Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.     

Involvement of single-strand breaks in complex formation between single-stranded DNA and nucleoids of Bacillus subtilis

Summary RNase-unfolded chromosomes of competent Bacillus subtilis are able to take up single-stranded homologous donor DNA fragments in vitro to form donor-recipient DNA complexes (Van Randen and Venema 1981). The unfolded chromosomes behave as supercoiled DNA molecules. X-irradiation increased the formation of unstable and stable complexes between donor and recipient DNA during incubation at 37° C. The complex-forming ability of the unfolded chromosomes increased linearly with increasing X-ray dose, even after complete relaxation of the unfolded chromosomes had occurred. Limited DNase I action increased the complex-forming ability of the chromosomes as effectively as X-irradiation.Unstable donor-recipient DNA complexes can be distinguished from stable ones by their dissociation upon density gradient centrifugation in CsCl at pH 11.2. They are stable at pH 10 (Van Randen et al. 1982a). At an intermediate pH value during isopycnic centrifugation, a fraction of the unstable complexes were stable, suggesting that a range of stabilities existed among the unstable complexes. The donor moiety of the stable donor-recipient DNA complexes was far more resistant to nuclease S1 treatment than that of the unstable ones.     

On enzymic clotting processes V: Rate equations for the case of arbitrary rate of production of the clotting species

The rate theory for enzyme-triggered coagulation reactions, such as the clotting of fibrin or casein, is extended to the case of an arbitrary rate of production of the clotting species. It is shown that the general expression for the growth of the weight-average molecular weight of the clotting product, -M_w, is given by -M_w = M₁{1 + k_s {∫₀^tP(t)² dt}/P(t)}, where M₁ is the “monomer” molecular weight, k_s the smoluchowskian flocculation rate constant and P(t) the total number of monomers produced by the enzyme in t. In the purely smoluchowskian case P(t) stands for the total number of monomers at the beginning of the clotting process. Numerical examples in which the rate of enzymic production is governed by complete Michaelis-Menten kinetics, are compared to cases in which this rate equals V_max- It is shown that after exhaustion of the substrate the system continues to coagulate in a purely smoluchowskian way. Turbidimetric experiments on the clotting of micelles of whole and κ-casein are presented which suggest inactivation of the enzyme by non-productive binding in the flocs formed.     

Multiplex PCR combining transgene and S-allele control primers to simultaneously confirm cultivar identity and transformation in apple

Many protocols for genetic transformation result in the regeneration of both transformed shoots and untransformed ones known as escapes. Here we describe a multiplex PCR technique for use with apple (Malus 2 domestica Borkh.), which simultaneously demonstrates the presence of both a transgene sequence and an endogenous gene using a single PCR reaction. Common transgene-specific primers were successfully used in combination with either the conserved X1 primers or with different S-allele primers. When primers matched to S-allele sequences are used during multiplex PCR, the presence of the PCR product proves that amplification occurred successfully but can also confirm the identity of the cultivar used in the experiment. This is particularly useful as a protection against mislabeling of cultivars during subculturing and other laboratory and greenhouse operations.     

Der Stoffwechsel der Honigbiene während des Fliegens

Zusammenfassung Es wurde der Stoffwechsel von Bienen studiert, in der Ruhe und während des Fluges.Die Stoffwechselbestimmung wurde für jedes Tier gesondert, also individuell ausgeführt. Der respiratorische Quotient wurde jedesmal mitbestimmt.Zur Kontrolle wurde auch mit einem kleinen Schwarm gearbeitet. Der Gewichtsverlust der Tiere während des Fluges wurde direkt gemessen.Die Ökonomie des Bienenfluges wird verglichen mit der des mechanischen menschlichen Fluges, wie dieser zur Zeit stattfindet.Wir sind Fräulein B. W. Grutterink, die die mühsame Arbeit der Gasanalyse übernahm, zu großem Dank verpflichtet.     

