can be used as a reference to support the Coelogyne breeding and conservation program.

Flavonoid Biosynthesis Pathway May Indirectly Affect Outcrossing Rate of Cytoplasmic Male-Sterile Lines of Soybean.

(1) Background: Cytoplasmic male sterility (CMS) is important for exploiting heterosis. Soybean (Glycine max L.) has a low outcrossing rate that is detrimental for breeding sterile lines and producing hybrid seeds. Therefore, the molecular mechanism controlling the outcrossing rate should be elucidated to increase the outcrossing rate of soybean CMS lines; (2) Methods: The male-sterile soybean lines JLCMS313A (with a high outcrossing rate; HL) and JLCMS226A (with a low outcrossing rate; LL) were used for a combined analysis of the transcriptome (RNA-seq) and the targeted phenol metabolome; (3) Results: The comparison between HL and LL detected 5946 differentially expressed genes (DEGs) and 81 phenolic metabolites. The analysis of the DEGs and differentially abundant phenolic metabolites identified only one common KEGG pathway related to flavonoid biosynthesis. The qRT-PCR expression for eight DEGs was almost consistent with the transcriptome data. The comparison of the cloned coding sequence (CDS) regions of the SUS, FLS, UGT, and F3H genes between HL and LL revealed seven single nucleotide polymorphisms (SNPs) only in the F3H CDS. Moreover, five significant differentially abundant phenolic metabolites between HL and LL were associated with flavonoid metabolic pathways. Finally, on the basis of the SNPs in the F3H CDS, one derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to distinguish between HL and LL soybean lines; (4) Conclusions: The flavonoid biosynthesis pathway may indirectly affect the outcrossing rate of CMS sterile lines in soybean.

A Novel PCR-Based Functional Marker of Rice Blast Resistance Gene Pi25

Rice blast is arguably the most devastating fungal disease of rice. Utilization of resistance genes to breed resistant cultivars is one of the most economical and environmentally friendly approaches to combat the disease. Pi25, a major resistance gene conferring broad-spectrum resistance to both leaf and neck blast, is an ideal gene resource to improve the resistance of rice varieties to blast. Recently, several allele-specific markers were developed. However, they were deficiently efficient due to either an additional process of restriction enzyme digestion for cleaved amplified polymorphic sequence (CAPS) markers or the risk of false-positive error in identifying susceptible Tetep allele (Pi25TTP) for PCR-based markers. In this study, based on a conserved single nucleotide polymorphism (SNP) between resistant and susceptible alleles, a tetra-primer amplification refractory mutation system (ARMS)-PCR marker was developed. The new marker, namely Pi25-2687R3, could effectively distinguish the resistant Gumei 2 (GM2) allele (Pi25GM2) and the susceptible allele Pi25TTP. Moreover, a perfect consistency of genotyping was exhibited between Pi25-2687R3 and published CAPS marker CAP3/Hpy 99I. A more accurate genotyping was also displayed compared to the previous PCR-based SNP marker Pi25-2566. Our finding proved that Pi25-2687R3 could achieve the same result as CAP3/Hpy 99I with less workload and cost and could promote the accuracy in the identification of genotypes superior to Pi25-2566. This study provided a quick and reliable functional marker for discriminating Pi25 alleles, which would be a valuable tool for genotypic assay and rice molecular breeding of blast resistance.

Fine mapping of the major gene BhHLS1 controlling seed size in wax gourd (Benincasa hispida).

The seed size of wax gourds is an important agronomic trait; however, the associated genes have not yet been reported. In this study, we used a high-density genetic map constructed based on F8 recombinant inbred line populations derived from a cross between MY-1 (large seed) and GX-71 (small seed) strains to detect quantitative trait locis (QTLs) for seed-size-related traits in wax gourd over a two-year period. Two stable QTLs (qSL10 and qSW10) for seed length (SL) and seed width (SW) on chromosome 10 were repeatedly detected over two years (2021-2022). qSL10 had a phenotypic variation rate of 75.30% and 80.80% in 2021 and 2022, respectively. Whereas, qSW10 had a phenotypic variation rate of 66.60% and 73.80% in 2021 and 2022, respectively. Further, a single nucleotide polymorphism mutation was found to cause early termination of Bch10G006400 (BhHLS1) translation in GX-71 through sequencing analysis of candidate genes. Based on gene functional annotation and quantitative real-time PCR analyses, BhHLS1 encoded a probable N-acetyltransferase HLS1-like protein and its expression level was significantly different between parents. Therefore, BhHLS1 is a major candidate gene associated with a one-factor polymorphism regulating the SL and SW of wax gourds. Finally, based on variation in the BhHLS1 sequence, a cleaved amplified polymorphic sequence marker was developed for the molecular marker-assisted breeding of wax gourds. Overall, this study is of great significance for the genetic improvement of seed size, verification of gene functions, and cultivation of specific germplasm resources for wax gourds.

