Characterization of the cryptic interspecific hybrid Lemna × mediterranea by an integrated approach provides new insights into duckweed diversity.

Lemnaceae taxonomy is challenged by the particular morphology of these tiny free-floating angiosperms. Although molecular taxonomy has helped clarify the phylogenetic history of this family, some inconsistency with morphological data leads to frequent misclassifications in the genus Lemna. Recently, the finding that Lemna japonica is an interspecific hybrid between Lemna minor and Lemna turionifera, provided a clear explanation to one of such taxonomic questions. Here we demonstrated that L. minor is also capable to hybridize with Lemna gibba, generating a cryptic but widespread taxon in the Mediterranean area. The nothotaxon Lemna × mediterranea is described and compared with clones of the putative parental species L. minor and L. gibba. Genetic analysis by nuclear and plastid markers, as well as genome size measurement, revealed that two different cytotypes, diploid and triploid, originated by at least two independent hybridization events. Despite high overall similarity, morphometrical, physiological and biochemical analyses showed an intermediate position of L. × mediterranea between its parental species in most qualitative and quantitative characters, and also separation of the two hybrid cytotypes by some criteria. These data provide evidence that hybridization and polyploidization, driving forces of terrestrial plant evolution, contribute to the duckweed genetic diversity and may have shaped the phylogenetic history of these mainly asexual, aquatic plants.

Decision-making regarding immediate vs. interval postpartum levonorgestrel intrauterine device insertion timing.

BACKGROUND: Understanding decision-making for contraception initiation timing postpartum may help guide patients in selecting a contraceptive method most aligned with their reproductive goals. The objective of this study was to explore the decision-making process in patients who chose immediate postplacental (IPP) levonorgestrel intrauterine device (LNG IUD) insertion versus interval insertion at the postpartum follow-up visit. METHODS: We recruited English-speaking, reproductive-aged adult postpartum participants who received either an IPP or interval postpartum LNG IUD from September to December 2017 at MetroHealth Medical Center. We conducted interviews over the phone utilizing a pilot-tested, semi-structured interview guide. Interview topics included past experiences with contraception, provider counseling, intrapartum factors, and current experiences after IUD insertion. RESULTS: We interviewed 20 participants (12 IPP and eight interval IUD recipients). Participants receiving an IPP IUD described convenience, desire for immediate contraception, pain control and availability of alternative contraception options as influential for their decision. Patients who received an interval IUD performed outside research, focused on the events surrounding delivery, and generally favored additional recovery time before obtaining an IUD. Patients who received interval IUDs were often not aware that IPP IUDs were available. Early, frequent, and comprehensive counseling was viewed favorably when compared to counseling upon arrival to the laboring unit. While overall there was congruence of participant expectations and experiences, unexpected expulsion affected desire for future IUD use in some participants. CONCLUSION: Providers should be mindful that prior experience and knowledge as well as delivery room considerations affect insertion timing decision-making. TRIAL REGISTRATION: N/A.

Tubulin-Based Polymorphism (TBP) in Plant Genotyping.

Tubulin-based polymorphism (TBP) is an intron length polymorphism (ILP) method widely applicable to any plant species and particularly suitable for a first and rapid classification of any plant genome. It is based on the selective, polymerase chain reaction (PCR)-based amplification of the two introns present at conserved positions within the coding sequences of plant ß-tubulin genes. Amplification releases a simple yet distinctive genomic profile.

Strategies for Intraspecific Genotyping of Duckweed: Comparison of Five Orthogonal Methods Applied to the Giant Duckweed Spirodela polyrhiza.

The predominantly vegetative propagating duckweeds are of growing commercial interest. Since clonal accessions within a respective species can vary considerably with respect to their physiological as well as biochemical traits, it is critical to be able to track the clones of species of interest after their characterization. Here, we compared the efficacy of five different genotyping methods for Spirodela polyrhiza, a species with very low intraspecific sequence variations, including polymorphic NB-ARC-related loci, tubulin-gene-based polymorphism (TBP), simple sequence repeat variations (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq), and low-coverage, reduced-representation genome sequencing (GBS). Four of the five approaches could distinguish 20 to 22 genotypes out of the 23 investigated clones, while TBP resolved just seven genotypes. The choice for a particular method for intraspecific genotyping can depend on the research question and the project budget, while the combination of orthogonal methods may increase the confidence and resolution for the results obtained.

