​Development of iTAG

In 2009 we developed a plan to utilize the power of barley genetics to create a teaching and training resource for high school and undergraduate classrooms. In 2010, NSF Award No. 0922746 was used to begin building this resource, Inheritance of Traits and Genes (iTAG), as summer training for the RET (Research Experience for Teachers) program. Between 2010 and 2014 iTAG was implemented in 35 classrooms and impacted >1,000 students in both rural and urban communities in the United States. Feedback was collected from students and instructors, and iTAG was revised. In 2014, a second NSF Award (No. 1339348) was used to fund additional RETs and iTAG Instructor Workshops, helping to further refine the curriculum. Subsequently, between 2015 and 2016 iTAG was implemented in 53 high school classes, impacting >1,400 students with similar demographics as before. In 2020, a new NIFA Award (No. 2020-67013-31184) sponsored an iTAG RET in 2021 and 2022 and then a Community College & University Instructors Workshop in 2023. In addition to 12 years of 6-week RET training, we sponsored week-long workshops in 2015, 2016, 2017, and 2023 to train four cohorts of instructors from across the United States. In order to promote diversity, equity, inclusion, and accessibility and widespread dissemination to rural and underserved populations, we engage the instructors throughout the school year to provide NSF-funded thermal cyclers, microcentrifuges, gel boxes, transilluminators, pipeteman, seed, and reagents needed to complete the program. This advanced equipment package provides the students with science lab experiences that they normally would not have access to, especially in districts with limited resources. iTAG barley has been used successfully by nearly 50 instructors across the United States in >200 high school and community college biology classes from 2010 to 2023, impacting a total of 4,964 students, of which one-third were from underrepresented groups from urban to rural communities. Despite the recent pandemic, we engaged iTAG instructors to train 304 new students, of which 79 went on to study in STEM disciplines.

​ Figure 1. Comparison of the hooded (A, DH10) and awned (B, DH49) phenotypes in Oregon Wolfe barley. These phenotypes are controlled by the interaction of two genes, Kap (Müller et al., 1995; Roig et al., 2004) and Lks2 (Yuo et al., 2012), respectively.​

Introduction

One of the basic concepts in biology is that an organism's physical traits are controlled by genes that are encoded in its DNA. In other words, one's genotype for a particular trait controls the phenotype that is expressed. Students and novice researchers often struggle understanding this connection between DNA and physical characteristic. iTAG Barley is a module of laboratory and classroom activities designed to connect visible traits (phenotype) to identifiable differences in the DNA sequence of genes.

In this workbook, we focus on three traits to illustrate basic concepts in plant development, domestication, and disease resistance. Students plant and grow barley plants so that phenotypic variation can be observed firsthand. For the first learning module, students describe the “awned" and “hooded" phenotypes and use common biotechnology methods to investigate the differences in the DNA sequence at one gene (Kap) that influence the development of barley spikelets (Fig. 1). Students completing this module learn basic molecular biology techniques of DNA extraction, polymerase chain reaction (PCR), and gel electrophoresis and interpret data from different plant phenotypes to document DNA polymorphisms among plants with different phenotypes.

The Educational Value of Oregon Wolfe Barley

The Oregon Wolfe barley (OWB) population is a model resource for genetics research and instruction. This collection of doubled haploid (DH) lines was developed from an F₁ of a cross between dominant and recessive marker stocks advanced by Dr. Robert Wolfe at Federal Agriculture Research in Alberta, Canada. These DH lines originate from a wide cross and have exceptionally diverse and dramatic phenotypes, making the OWB population attractive for teaching basic plant development, Mendelian and molecular genetics, and genomics in high school, community college, or first-year university biology (Cistué et al., 2011; Giménez et al., 2021; Szűcs et al., 2009). Dr. Pat Hayes at Oregon State University further selected the Informative and Spectacular Subset (ISS) from which these lessons are based (Hayes, 2011, 2023a, 2023c; Hayes and Stein, 2003).

Using the iTAG Barley module, students can observe the OWB spikes for seed-coat color, two row versus six row (encoded by Vrs1, a domestication trait where two row is dominant and six row is recessive) (Komatsuda et al., 2007), hooded versus nonhooded (Kap: dominant allele encoded by BKn3, a homoeotic mutation where the awn is replaced by a duplicate spikelet) (Müller et al., 1995; Roig et al., 2004; Williams-Carrier et al., 1997), and long awn versus short awn (long awn is dominant and encoded by Lks2) (Yuo et al., 2012) traits. Lastly, the OWB population has lines that are resistant or susceptible to powdery mildew disease due to traits encoded by different alleles of Mildew locus a (Mla) (Bettgenhaeuser et al., 2021; Halterman et al., 2001; Seeholzer et al., 2010; Wei et al., 2002). Additional details and resources can be found at Oregon Wolfe Barley Data and GrainGenes Tools—an archive and resource hub: https://wheat.pw.usda.gov/ggpages/maps/OWB (Hayes, 2011); and Barley World: https://barleyworld.org from Oregon State University.