New branched Porolithon species (Corallinales, Rhodophyta) from the Great Barrier Reef, Coral Sea, andLord Howe Island.

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.

Identification of a novel locus C2 controlling canary yellow flesh color in watermelons.

The flesh color of watermelon is an important trait that is determined by carotenoid composition and affects consumers' fruit desirability. Although a complete dominant control by C locus (Cllcyb) for canary yellow flesh (CY) over red flesh has been reported, red and CY colors frequently appear as a mixed pattern in the same flesh (incomplete canary yellow, ICY) in F1 and inbred lines carrying dominant C alleles. Therefore, we examined the genetic control of the mixed color pattern in ICY using whole-genome resequencing of three ICY (ICY group) and three CY inbred lines (CY group), as well as genetic linkage mapping of an F2 population. The segregation pattern in 135 F2 plants indicated that CY is controlled by a single locus (named C 2) dominant over ICY. The whole-genome resequencing of ICY and CY inbred lines revealed an ICY/CY-specific region of approximately 27.60-27.88Mb on Chr. 2 that was polymorphic between the ICY and CY groups. Our genetic map, using nine cleaved amplified polymorphic sequence markers developed based on the single-nucleotide polymorphisms from the ICY/CY-specific region, confirmed that C 2 is located on Chr. 2 and cosegregated with the marker (M7) derived from a non-synonymous single-nucleotide polymorphism of the pentatricopeptide repeat (PPR) gene (ClPPR, Cla97C02G039880). Additionally, 27 watermelon inbred lines of ICY, CY, and red flesh were evaluated using previously reported Cllcyb (C locus)-based markers and our C 2 locus-linked ClPPR-based marker (M7). As a result, dominant alleles at the C 2 locus were required to produce CY, in addition to dominant alleles at the C locus, while a recessive homozygous genotype at the C locus gave the red flesh irrespective of the genotype at the C 2 locus. Using a ClPPR-based cleaved amplified polymorphic sequence developed in this study and Cllcyb-based markers, watermelon cultivars with CY, ICY, and red flesh could be successfully discerned, implying that the combined use of these markers will be efficient for marker-assisted selection of flesh color in watermelon breeding.

Identification of G4 motifs of various stem cell markers and their biophysical and biochemical characterization

Regulatory regions in the human genome, enriched in guanine-rich DNA sequences have a remarkable enrichment of G-rich sequences having a tendency to fold into G-quadruplex structures. To identify the G-quadruplex forming motifs in regulatory regions of stem cell markers, gene sequences of various stem cell markers were downloaded and analyzed to see the abundance of G-rich sequences. We observed the enrichment of G-rich sequences in stem cell markers (CD13, CD19, CD24 and CD38) which could possibly play a critical role in its regulation. We used Circular Dichroism (CD), UV-Thermal denaturation (UV-T m) and polyacrylamide gel electrophoresis (PAGE) to demonstrate the formation of a G-quadruplex by G-rich sequences present in these stem cell markers. We observed that these G-rich sequences containing minimum consecutive G3 stretch separated by loop length ranging from one to three bases long adopt G-quadruplexes with different molecularity involving two-strands, three-strand and four-strand with parallel and antiparallel conformation. Interestingly, we proposed the formation of three-stranded G-quadruplex by CD13 in 100 mM Na+, CD19 in 100 mM K+, 100 mM K+ with 40 wt% PEG 200, and CD38 in 100 mM K+ + 40 wt% PEG 200. The formation of such diverse G-quadruplex structures in the regulatory regions leaves the fair possibility of recognition by regulatory factors to modulate the gene expression. First time, this study may give insight into the structural polymorphism of G4 forming motifs in different stem cell markers to design the best suitable ligand and to target them for therapeutic development. Communicated by Ramaswamy H. Sarma

