Real-Time Quantitative PCR - Primer Design, Reference Gene Selection, Calculations and Statistics

Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology

Genetic diversity and population structure analysis of barley landraces from Shanghai region using genotyping-by-sequencing

Barley (Hordeum vulgare L.) is an important economic crop for food, feed and industrial raw materials. In the present research, 112 barley landraces from the Shanghai region were genotyped using genotyping-by-sequencing (GBS), and the genetic diversity and population structure were analyzed. The results showed that 210,268 Single Nucleotide Polymorphisms (SNPs) were present in total, and the average poly-morphism information content (PIC) was 0.1642. Genetic diversity and population structure analyses suggested that these barley landraces were differentiated and could be divided into three sub-groups, with morphological traits of row-type and adherence of the hulls the main distinguishing factors between groups. Genotypes with similar or duplicated names were also investigated according to their genetic backgrounds and seed appearances. This study provided valuable information on barley landraces from the Shanghai region, and showed that all these barley landraces should be protected and used for future breeding program

Genome-wide identification of sucrose nonfermenting-1-related protein kinase (SnRK) genes in barley and RNA-seq analyses of their expression in response to ABA treatment

Background: Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) play important roles in regulating metabolism and stress responses in plants, providing a conduit for crosstalk between metabolic and stress signalling, in some cases involving the stress hormone, abscisic acid (ABA). The burgeoning and divergence of the plant gene family has led to the evolution of three subfamilies, SnRK1, SnRK2 and SnRK3, of which SnRK2 and SnRK3 are unique to plants. Therefore, the study of SnRKs in crops may lead to the development of strategies for breeding crop varieties that are more resilient under stress conditions. In the present study we describe the SnRK gene family of barley (Hordeum vulgare), the widespread cultivation of which can be attributed to its good adaptation to different environments Results: The barley HvSnRK gene family was elucidated in its entirety from publicly-available genome data and found to comprise 50 genes. Phylogenetic analyses assigned six of the genes to the HvSnRK1 subfamily, 10 to HvSnRK2 and 34 to HvSnRK3. The search was validated by applying it to Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome data, identifying 50 SnRK genes in rice (four OsSnRK1, 11 OsSnRK2 and 35 OsSnRK3) and 39 in Arabidopsis (three AtSnRK1, 10 AtSnRK2 and 26 AtSnRK3). Specific motifs were identified in the encoded barley proteins, and multiple putative regulatory elements were found in the gene promoters, with light-regulated elements (LRE), ABA response elements (ABRE) and methyl jasmonate response elements (MeJa) the most common. RNA-seq analysis of the roots of seedlings grown with and without ABA treatment showed that many of the genes responded to ABA, some positively, some negatively and some with complex time-dependent responses. Conclusions: The barley HvSnRK gene family is large, comprising 50 members, subdivided into HvSnRK1 (6 members), HvSnRK2 (10 members) and SnRK3 (34 members), showing differential positive and negative responses to ABA

Genetic diversity and genome-wide association analysis of the hulled/naked trait in a barley collection from Shanghai Agricultural Gene Bank.

Barley is one of the most important cereal crops in the world, so the research and use of barley germplasm are very important for global food security. Although a large number of barley germplasm have been collected globally, their specific genetic compositions are not well understood, and in many cases their origins are disputed. In this study, barley germplasm from Shanghai Agricultural Gene Bank were genotyped by genotyping-by-sequencing (GBS) technology and their genetic relationships were investigated. In addition, genome-wide association study (GWAS) was carried out for the hulled/naked grain trait. The results showed that these barley germplasm could be roughly divided into three categories according to phylogenetic analysis, which was generally consistent with the classification of the traits of row type and hulled/naked grain, among which the combination of the two-rowed and naked traits was relatively rare. Population structure analysis showed that the whole barley population could be divided into four sub-populations (SPs), the main difference from previous classifications being that that the two-rowed and hulled genotypes were sub-divided into two SPs. The GWAS analysis of the hulled/naked trait showed that many associated loci were unrelated to the nud locus, indicating that there might be new loci controlling the trait. A KASP marker was developed for one exon type SNP on chromosome 7. Genotyping based on the KASP assay was consistent with that based on SNPs, indicating that the gene of this locus might be associated with the hulled/naked trait. The above work not only lays a good foundation for the future utilization of this barley germplasm population, but also provides new loci and candidate genes for the hulled/naked trait

Novel low-nitrogen stress-responsive long non-coding RNAs (lncRNA) in barley landrace B968 (Liuzhutouzidamai) at seedling stage

Background: Reducing the dependence of crop production on chemical fertilizer with its associated costs, carbon footprint and other environmental problems is a challenge for agriculture. New solutions are required to solve this problem, and crop breeding for high nitrogen use efficiency or tolerance of low nitrogen availability has been widely considered to be a promising approach. However, the molecular mechanisms of high nitrogen use efficiency or low-nitrogen tolerance in crop plants are still to be elucidated, including the role of long non-coding RNAs (lncRNAs. Results: In this study, we identified 498 lncRNAs in barley (Hordeum vulgare) landrace B968 (Liuzhutouzidamai), of which 487 were novel, and characterised 56 that were responsive to low-nitrogen stress. For functional analysis of differentially-expressed lncRNAs, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of co-expressed and co-located protein-coding genes were analyzed, and interactions with annotated co-expressed protein coding genes or micro RNAs (miRNAs) were further predicted. Target mimicry prediction between differentially-expressed lncRNAs and miRNAs identified 40 putative target mimics of lncRNAs and 58 target miRNAs. Six differentially-expressed lncRNAs were further validated by qPCR, and one in particular showed consistent differential expression using both techniques. Expression levels of most of the lncRNAs were found to be very low, and this may be the reason for the apparent inconsistency between RNA-seq and qPCR data. Conclusions: The analysis of lncRNAs that are differentially-expressed under low-nitrogen stress, as well as their coexpressed or co-located protein coding genes and target mimics, could elucidate complex and hitherto uncharacterised mechanisms involved in the adaptation to low-nitrogen stress in barley and other crop plants