A Combinatorial Q-Locus and Tubulin-Based Polymorphism (TBP) Approach Helps in Discriminating Triticum Species.

The simple and straightforward recognition of Triticum species is not an easy task due to their complex genetic origins. To provide a recommendation, we have compared the performance of different PCR-based methods relying on the discrimination ability of the Q- and γ-gliadin (GAG56D) genes, as well as TBP (Tubulin-Based Polymorphism), a method based on the multiple amplification of genes of the ß-tubulin family. Among these approaches, the PCR-RFLP (Restriction Fragment Length Polymorphism) assay based on a single-nucleotide polymorphism (SNP) present in the Q gene is the only one capable of fully discerning hexaploid spelt and common wheat species, while both γ-gliadin and TBP fail with similar error frequencies. The Q-locus assay results in the attainment of either a single fragment or a doublet, depending on the presence of a suitable restriction site, which is affected by the mutation. This dual pattern of resolution limits both the diagnostic effectiveness, when additional Triticum species are assayed and compared to each other, and its usefulness, when commercially available flours are analyzed. These limitations are overtaken by flanking the Q-locus assay with the TBP analysis. In this way, almost all of the Triticum species can be accurately identified.

Postpartum Contraceptive Decision-Making of Parous Teens-A Qualitative Study.

STUDY OBJECTIVE: Approximately 25% of teens in the United States will become pregnant before the age of 18, and within 2 years, more than 31% will have a repeat pregnancy. Acknowledging that some adolescents might seek or be ambivalent toward rapid repeat pregnancy, compared with their counterparts, not using a long-acting reversible contraception method increases a teen's risk of another pregnancy in 2 years by more than 35 times. We seek to better understand the influences and factors surrounding adolescent postpartum contraceptive decision-making following the index delivery. DESIGN: We completed a qualitative study via focused, semistructured interviews during an inpatient postpartum course. The interview guide was modeled after those used in other studies of adolescent contraceptive decision-making, beta tested, and developed iteratively. Interviews were completed, transcribed, coded, and analyzed with the assistance of Dedoose. SETTING: The study was conducted at MetroHealth Medical Center in Cleveland, Ohio. PARTICIPANTS: Parous adolescents aged 13-19 RESULTS: We performed 12 interviews prior to reaching theoretical saturation. Themes were identified related to the participants' prior experiences with contraception and prior and current pregnancies. The participants' contraceptive choices were influenced by personal relationships, varying levels of autonomy, misperceptions, and changing contraceptive needs. CONCLUSIONS: We found that adolescents' contraceptive decision-making was influenced by their social networks and community, including their parents and friends. Mothers played a key role as adolescents transitioned to gaining more autonomy over their reproductive decisions. Providers should consistently present adolescents with comprehensive contraceptive options as a component of preventive health care.

New Insights into Interspecific Hybridization in Lemna L. Sect. Lemna (Lemnaceae Martinov).

Duckweeds have been increasingly studied in recent years, both as model plants and in view of their potential applications as a new crop in a circular bioeconomy perspective. In order to select species and clones with the desired attributes, the correct identification of the species is fundamental. Molecular methods have recently provided a more solid base for taxonomy and yielded a consensus phylogenetic tree, although some points remain to be elucidated. The duckweed genus Lemna L. comprises twelve species, grouped in four sections, which include very similar sister species. The least taxonomically resolved is sect. Lemna, presenting difficulties in species delimitation using morphological and even barcoding molecular markers. Ambiguous species boundaries between Lemna minor L. and Lemna japonica Landolt have been clarified by Tubulin Based Polymorphism (TBP), with the discovery of interspecific hybrids. In the present work, we extended TBP profiling to a larger number of clones in sect. Lemna, previously classified using only morphological features, in order to test that classification, and to investigate the possible existence of other hybrids in this section. The analysis revealed several misidentifications of clones, in particular among the species L. minor, L. japonica and Lemna gibba L., and identified six putative 'L. gibba' clones as interspecific hybrids between L. minor and L. gibba.