Influence of season and microclimate on fertility of dairy cows in a hot-arid environment

Records were obtained over a 3 year period from six Holstein dairy farms of 300 to 500 cows each in the Phoenix, Ariz. area. Dairies were selected on the basis of similar management practices, herd size, milk production and facilities (with the exception of cooling systems). Microclimatic modifications (two dairies each) were shade only (approximately 3.7 m²/cow), evaporative-cooled shades and low-pressure water foggers under the shades. Data were categorized by season of calving (spring, Feb.–May; summer, June–Sept.; and fall, Oct.–Jan.). Traits evaluated were calving interval, days open and services/conception. Calving interval was shortest for cows calving in the spring (378 days), intermediate in fall (382 days) and longest in summer (396 days). Similar seasonal trends were observed for days open (103, 103 and 119 days, respectively) and services/conception (1.54, 1.81 and 1.93, respectively). All differences between spring and summer were significant (P < 0.05). Calving interval and days open were less for evaporative-cooled groups (374 and 98 days, respectively), with no difference between shade only and foggers (391 and 392 days, 112 and 116 days, respectively). Services/conception were similar for all groups (1.72 to 1.79). A significant interaction between microclimate and season for services/conception could be interpreted as (i) smaller season differences for evaporative-cooled groups than for shade or foggers, or (ii) a change in the ranking of control and fogger groups during summer versus fall. Evaporative cooling was more effective than fogging for reducing the detrimental effects of seasonal high temperatures on fertility.     

On enzymatic clotting processes. I. Kinetics of enzyme-triggered coagulation reactions

The kinetics of enzymatic clotting reactions such as the clotting of blood or milk, is analyzed. The appearance of a lag phase in the clotting is shown to be due to the difference in reaction order of enzymatic production and flocculation. The weight-average particle weight of the product formed is found to be a quadratic function of the reaction time. The condition for the clotting time is t square root of ksV/2 = C, where t is the clotting time, ks the flocculation rate constant, V the maximum rate of enzymatic product formation and C a constant. In agreement with this result double-logarithmic plots of t versus enzyme dilution are always observed to be linear over a wide range of enzyme concentrations. No statistical evidence could be produced for the widely held belief that the clotting time should be inversely proportional to the enzyme concentration. Varying exponents of the latter in its relation to the clotting time are discussed in terms of von Smoluchowski's theory of the slow coagulation of colloidal particles.     

Vergleichende Untersuchungen über das periphere Nerven-Muskelsystem von Crustaceen