In a series of three different exercises, students perform PCR to amplify the Kap, Vrs1 (HvHox1), and Mla genes using DNA they isolate from plants with phenotypic differences; complete gel electrophoresis using agarose gels; and document their results by estimating PCR product size by comparing PCR products with size markers. They then discuss their results and describe differences in the DNA at each gene locus and differences in whole plant phenotypes in the OWB population. Instructors can then lead a discussion of cosegregation and how researchers associate genotype and phenotype.

Module Objectives

• After completing iTAG Barley students will
• Understand the role of DNA in an organism.
• Understand the relationship between a genotype and a phenotype, including homoeotic mutations, epistatic interactions, and the impact of phenotype on yield.
• Experience science as it is done in a research laboratory.
• Understand that science takes time.
  

Next Generation Science Standards

While these exercises are designed for early-career undergraduate students, they can also be adapted for use in high school and address the following next generation science standards (NGSS) for high school:

• Science and Engineering Practices (SEP): Asking Questions and problem solving, analyzing and interpreting data
• Life Science Core Ideas: Variation of traits
• Nature of Science: Science uses a variety of methods

Genetics Learning Framework (Genetics Society of America)

In 2015, the Genetics Society of America (GSA) Education Committee approved a comprehensive learning framework to guide educators designing and implementing undergraduate courses in general biology and general genetics. The exercises in the iTAG manual align with this framework in several ways:

• Molecular Biology of Gene Function
• “Explain how the genetic code relates transcription to translation."
• “Discuss how various factors might influence the relationship between genotype and phenotype."
• Gene Expression and Regulation
• “Defend how most cells can have the same genetic content and yet have different functions in the body."

Core Questions Addressed

Combining the high school (NGSS) and undergraduate (GSA framework) educational goals, we have addressed four core questions, linked them to concepts from both high school and undergraduate biology coursework, and connected these exercises to other coursework that undergraduate students may study in genetics, general biology, and/or biochemistry:

Project Overview

Students begin by planting a population of OWB. By placing the responsibility of planting, watering, and fertilizing on the students, they develop a vested interest in the plants.

Because one of the goals of this module is for students to understand the relationship between an individual's genotype and phenotype, they start by the amplification of a single gene (Kap) using PCR. The genotype of each plant in the population can be compared to the phenotype to observe cosegregation of DNA differences with differences in phenotype. This connects molecular differences to phenotypic outcomes. The primers utilized to amplify the Kap gene are found on either side of a tandem duplication (Kap allele) or not (kap allele). The presence or absence of this insertion allows us to identify the two alleles using gel electrophoresis. After running gels, students use GelGreen DNA stain and ultraviolet transilluminators to visualize bands of DNA. The GelGreen is both nontoxic and light insensitive, making it safe and convenient to use. Once the gel data has been documented, the class discusses the role of gene interactions (epistasis) on the development of a phenotype.

The second project also uses PCR, but this time students amplify the Vrs1 gene. The two alleles we investigate (Vrs1 and vrs1) differ in sequence, which cannot be detected by PCR alone with the primers we use. Students cut their PCR products with a restriction enzyme digest and use electrophoresis to distinguish the two alleles.

The last project of this trio of experiments involves plant pathology. One of the traits we look at is encoded by the Mla locus and confers resistance or susceptibility to powdery mildew, a fungal disease (Halterman et al., 2001, 2003; Halterman and Wise, 2004, 2006; Seeholzer et al., 2010; Wei et al., 2002).

Transfer of Concepts: Genotype to Phenotype

Barley is the experimental organism in this module; however, the concepts can be applied to all plants. In many areas of the country, the economy is largely dependent on agriculture. Because genes determine traits, discussion of genetic engineering and its influence on agriculture is a simple but meaningful application. Students investigate naturally occurring traits (e.g., hooded versus awned, two row versus six row) that are associated with molecular differences that they can detect by closely examining the DNA at a particular genetic locus. This can be the basis for a discussion of genetically modified organisms (GMOs), selective breeding, and domestication of plants for agriculture.

While agriculture is vital to human civilization, students often struggle with plants as living organisms with heritable traits. Once students have completed the first module (investigation of the Kap gene locus), the instructor can lead a discussion linking the student findings, modeling crosses between plants with awned and hooded spikelets, and making predictions. The same principles that plant geneticists used to associate the Kap gene with the hooded phenotype are used regularly to associate genes with inherited human conditions such as cilantro-taste preference (Eriksson et al., 2012), sickle cell disorder, or Tay-Sachs disorder.

​ ​Figure 2. Central dogma of molecular biology.​

Connecting the Central Dogma of Molecular Biology to Barley Phenotypes

The central dogma of molecular biology states that information stored in DNA is used as a template in transcription, which forms different types of RNA (Fig. 2). One type, messenger RNA (mRNA), is used as the template for ribosomes to make proteins. Proteins serve many different functions in a cell. Some are signaling molecules, while others make up the structure of the cell or catalyze reactions.