Obstetrician-Gynecologists' Practices in Postpartum Sterilization Without a Valid Medicaid Consent Form.

OBJECTIVE: To explore the practices of obstetrician-gynecologists (ob-gyns) in the United States surrounding postpartum sterilization when the Medicaid consent form was not valid. METHODS: Using the American College of Obstetricians and Gynecologists' online directory, we conducted a qualitative study where we recruited ob-gyns practicing in 10 geographically diverse U.S. states for a qualitative study using semi-structured interviews conducted by telephone. We analyzed interview transcripts using the constant comparative method and principles of grounded theory. RESULTS: Thirty ob-gyns (63% women, 77% nonsubspecialized, and 53% academic setting) were interviewed. Although most physicians stated that they did not perform sterilizations without a valid Medicaid sterilization form, others noted that they sometimes did due to a sense of ethical obligation toward their patient's health, being in a role with more authority or seniority, interpreting the emergency justification section of the form more broadly, or backdating the form. The physicians who said that they never went ahead without a signed form tended to work at large institutions and were concerned with losing funding and engaging in potentially illegal or fraudulent behavior. CONCLUSION: Physicians' varied behaviors related to providing postpartum sterilization without a valid Medicaid consent form demonstrate that the policy is in need of revision. Unclear terminology and ramifications of the Medicaid sterilization policy need to be addressed to ensure equitable care.

Duckweed Species Genotyping and Interspecific Hybrid Discovery by Tubulin-Based Polymorphism Fingerprinting.

Duckweeds (Lemnaceae) are the smallest and fastest-growing angiosperms. This feature, together with high starch production and good nutritional properties, makes them suitable for several applications, including wastewater treatment, bioenergy production, or feed and food supplement. Due to their reduced morphology and great similarity between diverse species, taxonomic identification of duckweeds is a challenging issue even for experts. Among molecular genotyping methods, DNA barcoding is the most useful tool for species identification without a need for cluster analysis. The combination of two plastid barcoding loci is now considered the gold standard for duckweed classification. However, not all species can be defined with confidence by these markers, and a fast identification method able to solve doubtful cases is missing. Here we show the potential of tubulin-based polymorphism (TBP), a molecular marker based on the intron length polymorphisms of ß-tubulin loci, in the genomic profiling of the genera Spirodela, Landoltia, and Lemna. Ninety-four clones were analyzed, including at least two representatives of each species of the three genera, with a special focus on the very heterogeneous species Lemna minor. We showed that a single PCR amplification with universal primers, followed by agarose gel analysis, was able to provide distinctive fingerprinting profiles for 10 out of 15 species. Cluster analysis of capillary electrophoresis-TBP data provided good separation for the remaining species, although the relationship between L. minor and Lemna japonica was not fully resolved. However, an accurate comparison of TBP profiles provided evidence for the unexpected existence of intraspecific hybrids between Lemna turionifera and L. minor, as further confirmed by amplified fragment length polymorphism and sequence analysis of a specific ß-tubulin locus. Such hybrids could possibly correspond to L. japonica, as originally suggested by E. Landolt. The discovery of interspecific hybrids opens a new perspective to understand the speciation mechanisms in the family of duckweeds.

Attitudes and beliefs of obstetricians-gynecologists regarding Medicaid postpartum sterilization - A qualitative study.

