A panel of 89 local commercial cultivars of bread wheat was tested in field trials in the dry conditions of Northern Kazakhstan. Two distinct groups of cultivars (six cultivars in each group), which had the highest and the lowest grain yield under drought were selected for further experiments. A dehydration test conducted on detached leaves indicated a strong association between rates of water loss in plants from the first group with highest grain yield production in the dry environment relative to the second group. Modern high-throughput Amplifluor Single Nucleotide Polymorphism (SNP) technology was applied to study allelic variations in a series of drought-responsive genes using 19 SNP markers. Genotyping of an SNP in the TaDREB5 (DREB2-type) gene using the Amplifluor SNP marker KATU48 revealed clear allele distribution across the entire panel of wheat accessions, and distinguished between the two groups of cultivars with high and low yield under drought. Significant differences in expression levels of TaDREB5 were revealed by qRT-PCR. Most wheat plants from the first group of cultivars with high grain yield showed slight up-regulation in the TaDREB5 transcript in dehydrated leaves. In contrast, expression of TaDREB5 in plants from the second group of cultivars with low grain yield was significantly down-regulated. It was found that SNPs did not alter the amino acid sequence of TaDREB5 protein. Thus, a possible explanation is that alternative splicing and up-stream regulation of TaDREB5 may be affected by SNP, but these hypotheses require additional analysis (and will be the focus of future studies).

In dry conditions, tall and fast‐growing wheat plants with good tolerance to drought may offer higher grain yields than ‘Green revolution’ wheat.

BackgroundKASP (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.MethodsAmplifluor-like SNP and KASP analysis was carried out using published and own design of Universal probes (UPs) and Gene-specific primers (GSPs).ResultsAdvantages 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1197-x) contains supplementary material, which is available to authorized users.

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.

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

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The responses to temperature (vernalization) and day length (photoperiod) in wheat (Triticum aestivum L.) are entirely defined by allelic differences at the respective VRN-1 and PPD-1 loci. These two physiological factor greatly affect wheat phenology. Using molecular markers, a panel of 61 diverse bread wheat varieties were genotyped for VRN-1 and PPD-1 alleles, and the traits were evaluated under autumn and spring sowing conditions to investigate the impact of the alleles and their combinations on phenological stage, morphological traits, and yield traits. The results revealed that the earlier heading (HD), and flowering (FD) conferred by insensitive alleles for VRN-1 and PPD-1 genes was consistent regardless of the variation under the two sowing conditions. The effects of a single dominant allele of VRN-1 were similar, with an additive effects when combined. The effects of a single PPD-1 insensitive allele differed, with Ppd-D1a stronger than Ppd-B1a, and Ppd-A1a was the least, with an additive effects when combined. Secondly, varieties sown under spring condition recorded a larger flag leaf area when compared to those sown under autumn condition regardless of the single and combined influence at the VRN-1 and PPD-1 loci. When compared to other genes, PPD-D1 had a consistent and significant effect on flag leaf area, spike length, and plant height across both sowing conditions. Furthermore, the sensitive allele Ppd-D1b had larger biomass across both sowing conditions, but this did not translate into increased yield because the Ppd-D1a insensitive allele recorded higher yield. The impact of PPD-D1 gene on grain yield was evident across both sowing conditions. Overall, according to the findings of this study, VRN-1 and PPD-1 genes impact in modulating phenology stages, morphological, spike and yield traits was dependent on the sowing conditions and also on genetic background with the spring and insensitive alleles poised to be favored for selection.