A gene is a functional segment of DNA that encodes a protein (e.g., an enzyme) or RNA molecule (e.g., tRNA). It contains the instructions to make its gene product. Since the OWB plants are DH lines, they have two identical copies of every gene (on two homologous chromosomes). We call the location of each gene its genetic locus, typically by describing where it is on the chromosome on which it is found. In diploid (2N) species (like humans), there are also two copies of each gene locus. The information at a given gene locus is similar for the two chromosomes but not necessarily identical. These alternate forms of DNA at the same genetic locus are called alleles. For example, in peas there is a locus for flower color, but this locus may encode a purple (P) or white (p) allele. By convention, we abbreviate the dominant allele with a capital letter (AA or BB) and the recessive with a lowercase letter (aa or bb). The genotype of an organism lists the alleles present at a given locus. For example, AA, Bb, and cc are genotype abbreviations for three different genes (A, B, and C), and the individual in question has two copies of the dominant allele for the A locus (AA), one copy of the dominant and one copy of the recessive (Bb) allele, and two copies of the recessive (cc) allele. Often the dominant allele encodes a functional protein, so only one copy of this allele is needed to see an observable trait (phenotype). The recessive allele often has an error that makes the protein that is produced nonfunctional, which means both copies of the genetic locus need to encode the recessive allele for it to be observed.

​ Figure 3. Genetic basis for the hooded phenotype. There is a 305-bp insertion in the dominant Kap allele that is not found in the recessive kap allele. This diagram illustrates the 305-bp tandem duplication within the fourth intron of the dominant allele (Kap). Exons are depicted by green boxes and introns by solid black ​​lines. ATG start and TAG stop codons are shown. PCR primers flanking this region can be used to amplify 1,247- and 1,552-bp fragments for the recessive (kap) and dominant (Kap) alleles, respectively. Adapted from Giménez et al. (2021). ​

​ Figure 4. Kap-Lks2 interactions. Plants that carry two copies of the recessive allele for lks2 will not produce a protein needed to make either a long awn or hooded phenotype. This means a plant could have the dominant Kap allele (Kap/_) and still not show the hooded phenotype if it lacks the dominant Lks2 allele (lks2/lks2). The use of the nomenclature “Lks2/_" or “Kap/_" indicates that the plant has at least one copy of the dominant allele, but we might not know from the data provided whether the plant is homozygous dominant (Lks2/Lks2 or Kap/Kap) versus heterozygous (Lks2/lks2 or Kap/kap).​

The Hooded Phenotype and Genotype

Barley plants with the hooded phenotype have an additional spikelet (the barley flower) in the place of an awn as their spikes develop. The development of a structure in an inappropriate place is an example of a class of mutations called homoeotic mutations because they are due to alterations in genes that regulate development. Homoeotic mutations can be found in both animals and plants.

When they compared the DNA sequence of the Kap gene in awned and hooded barley plants, researchers found that the hooded phenotype is associated with a 305-bp duplication found within the Kap (hooded) allele that is not present in the kap (nonhooded) allele. Surprisingly, this inserted DNA segment is found within an intron at this genetic locus (Fig. 3). Heterozygous plants will display the hooded phenotype, making the Kap allele dominant to the kap allele. The Kap gene is a transcription factor that regulates shoot and awn development. The presence of the duplication in the Kap allele causes a small flower to form where an awn normally forms during spike development in barley plants.

The Kap gene and Lks2 gene loci each encode proteins that influence the production of barley spikes (Fig. 4). The Lks gene locus determines whether the barley grain will have a long awn (Lks/_) or short awn (lks2/lks2). There is a complicating factor in plants with the Kap allele—they will form a spikelet instead of an awn if there is a dominant Lks2 allele present. Recessive lks2/lks2 plants may have the dominant Kap allele but will not show it in their phenotype, because the lks2 gene masks the Kap phenotype (hooded). We call this interaction epistasis, where the gene products from two or more genetic loci influence a single trait (in this case, the appearance of barley spikes) (Hayes, 2023b).​

​​Experiment 1​​Experiment 2​​Experiment 3
​iTAG Instructor’s Planning Resources
​​AppendixGlossary​​​

​Acknowledgments​

We thank the Iowa State University Research Experience for Teachers (RET) for connecting teachers with career scientists to further their professional development and Dr. Pat Hayes of Oregon State University for OWB-ISS population resources. This program was funded, in part, by National Science Foundation–Plant Genome Research Program Grant 13-39348; USDA-Agricultural Research Service Projects 3625-21000-067-00D and 5030-21220-068-000-D to RPW; USDA-National Institute of Food and Agriculture Grant 2020-67013-31184 to RPW, NB, and NP; and Iowa Agriculture and Home Economics Experiment Station (IAHEES) Project 4208 to NP and NB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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