OBJECTIVE: To explore the attitudes and beliefs of obstetrician-gynecologists in the United States (US) regarding the Medicaid postpartum sterilization policy. STUDY DESIGN: We recruited obstetrician-gynecologists practicing in ten geographically diverse US states for a qualitative study using the American College of Obstetricians and Gynecologists directory. We conducted semi-structured interviews via telephone, professionally transcribed, and analyzed using the constant comparative method and principles of grounded theory. RESULTS: We interviewed thirty obstetrician-gynecologists (63.3% women, 76.7% non-subspecialized, and 53.3% academic setting). Participants largely described the consent form as unnecessary, paternalistic, an administrative hassle, a barrier to desired patient care, and associated with worse health outcomes. Views on the waiting period's utility and impact were mixed. Many participants felt the sterilization policy was discriminatory. However, some participants noted the policy's importance in terms of the historical basis, used the form as a counseling tool to remind patients of the permanence of sterilization, felt the policy prompted them to counsel regarding sterilization, and protected patients in contemporary medical practice. CONCLUSION: Many physicians shared concerns about the ethics and clinical impact of the Medicaid sterilization policy. Future revisions to the Medicaid sterilization policy must balance prevention of coercion with reduction in barriers to those desiring sterilization in order to maximize reproductive autonomy. IMPLICATIONS: Obstetrician-gynecologists are key stakeholders of the Medicaid sterilization policy. Obstetrician-gynecologists largely believe that revision to the Medicaid sterilization policy is warranted to balance reduction of external barriers to desired care with a process that enforces the need for counseling regarding contraception and reviewing patient preference for sterilization throughout pregnancy in order to minimize regret.

aTBP: A versatile tool for fish genotyping.

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.

On the applicability of the Tubulin-Based Polymorphism (TBP) genotyping method: a comprehensive guide illustrated through the application on different genetic resources in the legume family.

BACKGROUND: Plant discrimination is of relevance for taxonomic, evolutionary, breeding and nutritional studies. To this purpose, evidence is reported to demonstrate TBP (Tubulin-Based-Polymorphism) as a DNA-based method suitable for assessing plant diversity. RESULTS: Exploiting one of the most valuable features of TBP, that is the convenient and immediate application of the assay to groups of individuals that may belong to different taxa, we show that the TBP method can successfully discriminate different agricultural species and their crop wild relatives within the Papilionoideae subfamily. Detection of intraspecific variability is demonstrated by the genotyping of 27 different accessions of Phaseolus vulgaris. CONCLUSIONS: These data illustrate TBP as a useful and versatile tool for plant genotyping. Since its potential has not yet been fully appreciated by the scientific community, we carefully report all the experimental details of a successful TBP protocol, while describing different applications, so that the method can be replicated in other laboratories.

Flax tubulin and CesA superfamilies represent attractive and challenging targets for a variety of genome- and base-editing applications.

Flax is both a valuable resource and an interesting model crop. Despite a long history of flax genetic transformation only one transgenic linseed cultivar has been so far registered in Canada. Implementation and use of the genome-editing technologies that allow site-directed modification of endogenous genes without the introduction of foreign genes might improve this situation. Besides its potential for boosting crop yields, genome editing is now one of the best tools for carrying out reverse genetics and it is emerging as an especially versatile tool for studying basic biology. A complex interplay between the flax tubulin family (6 α-, 14 ß-, and 2 γ-tubulin genes), the building block of microtubules, and the CesA (15-16 genes), the subunit of the multimeric cellulose-synthesizing complex devoted to the oriented deposition of the cellulose microfibrils is fundamental for the biosynthesis of the cell wall. The role of the different members of each family in providing specificities to the assembled complexes in terms of structure, dynamics, activity, and interaction remains substantially obscure. Genome-editing strategies, recently shown to be successful in flax, can therefore be useful to unravel the issue of functional redundancy and provide evidence for specific interactions between different members of the tubulin and CesA gene families, in relation to different phase and mode of cell wall biosynthesis.

Preliminary Results, Perspectives, and Proposal for a Screening Method of In Vitro Susceptibility of Prototheca Species to Antimicrotubular Agents.

