Phenotypic and Genetic Characterization for Incompatible Cross-Match Cases in the Feline AB Blood Group System

The feline AB blood group system (blood types A, B, and AB) encoding the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene is the most significant in transfusion medicine and hemolysis of the newborn for cats. Blood typing and cross-matching in pre-transfusion testing are crucial to determining blood compatibility and thus prevent hemolytic transfusion reactions. We here performed serological and genetic investigations to characterize blood samples from cats with discordant results for card agglutination (CARD) and the alloantibody agglutination test for blood typing in two cats (subjects K and R). Subject K showed incompatible cross-matching in pre-transfusion testing. Red blood cells from subjects K and R determined blood type B from the CARD method showed blood type AB by alloanti-A and alloanti-B antibodies in agglutination testing. Genomic DNA sequencing of the coding region (exons 1a to 14) for the cat CMAH gene showed that subject K had four mutations with heterozygosity at c.139C>T, c.179G>T, c.327A>C, and c.364C>T. Similarly, the CMAH gene of subject R carried six mutations with heterozygosity at c.142G>A, c.187A>G, c.268T>A, c.327A>C, c.773G>A and c.1603G>A, representing a new diplotype including a novel synonymous single nucleotide polymorphism (SNP) in exon 7 (c.773 G>A: Arg258Gln). The CMAH diplotype in subjects K and R was different from major diplotype in blood type B cats. This study is the first to report CMAH variants in cats with discordant blood types between CARD and TUBE methods. These results could assist in the classification of feline AB blood types for transfusion medicine to avoid blood incompatibilities.

De novo hydroponics system efficiency for the cuttings of alfalfa (Medicago sativa L.)

The legume plant alfalfa (Medicago sativa L.) is a widely cultivated perennial forage due to its high protein content, palatability, and strong adaptability to diverse agro-ecological zones. Alfalfa is a self-incompatible cross-pollinated autotetraploid species with tetrasomic inheritance. Therefore, maintaining excellent traits through seed reproduction is a prime challenge in alfalfa. However, the cutting propagation technology could enable consistent multiplication of quality plants that are genetically identical to the parent plant. The current study aimed to develop a simple, cost-effective, reproducible, and efficient hydroponic cutting method to preserve alfalfa plants and for molecular research. In this study, alfalfa landrace ‘Wudi’ was grown in hydroponics for 30 days and used as source material for cuttings. The top, middle and bottom sections of its stem were used as cuttings. The rooting rate, root length, and stem height of the different stem sections were compared to determine the best segment for alfalfa propagation in four nutrient treatments (HM, HM + 1/500H, HM + 1/1000H and d HM + 1/2000H). After 21 days of culture, the rooting rates of all the three stem types under four cutting nutrient solutions were above 78%. The rooting rate of the middle and bottom parts in HM + 1/1000 H and HM + 1/2000 H nutrient solutions reached more than 93%, with a higher health survey score (> 4.70). In conclusion, this study developed a de novo cutting propagation method that can be used to conserve and propagate germplasm in breeding programs and research. This method is a new report on the cutting propagation of alfalfa by hydroponics, which could supplement the existing cutting propagation methods.

Pollination Timing and Hybrid Seed Production of Neoregelia

Neoregelia cultivars have been used in many areas for landscaping and indoors in a variety of creative ways, but scientific reports of their pollination and hybridization are presently limited. Cross-combinations of Neoregelia cultivars were created to define conditions for pollination timing and to evaluate cross-compatibility. Neoregelia cultivars have short-lived flowers. Hybrid seeds were obtained only when cross-pollination was performed before 1200 hr. Results of 19 cross-combinations including six reciprocal crosses revealed that hybrid seeds were obtained in the female parents with a 1.9- to 2.0-cm style length, but not in those with a 2.6- to 3.0-cm style length. The pollen tube penetrated the ovule as early as 1 day after pollination in the compatible cross, whereas swollen pollen tubes were observed at half and two-thirds of the style in the incompatible cross. Removal of 50% of the style length of the female parents could overcome the fertilization barrier for those incompatible crosses and hybrid seeds could be successfully obtained.

US military spending will increase for NATO’s east

Significance The United States has stationed 600 paratroopers in the Baltic countries and will defend their airspace for the duration of the Zapad-2017 exercise in a bid to reassure NATO allies of Washington’s commitment to their security. Longer term, Washington's European Deterrence Initiative (EDI) is paying for a forward presence of additional US forces in Europe, construction of additional infrastructure and upgrades to existing facilities. Impacts The US military will seek to conclude detailed hosting agreements with Eastern European NATO countries. Poland and South Korea are likely candidates for an expanded permanent US forward presence. Repeated US congressional budget standoffs will worry allies awaiting longer-term infrastructure projects. Stationing US tactical nuclear weapons on NATO’s eastern borders would be more provocative to Moscow than EDI-funded rotations. Replacing incompatible cross-border railway gauges between the Baltic states and Poland will take over a decade.