Identification of male-specific markers by 2b-RAD sequencing reveals an XX/XY sex-determination system in cultured lined seahorse, Hippocampus erectus

Seahorses (Hippocampus spp.), a group of male ovoviviparous fish, are of high commercial value in traditional Chinese medicine and aquarium trades. The lined seahorse (Hippocampus erectus), an easily domesticated species, was introduced to China and quickly became a popular farmed species to relieve the conflict between supply and demand. Although the sexual dimorphism in size and immunocompetence has been observed, the sex determination mechanism in the lined seahorse remains unclear. In the present study, we applied the 2b-RAD-seq approach for the first time to successfully identify 6 male-specific tags and 4 sex-specific SNP loci in the lined seahorse, which were further validated in 108 individuals by PCR amplification based on the flanking sequences of sex-specific markers. The male-specific tags and heterozygous SNP loci indicate that a male heterozygous XX/XY sex-determination system is employed in the lined seahorse. Based on the annotation and analysis of the sex-specific sequences, two sex-related candidate genes, lrriq1 and il34, were identified. The results provide novel insights into sex-determination mechanisms and promising methods for all-male breeding of Hippocampus erectus.

Using morphological, chemical, and molecular data to study the diversity of Xanthoparmelia species from South Africa (Ascomycota, Parmeliaceae)

AbstractThere is still a high diversity of lichen-forming fungi that remains undescribed, especially cryptic lineages at the species level. Integrating morphological, chemical, and DNA sequence data has proved useful in corroborating species descriptions and delimitations. Here we reviewed morphological features, secondary metabolites and the DNA sequences of ITS, mtSSU and nuLSU markers to study the diversity of Xanthoparmelia in southern Africa. A total of 37 species were recorded. Three of these appear undescribed, and we therefore describe them here as new: Xanthoparmelia nimisii, with a sorediate thallus and broad lobes, is well supported as a clade separate from X. annexa; X. pseudochalybaeizans with a white medulla is phylogenetically distinct from the otherwise similar X. chalybaeizans; and X. sipmaniana, well supported as a separate clade from the similar X. hypoprocetrarica. In addition, the separation of Xanthoparmelia capensis and X. tinctina requires further studies.

Toward Integrating EBPR and the Short-Cut Nitrogen Removal Process in a One-Stage System for Treating High-Strength Wastewater.

Enhanced biological phosphorus removal (EBPR) is an economical and sustainable process for phosphorus removal from wastewater. Despite the widespread application of EBPR for low-strength domestic wastewater treatment, limited investigations have been conducted to apply EBPR to the high-strength wastewaters, particularly, the integration of EBPR and the short-cut nitrogen removal process in the one-stage system remains challenging. Herein, we reported a novel proof-of-concept demonstration of integrating EBPR and nitritation (oxidation of ammonium to nitrite) in a one-stage sequencing batch reactor to achieve simultaneous high-strength phosphorus and short-cut nitrogen removal. Excellent EBPR performance of effluent 0.8 ± 1.0 mg P/L and >99% removal efficiency was achieved fed with synthetic high-strength phosphorus wastewater. Long-term sludge acclimation proved that the dominant polyphosphate accumulating organisms (PAOs), Candidatus Accumulibacter, could evolve to a specific subtype that can tolerate the nitrite inhibition as revealed by operational taxonomic unit (OTU)-based oligotyping analysis. The EBPR kinetic and stoichiometric evaluations combined with the amplicon sequencing proved that the Candidatus Competibacter, as the dominant glycogen accumulating organisms (GAOs), could well coexist with PAOs (15.3-24.9% and 14.2-33.1%, respectively) and did not deteriorate the EBPR performance. The nitrification activity assessment, amplicon sequencing, and functional-based gene marker quantification verified that the unexpected nitrite accumulation (10.7-21.0 mg N/L) in the high-strength EBPR system was likely caused by the nitritation process, in which the nitrite-oxidizing bacteria (NOB) were successfully out-selected (<0.1% relative abundance). We hypothesized that the introduction of the anaerobic phase with high VFA concentrations could be the potential selection force for achieving nitritation based on the literature review and our preliminary batch tests. This study sheds light on developing a new feasible technical route for integrating EBPR with short-cut nitrogen removal for efficient high-strength wastewater treatment.