Microorganisms belonging to the genus Prototheca are achlorophyllous microalgae, occasionally behaving as environmental pathogens that cause severe mastitis in milk cows, as well as localized or systemic infections in humans and animals. Among the different species belonging to the genus, Prototheca zopfii genotype 2 (recently reclassified as P. bovis) and P. blaschkeae are most commonly associated with bovine mastitis. To date, no pharmacological treatment is available to cure protothecal mastitis, and infected animals must be quarantined to avoid spreading the infection. The few antibiotic and antifungal drugs effective in vitro against Prototheca give poor results in vivo This failure is likely due to the lack of specificity of such drugs. As microalgae are more closely related to plants than to bacteria or fungi, an alternative possibility is to test molecules with herbicidal properties, in particular, antimicrotubular herbicides, for which plant rather than animal tubulin is the selective target. Once a suitable test protocol was set up, a panel of 11 antimicrotubular agents belonging to different chemical classes and selective for plant tubulin were tested for the ability to inhibit growth of Prototheca cells in vitro Two dinitroanilines, dinitramine and chloralin, showed strong inhibitory effects on P. blaschkeae at low micromolar concentrations, with half-maximal inhibitory concentrations (IC50) of 4.5 and 3 µM, respectively, while both P. zopfii genotype 1 (now reclassified as P. ciferrii) and P. bovis showed susceptibility to dinitramine only, to different degrees. Suitable screening protocols for antimitotic agents are suggested.

Tubulin-Based DNA Barcode: Principle and Applications to Complex Food Matrices.

The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods.

Untargeted DNA-based methods for the authentication of wheat species and related cereals in food products.

New food commodities, particularly pasta, bread and cookies, made with mixed flours containing ancient wheat species and other cereals, have become popular in recent years. This calls for analytical methods able to determine authenticity of these products. Most DNA-based methods for the authentication of foodstuff rely on qPCR assays specifically targeting each plant species, not allowing the identification of unsearched ingredients. Moreover, the discrimination among closely related plant species, particularly congeneric ones like Triticum spp, remains a challenging task. DNA fingerprinting through tubulin-based polymorphism (TBP) and a new assay, TBP light, have been optimized for the authentication of different wheat and farro species and other cereals and tested on a set of commercial food products. The assay has a sensitivity of 0.5-1% w/w in binary mixtures of durum wheat in einkorn or emmer flour and was able to authenticate the composition of test food sample and to detect possible adulterations.

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples.

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Interlaboratory Comparison of Methods Determining the Botanical Composition of Animal Feed.

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project with the aim of addressing the requirements stated in European Union Regulation No. 767/2009, concerning market placement and use of feed of known and ascertained botanical composition. Accordingly, an interlaboratory trial was set up to compare the performance of different assays based either on optical microscope or DNA analysis for the qualitative and quantitative identification of the composition of compound animal feeds. A tubulin-based polymorphism method, on which the Feed-Code platform was developed, provided the most accurate results. The present study highlights the need for the performance of ring trials for the determination of the botanical composition of animal feeds and raises an alarm on the actual status of analytical inaccuracy.

Evolutionary characterization and transcript profiling of ß-tubulin genes in flax (Linum usitatissimum L.) during plant development.

BACKGROUND: Microtubules, polymerized from alpha and beta-tubulin monomers, play a fundamental role in plant morphogenesis, determining the cell division plane, the direction of cell expansion and the deposition of cell wall material. During polarized pollen tube elongation, microtubules serve as tracks for vesicular transport and deposition of proteins/lipids at the tip membrane. Such functions are controlled by cortical microtubule arrays. Aim of this study was to first characterize the flax ß-tubulin family by sequence and phylogenetic analysis and to investigate differential expression of ß-tubulin genes possibly related to fibre elongation and to flower development. RESULTS: We report the cloning and characterization of the complete flax ß-tubulin gene family: exon-intron organization, duplicated gene comparison, phylogenetic analysis and expression pattern during stem and hypocotyl elongation and during flower development. Sequence analysis of the fourteen expressed ß-tubulin genes revealed that the recent whole genome duplication of the flax genome was followed by massive retention of duplicated tubulin genes. Expression analysis showed that ß-tubulin mRNA profiles gradually changed along with phloem fibre development in both the stem and hypocotyl. In flowers, changes in relative tubulin transcript levels took place at anthesis in anthers, but not in carpels. CONCLUSIONS: Phylogenetic analysis supports the origin of extant plant ß-tubulin genes from four ancestral genes pre-dating angiosperm separation. Expression analysis suggests that particular tubulin subpopulations are more suitable to sustain different microtubule functions such as cell elongation, cell wall thickening or pollen tube growth. Tubulin genes possibly related to different microtubule functions were identified as candidate for more detailed studies.