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.

Effect of a novel dwarfing mutant site on chromosome 4B on agronomic traits in common wheat.

The introduction of dwarfing genes triggered a wave of "green revolution". A number of wheats dwarfing genes have been reported in previous studies, and only a small fraction of these have been applied to production practices. Therefore, the development of novel dwarfing genes for wheat is of great value. In this study, a novel dwarfing site, Rht-yz, identified in the Yanzhan mutation, is located on chromosome 4B (30-33MB) and its mechanism of action is different from that of Rht-B1b (C-T mutation), but whether it affects the Rht-B1a (TraesCS4B02G043100) or other genes is unclear. Exogenously applied GA3 experiments showed that Rht-yz is one of the gibberellin-insensitive dwarf genes. The effects of the dwarf gene Rht-yz on agronomic traits in wheat were evaluated in the field using Yanzhan, Yanzhan mutations, F2:3 and F3:4 lines. The results showed that Rht-yz improved lodging resistance by reducing plant height, increasing diameter, wall thickness and mechanical strength of the basal stem. In terms of yield traits, Rht-yz had negative effects on tiller number plant-1, biomass plant-1 and yield plant-1, but had no significant effect on harvest index, 1000-kernel weight and spike traits. In addition, Rht-yz significantly increased crude protein, wet gluten and starch content. Therefore, the rational use of the new dwarfing site Rht-yz has potential and value in dwarf wheat breeding.

SNP Genotyping with Amplifluor-Like Method.

For SNP genotyping, amplification of fluorescence (Amplifluor) is a popular and actively developing method in the plant sciences. The "Amplifluor-like" is a "home-made" modification of the original commercial Amplifluor method. Amplifluor-like genotyping requires two essential components: (1) two allele-specific forward primers targeting the SNP site with one common reverse primer; and (2) a universal part with two non-allele-specific molecular probes containing one of the two used fluorophores and a quencher. Allele discrimination is based on the fluorescence score, where the dominance of one dye over the other confirms the presence of each specific SNP allele. The Amplifluor-like method is similar to commercial KASP and original Amplifluor methods but is much cheaper because all components can be ordered as regular and modified oligos. The easily adaptable Amplifluor-like method can be modified by any researcher to make it suitable for available instruments, reagents and conditions in low-budget laboratories for SNP genotyping of any plant species with identified genetic polymorphism.

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

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.

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.

Corrigendum: Expression Level of the DREB2-Type Gene, Identified with Amplifluor SNP Markers, Correlates with Performance, and Tolerance to Dehydration in Bread Wheat Cultivars from Northern Kazakhstan.

[This corrects the article on p. 1736 in vol. 7, PMID: 27917186.].

Expression Level of the DREB2-Type Gene, Identified with Amplifluor SNP Markers, Correlates with Performance, and Tolerance to Dehydration in Bread Wheat Cultivars from Northern Kazakhstan.

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).