S-RNase proteins: functional studies of the 120kDa glycoprotein and SRNase oligomerization

Flowering plants control fertilization through pollen-pistil interactions. Self-incompatibility (SI) is a well-studied pollen-pistil interaction that promotes cross-pollination. SI is controlled by a multi-haplotype locus called the S-locus. In Nicotiana alata, S-RNase is a product of the S-locus and regulates specificity in the pistil, while S-locus F-box protein (SLF) controls specificity in the pollen. The interaction between S-RNase and SLF determines whether the pollination is compatible or incompatible. In an incompatible cross, the ribonuclease activity of S-RNase inhibits pollen tube growth. Genetic experiments indicate that, in addition to S-RNase and SLF, non-S-factors are also required for SI. S-RNase binding proteins represent potential non-S-factors required for SI. Using affinity chromatography, we found that S-RNase selfassociates and three homologous stylar glycoproteins - the 120kDa glycoprotein (120K), N. alata pistil extensin-like protein III (NaPELP III), and N. alata transmitting tract specific glycoprotein (NaTTS) - bind directly to S-RNase. I studied the oligomerization of S-RNase in detail and found that self-association is dependent on S-haplotype and buffer conditions. I determined that the components of the S-RNase complex account for 30% of soluble pistil protein. 120K is the most likely candidate for a non-S-factor because it enters the cytoplasm of growing pollen tubes and shows polymorphism when SI and self-compatible Nicotiana species are compared. To test its role in SI, I suppressed 120K expression using RNAi. Suppressing 120K caused a breakdown of SI, confirming that it functions in SI.

Citrus pollen tube development in cross-compatible gynoecia, self-incompatible gynoecia, and in vitro

The route of 'Orlando' tangelo (Citrus paradisi Macf. × C. reticulata Blanco) pollen tubes was traced and compared in self-incompatible pollinations and cross-compatible pollinations with 'Dancy' tangerine (C. reticulata Blanco). In both crosses, 'Orlando' pollen germinated in the stigmatic exudate and grew between the papillae on the stigma surface and inter-cellularly between the parenchymatous cells until reaching a stylar canal by 3 days. However, in the incompatible pollination, irregular deposition of callose occurred in the pollen tube walls as early as 1 day after pollination. By day 6, pollen tubes were in the upper portion of the ovary in the compatible pollination, whereas most pollen tubes from the incompatible pollination were still in the upper style. 'Orlando' pollen tube growth rate decreased substantially by day 3 in both the self-incompatible pollination and in vitro but increased rapidly after day 3 in the compatible combination. The generative cell divided between 1 and 3 days after pollination in the compatible cross. Generative cell division was observed by day 3 in only a few pollen tubes in the incompatible cross and in cultured tubes. Compatible pollen tubes grew slowly for the first 3 days after pollination, during which time generative cells divided and then grew rapidly until fertilization. In contrast, incompatible pollen tubes showed morphological features indicative of an incompatibility reaction by 1 day after pollination and grew slowly for a period of 6 days, and then ceased growth.

A quantitative and structural comparison of Citrus pollen tube development in cross-compatible and self-incompatible gynoecia

Pollen tube development in Orlando tangelo (Citrus paradisi Macf. × C. reticulata Blanco.) was compared within and between cross-compatible pollinations of Orlando pollen on Dancy tangerine (C. reticulata Blanco.) stigmas and self-incompatible pollinations on Orlando tangelo stigmas. Orlando and Dancy gynoecia were morphologically similar but differed slightly in stigma, style, and ovary lengths. Orlando pollen tube development was studied 1, 3, 6, 9, and 12 days after both cross- and self-pollination to record the number of pollen tubes at each of five levels: stigma surface, upper style, lower style, ovary, and entrance into ovules. In the incompatible cross (self-pollinated Orlando), the stigma was the primary region of pollen tube arrest. In the compatible cross (Orlando pollen on Dancy), some pollen tubes penetrated ovules between 9 and 12 days after cross pollination; however, other pollen tubes were arrested in the stigma. Pollen tubes that successfully penetrated ovules in the compatible cross differed morphologically from pollen tubes arrested in both the compatible and incompatible situations. Successful compatible pollen tubes were straight with thin-walled tips and regularly spaced callose plugs behind the growing tips. Many pollen tube abnormalities associated with the self-incompatible pollination of Orlando were also present among arrested pollen tubes from the compatible cross.