Modified "Allele-Specific qPCR" Method for SNP Genotyping Based on FRET

Peer reviewe

Genes encoding transcription factors TaDREB5 and TaNFYC-A7 are differentially expressed in leaves of bread wheat in response to drought, dehydration and ABA

Two groups of six spring bread wheat varieties with either high or low grain yield under the dry conditions of Central and Northern Kazakhstan were selected for analysis. Experiments were set up with the selected wheat varieties in controlled environments as follows: (1) slowly progressing drought imposed on plants in soil, (2) rapid dehydration of whole plants grown in hydroponics, (3) dehydration of detached leaves, and (4) ABA treatment of whole plants grown in hydroponics. Representatives of two different families of transcription factors (TFs), TaDREB5 and TaNFYC-A7, were found to be linked to yield-under-drought using polymorphic Amplifluor-like SNP marker assays. qRT-PCR revealed differing patterns of expression of these genes in the leaves of plants subjected to the above treatments. Under drought, TaDREB5 was significantly up-regulated in leaves of all high-yielding varieties tested and down-regulated in all low-yielding varieties, and the level of expression was independent of treatment type. In contrast, TaNFYC-A7 expression levels showed different responses in the high- and low-yield groups of wheat varieties. TaNFYC-A7 expression under dehydration (treatments 2 and 3) was higher than under drought (treatment 1) in all high-yielding varieties tested, while in all low-yielding varieties the opposite pattern was observed: the expression levels of this gene under drought were higher than under dehydration. Rapid dehydration of detached leaves and intact wheat plants grown in hydroponics produced similar changes in gene expression. ABA treatment of whole plants caused rapid stomatal closure and a rise in the transcript level of both genes during the first 30 min, which decreased 6 h after treatment. At this time-point, expression of TaNFYC-A7 was again significantly up-regulated compared to untreated controls, while TaDREB5 returned to its initial level of expression. These findings reveal significant differences in the transcriptional regulation of two drought-responsive and ABA-dependent TFs under slowly developing drought and rapid dehydration of wheat plants. The results obtained suggest that correlation between grain yield in dry conditions and TaNFYC-A7 expression levels in the examined wheat varieties is dependent on the length of drought development and/or strength of drought; while in the case of TaDREB5, no such dependence is observed.Lyudmila Zotova, Akhylbek Kurishbayev, Satyvaldy Jatayev, Gulmira Khassanova, Askar Zhubatkanov, Dauren Serikbay, Sergey Sereda, Tatiana Sereda, Vladimir Shvidchenko, Sergiy Lopato, Colin Jenkins, Kathleen Soole, Peter Langridge, and Yuri Shavruko

СОРБЦИЯ РЕНИЯ ИЗ СЕРНОКИСЛЫХ РАСТВОРОВ ИМПРЕГНАТАМИ, СОДЕРЖАЩИМИ ТРИАЛКИЛАМИН

The paper studies rhenium (VII) sorption from sulfuric acid solutions by impregnates based on macroporous polymer carriers (copolymers of styrene with divinylbenzene, weak acid cation exchange resion) containing commercial trialkylamine (ТAA). The study provides equilibrium and kinetic characteristics of rhenium recovery by the impregnate based on the macroporous weakly acidic cation exchange resin (K-TAA) having the best rhenium capacity. The maximum coefficient of rhenium distribution in the K-TAA impregnate is observed in sorption from pH = 2 solutions. The rhenium sorption isotherm is described by the Langmuir equation with the K = 29±2 ml/g constant. A limited solution volume method was used to obtain the integral kinetic curves of sorption with a half-reaction time value considered to calculate the effective coefficients of rhenium diffusion in the impregnate amounted to (3,8·10–11 (295 K) and 1,3·10–10 (308 K) m2/s). Kinetic results linearized by the equations of models (pseudo-first, pseudo-second order, Elovich and inner diffusion) showed that kinetic curves with the highest correlation degree are described by the pseudo-second order equation with the 0,00056 (295 K) and 0,00059 (308 K) g·mg–1·min–1 rate constants. The apparent activation energy of rhenium sorption (39±2 kJ/mol) was calculated using the Arrhenius equation. The K-TAA impregnate was tested for rhenium sorption from the eluate obtained by rhenium desorption from the Purolite A170, weak base anion exchange resin pre-saturated with rhenium from the complex pregnant solution for leaching of products derived from poor rhenium-containing copper sulfide raw materials processed.В статических условиях изучена сорбция рения (VII) из сернокислых растворов импрегнатами на основе макропористых полимерных носителей (сополимеров стирола с дивинилбензолом, слабокислотного катионита), содержащих технический триалкиламин (ТАА). Получены равновесные и кинетические характеристики сорбции рения импрегнатом К-ТАА на основе макропористого катионита, имеющего лучшие емкостные характеристики по рению. Максимальное значение коэффициента распределения рения в импрегнате К-ТАА наблюдается при сорбции из растворов с рН = 2. Изотерма сорбции рения описывается уравнением Ленгмюра с константой K = 29±2 мл/г. Методом ограниченного объема раствора при различных температурах получены интегральные кинетические кривые сорбции и с учетом времени полупревращения рассчитаны эффективные коэффициенты диффузии рения в импрегнате, составившие 3,8·10–11 (295 K) и 1,3·10–10 (308 K) м2/с. Обработка кинетических данных линеаризацией по уравнениям моделей псевдопервого, псевдовторого порядка, внутренней диффузии и Еловича показала, что кинетические кривые с наиболее высокой степенью корреляции описываются уравнением псевдовторого порядка с константами скорости 0,00056 (295 K) и 0,00059 (308 K) г·мг–1·мин–1. По уравнению Аррениуса рассчитано значение кажущейся энергии активации сорбции рения, составившее 39±2 кДж/моль. Осуществлена апробация импрегната К-ТАА для сорбции рения из элюата, полученного при десорбции рения со слабоосновного анионита Purolite A170, предварительно насыщенного рением из сложного по составу продуктивного раствора выщелачивания продуктов переработки бедного ренийсодержащего сульфидного медного сырья

Advantages of Amplifluor-like SNP markers over KASP in plant genotyping

Background: KASP (KBioscience Competitive Allele Specific PCR) and Amplifluor (Amplification with fluorescence) SNP markers are two prominent technologies based upon a shared identical Allele-specific PCR platform. Methods: Amplifluor-like SNP and KASP analysis was carried out using published and own design of Universal probes (UPs) and Gene-specific primers (GSPs). Results: Advantages of the Amplifluor-like system over KASP include the significantly lower costs and much greater flexibility in the adjustment and development of ‘self-designed’ dual fluorescently-labelled UPs and regular GSPs. The presented results include optimisation of ‘tail’ length in UPs and GSPs, protocol adjustment, and the use of various fluorophores in different qPCR instruments. The compatibility of the KASP Master-mix in both original and Amplifluor-like systems has been demonstrated in the presented results, proving their similar principles. Results of SNP scoring with rare alleles in addition to more frequently occurring alleles are shown. Conclusions: The Amplifluor-like system produces SNP genotyping results with a level of sensitivity and accuracy comparable to KASP but at a significantly cheaper cost and with much greater flexibility for UPs with self-designed GSPs.Satyvaldy Jatayev, Akhylbek Kurishbayev, Lyudmila Zotova, Gulmira Khasanova, Dauren Serikbay, Askar Zhubatkanov, Makpal Botayeva, Aibek Zhumalin, Arysgul Turbekova, Kathleen Soole, Peter Langridge and Yuri Shavruko