Advances in barley breeding for improving nitrogen use efficiency

Crop breeding for high nitrogen use efficiency (NUE) or tolerance to low nitrogen fertilization is thought to be an ideal solution to reduce the cost, carbon footprint, and other environmental problems caused by the excess use of nitrogen fertilizers. As a model plant for cereal crops, barley has many advantages, including good adaptability, a short growth period, and high natural stress resistance or tolerance. Therefore, research on improving NUE in barley is not only beneficial for nitrogen-efficient barley breeding but will also inform NUE improvement in other cereal crops. In this review, recent progress in understanding barley’s response to nitrogen nutrition, evaluation of NUE or low-nitrogen tolerance, quantitative trait loci (QTL) mapping and gene cloning associated with improving NUE, and breeding of nitrogen-efficient barley is summarized. Furthermore, several biotechnological tools that could be used for revealing the molecular mechanisms of NUE or breeding for improving NUE in barley are introduced, including GWAS, omics, and gene editing. The latest research ideas in unraveling the molecular mechanisms of improving NUE in other crops are also discussed. Thus, this review provides a better understanding of improving the NUE of barley and some directions for future research in this area

Rapid Generation and Analysis of a Barley Doubled Haploid Line with Higher Nitrogen Use Efficiency than Parental Lines by F1 Microspore Embryogenesis

Creating varieties with high nitrogen use efficiency (NUE) is crucial for sustainable agriculture development. In this study, a superior barley doubled haploid line (named DH45) with improved NUE was produced via F1 microspore embryogenesis with three rounds of screening in different nitrogen levels by hydroponic and field experiments. The molecular mechanisms responsible for the NUE of DH45 surpassing that of its parents were investigated by RNA-seq analysis. 1027 differentially-expressed genes (DEGs) were identified that were up- or down-regulated in DH45 under low nitrogen conditions but showed no significant differences in the parents. GO analysis indicated that genes involved in nitrogen compound metabolic processes were significantly enriched in DH45 compared with the parents. KEGG analysis showed the MAPK signaling pathway-plant to be highly enriched in DH45 relative to its parents, as well as genes involved in alanine, aspartate and glutamate metabolism, and arginine biosynthesis. In conclusion, our study revealed the potential to fix trait superiority in a line by combining crossing with F1 microspore culture technologies in future crop breeding, and also identified several candidate genes that are expressed in shoots and may enable barley to cope with low-nitrogen stress

The fundamental cycle of concept construction underlying various theoretical frameworks

In this paper, the development of mathematical concepts over time is considered. Particular reference is given to the shifting of attention from step-by-step procedures that are performed in time, to symbolism that can be manipulated as mental entities on paper and in the mind. The development is analysed using different theoretical perspectives, including the SOLO model and various theories of concept construction to reveal a fundamental cycle underlying the building of concepts that features widely in different ways of thinking that occurs throughout mathematical learning

Employability and higher education: contextualising female students' workplace experiences to enhance understanding of employability development

Current political and economic discourses position employability as a responsibility of higher education, which deploys mechanisms such as supervised work experience (SWE) to embed employability skills development into the undergraduate curriculum. However, workplaces are socially constructed complex arenas of embodied knowledge that are gendered. Understanding the usefulness of SWE therefore requires consideration of the contextualised experiences of it, within these complex environments. This study considers higher education's use of SWE as a mechanism of employability skills development through exploration of female students' experiences of accounting SWE, and its subsequent shaping of their views of employment. Findings suggest that women experience numerous, indirect gender-based inequalities within their accounting SWE about which higher education is silent, perpetuating the framing of employability as a set of individual skills and abilities. This may limit the potential of SWE to provide equality of employability development. The study concludes by briefly considering how insights provided by this research could better inform higher education's engagement with SWE within the employability discourse, and contribute to equality of employability development opportunity

The wheat SnRK1α family and its contribution to Fusarium toxin tolerance.

Deoxynivalenol (DON) is a mycotoxin produced by phytopathogenic Fusarium fungi in cereal grain and plays a role as a disease virulence factor. TaFROG (Triticum aestivum Fusarium Resistance Orphan Gene) enhances wheat resistance to DON and it interacts with a sucrose non-fermenting-1 (SNF1)-related protein kinase 1 catalytic subunit α (SnRK1α). This protein kinase family is central integrator of stress and energy signaling, regulating plant metabolism and growth. Little is known regarding the role of SnRK1α in the biotic stress response, especially in wheat. In this study, 15 wheat (Triticum aestivum) SnRK1α genes (TaSnRK1αs) belonging to four homoeologous groups were identified in the wheat genome. TaSnRK1αs are expressed ubiquitously in all organs and developmental stages apart from two members predominantly detected in grain. While DON treatment had either no effect or downregulated the transcription of TaSnRK1αs, it increased both the kinase activity associated with SnRK1α and the level of active (phosphorylated) SnRK1α. Down-regulation of two TaSnRK1αs homoeolog groups using virus induced gene silencing (VIGS) increased the DON-induced damage of wheat spikelets. Thus, we demonstrate that TaSnRK1αs contribute positively to wheat tolerance of DON and conclude that this gene family may provide useful tools for the improvement of crop biotic stress resistance