Zusammenfassung Mit kurz dauernden Gleichströmen wurde bei indirekter Reizung eine schnelle Kontraktion erzi lt, die eine sehr eigentümliche Alles-oder-Nichts-Relation zu erkennen gibt. Die Reizzeitwerte wurden bestimmt nach Maßgabe ihrer Lage auf. der Reizspannungskurve. Zu einer bestimmten Reihe von Werten (d. h. Strecke dieser Kurve) gehört jeweils eine einzige Zuckungshöhe. Überschreitet man die Grenze dieser Werte, so tritt ganz plötzlich eine höhere Kontraktion auf, die wiederum einer ganzen Zone von Reizwerten entspricht und innerhalb dieser Zone konstant bleibt. Es ließen sich auf diese Weise mehrere Stufen nachweisen. Wenn wir die Kontraktionen als Tetani also als Folge einer Summation auffassen, dann sehen wir, daß diese Summation einer ganz bestimmten Quantengesetzmäßigkeit der Reizintensitäten gehorcht.Neben der schnellen Zuckung mit ihren Stufen wurde die langsame Kontraktion untersucht. Sie tritt auf bei lang dauernden Gleichströmen Es handelt sich um Kurven, wie sie beim Wirbeltiere nach Veratrinvergiftung vorkommen: steiler Anstieg, dann Senkung, darauf ein zweiter flacher Gipfel. Man kann auch, die langsame Kontraktion allein erhalten, wenn man unter der Rheobase der schnellen Zuckung reizt [lange Latenzzeit, Kontraktion, die mit Stromstärke und Beizzeit an Höhe kontinuierlich (also nicht in Stufen) zunimmt.]Von diesen Kontraktionen wurden die Aktionsströme registriert. Bei den Elektrogrammen der schnellen Zuckung ergab sich die gleiche Alles-oder-Nichts-Relation wie bei den Mechanogrammen: die gleichen Stufen, zwar in Abhängigkeit von der Zone der Reizstärke, aber innerhalb der Zone von dieser unabhängig. Höhere Zonen der Reizintensität haben keine höheren Ausschläge zur Folge, wohl aber einen zweiten Gipfel (Chronaxie 6 ). Die Gipfel haben den Charakter einer Afterdischarge (zentrale Eigenschaften peripherer Nerven wirbelloser Tiere). Der zweite Gipfel tritt bei Überschreiten der Zonengrenze absolut plötzlich auf.Die langsame Kontraktion ergibt rhythmische (phasische) Ausschläge der Saite, auch wenn das Ganze die Form einer Veratrinkurve hat. Die Einzelausschläge dieses Rhythmus zeigen zu Anfang die größte Frequenz, sie nimmt mit der Zeit ab, während zugleich die Höhe der Ausschläge zunimmt. Die Frequenz der Ausschläge der langsamen Kontraktion ist abhängig von der Reizstärke (bis zu 150 pro Sekunde). Wenn man einer Reizung eine zweite in kurzem Intervall folgen läßt, so treten die späteren Phasen des Bildes (geringere Frequenz, höhere Ausschläge) viel schneller auf, die Latenz ist verkürzt (Nachwirkung).Es wurde ferner die Wirkung summierbarer Einzelreize bei indirekter Reizung untersucht. Es zeigte sich sehr ausgesprochene addition latente. Wenn man unterschwellige Reize wiederholt, so werden sie wirksam. Bei einem solchen unterschwelligen Reiz bleibt aber nicht nur jeder mechanische, sondern auch jeder elektrische Effekt des Muskels aus.Es ergaben sich Fälle von Aktionsströmen ohne Muskelkontraktion (Block zwischen Ort der Aktionsströme und des Kontraktions).Die Kontraktionen des Öffners gleichen hauptsächlich den langsamen Kontraktionen des Schließers (biologische Bedeutung). Zuweilen aber nehmen die, Kurven Veratrinform an, mit schneller Anfangszuckung, ein Beweis, daß ein einziger Axon beide Kontraktionsarten hervorrufen kann.Die Hemmung wurde untersucht. Öffnerhemmung gelang leicht, diejenige des Schließers nur ganz selten. Die Hemmung wird durch den als solche bekannten Henimungsaxon übertragen. Es wird vermutet, daß die Hemmung nicht die eigentliche Kontraktionserzeugung im Muskel betrifft, sondern den Zwischenprozeß, der sich durch Entstehen des Aktionsstromes zu erkennen gibt und zwar wird bei diesem die Bahnung (Zunahme der Seitenausschläge) unterdrückt. Die schnelle Zuckung scheint nicht gehemmt werden zu können (biologische Bedeutung). Eine Theorie aller dieser Erscheinungen wird versucht.Zum Schluß möchte ich meinen Dank aussprechen für die Gastfreundschaft, welche ich in dem Marine biological Laboratory in Plymouth gefunden habe. Besonders möchte ich den Herrn Dr. E. D. Allen unsd Dr. C. Yonge für ihr freundlichstes Entgegenkommen danken. Weiter bin ich den Kuratoren des Dondersfonds zu großem Dank verpflichtet für das Stipendium, welches mir den Aufenthalt in England möglich machte. Die Versuche wurden in den Jahren 1930–1931 ausgeführt.