Transposing from the Laboratory to the Classroom to Generate Authentic Research Experiences for Undergraduates

Large lecture classes and standardized laboratory exercises are characteristic of introductory biology courses. Previous research has found that these courses do not adequately convey the process of scientific research and the excitement of discovery. Here we propose a model that provides beginning biology students with an inquiry-based, active learning laboratory experience. The Dynamic Genome course replicates a modern research laboratory focused on eukaryotic transposable elements where beginning undergraduates learn key genetics concepts, experimental design, and molecular biological skills. Here we report on two key features of the course, a didactic module and the capstone original research project. The module is a modified version of a published experiment where students experience how virtual transposable elements from rice (Oryza sativa) are assayed for function in transgenic Arabidopsis thaliana. As part of the module, students analyze the phenotypes and genotypes of transgenic plants to determine the requirements for transposition. After mastering the skills and concepts, students participate in an authentic research project where they use computational analysis and PCR to detect transposable element insertion site polymorphism in a panel of diverse maize strains. As a consequence of their engagement in this course, students report large gains in their ability to understand the nature of research and demonstrate that they can apply that knowledge to independent research projects.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

几种农作物细胞质雄性不育恢复基因的定位和分子标记研究进展

利用杂种优势提高作物产量时, 生产杂交种的主要授粉控制系统是细胞质雄性不育及其恢复系统。在杂交品种的选育过程中, 优良恢复系选育至关重要。为了高效并准确地鉴定选择恢复材料, 同时更深入地研究恢复基因的作用机理, 近年来植物细胞质雄性不育恢复基因分子标记研究受到了广泛重视。本文综述了主要农作物水稻、油菜、小麦、棉花和玉米等细胞质雄性不育类型恢复基因的定位和分子标记研究进展, 并讨论了恢复基因的精确定位和分子标记鉴定在基因克隆和分子标记辅助选择育种中的意义和应用前景。     

几种农作物细胞质雄性不育恢复基因的定位和分子标记研究进展

利用杂种优势提高作物产量时,生产杂交种的主要授粉控制系统是细胞质雄性不育及其恢复系统。在杂交品种的选育过程中,优良恢复系选育至关重要。为了高效并准确地鉴定选择恢复材料,同时更深入地研究恢复基因的作用机理,近年来植物细胞质雄性不育恢复基因分子标记研究受到了广泛重视。本文综述了主要农作物水稻、油菜、小麦、棉花和玉米等细胞质雄性不育类型恢复基因的定位和分子标记研究进展,并讨论了恢复基因的精确定位和分子标记鉴定在基因克隆和分子标记辅助选择育种中的意义和应用前景。     

Production and characterization of maize chromosome 9 radiation hybrids derived from an oat-maize addition line

In maize (Zea mays L., 2n = 2x = 20), map-based cloning and genome organization studies are often complicated because of the complexity of the genome. Maize chromosome addition lines of hexaploid cultivated oat (Avena sativa L., 2n = 6x = 42), where maize chromosomes can be individually manipulated, represent unique materials for maize genome analysis. Maize chromosome addition lines are particularly suitable for the dissection of a single maize chromosome using radiation because cultivated oat is an allohexaploid in which multiple copies of the oat basic genome provide buffering to chromosomal aberrations and other mutations. Irradiation (gamma rays at 30, 40, and 50 krad) of a monosomic maize chromosome 9 addition line produced maize chromosome 9 radiation hybrids (M9RHs)-oat lines possessing different fragments of maize chromosome 9 including intergenomic translocations and modified maize addition chromosomes with internal and terminal deletions. M9RHs with 1 to 10 radiation-induced breaks per chromosome were identified. We estimated that a panel of 100 informative M9RHs (with an average of 3 breaks per chromosome) would allow mapping at the 0. 5- to 1.0-Mb level of resolution. Because mapping with maize chromosome addition lines and radiation hybrid derivatives involves assays for the presence or absence of a given marker, monomorphic markers can be quickly and efficiently mapped to a chromosome region. Radiation hybrid derivatives also represent sources of region-specific DNA for cloning of genes or DNA markers.     

Multiple phenotypes resulting from a mutagenesis screen for pharynx muscle mutations in Caenorhabditis elegans

We describe a novel screen to isolate pharyngeal cell morphology mutants in Caenorhabditis elegans using myo-2::GFP to rapidly identify abnormally shaped pharynxes in EMS (Ethyl Methanesulfonate) mutagenized worms. We observed over 83 C. elegans lines with distinctive pharyngeal phenotypes in worms surviving to the L1 larval stage, with phenotypes ranging from short pharynx, unattached pharynx, missing cells, asymmetric morphology, and non-adherent pharynx cells. Thirteen of these mutations have been chromosomally mapped using Single Nucleotide Polymorphisms (SNPs) and deficiency strain complementation. Our studies have focused on genetically mapping and functionally testing two phenotypes, the short pharynx and the loss of muscle cohesion phenotypes. We have also identified new alleles of sma-1, and our screen suggests many genes directing pharynx assembly and structure may be either pharynx specific or less critical in other tissues.     

Drosophila Transposon Insertions as Unknowns for Structured Inquiry Recombination Mapping Exercises in an Undergraduate Genetics Course

Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.INQUIRY-BASED learning in which students are engaged in open-ended, student-centered, hands-on activities is an important tool for training undergraduates to think like scientists (Colburn 2000; Handelsman et al. 2004). With this approach, students learn scientific subjects by interpreting and discussing experimental results in a fashion similar to that used by scientific researchers (NRC 2003). There are three main approaches to instruction via inquiry. In open inquiry, students formulate their own problem, as well as the procedures to investigate the problem. In guided inquiry, the instructor provides the problem and necessary materials, but the students devise an experimental procedure to investigate the problem. Finally, in structured inquiry, the instructor provides the problem, the materials, and the procedure, but the students are required to gather and interpret the experimental data independently, coming to their own conclusions (Welch et al. 2006). In each case, the instructor does not provide “the answer” to the problem. In the ideal case, the instructor does not even know what the answer will be prior to the student experiment, forcing the students to grapple with the information themselves. Inquiry-based laboratories can even be extended so that students are participating in novel research as part of their coursework (DebBurman 2002; Buckner et al. 2007), which been shown to improve undergraduate retention and student performance in biology lecture courses (Marcus et al. 2009).The process of inquiry has been identified as central to training students to understand fundamental approaches used in the field of genetics such as the design of controlled crosses and interpretation of experimental data (Cartier and Stewart 2000). Pukkila (2004) discusses effective methods by which inquiry-based learning can be incorporated into undergraduate genetics lecture courses with large enrollments and into recitation sections. However, the implementation of inquiry-based approaches in undergraduate genetics laboratories has not been discussed extensively.Teaching laboratories offer some advantages for inquiry learning because they generally contain small groups of students, facilitating a flexible and intimate learning environment with many interactions between students and the instructor, as well as among classmates. However, teaching laboratories associated with large lecture courses also offer some challenges, in particular how to deliver substantially similar experiences to laboratory sections taught by multiple instructors, as well as how to provide inquiry-based learning in a logistically manageable and cost-effective manner. For these reasons, most inquiry-based genetics laboratory exercises have used the structured inquiry approach, for example, using many Drosophila melanogaster strains with similar mutant phenotypes (e.g., white eyes and black bodies), but a variety of genotypes, in a series of standardized genetic mapping crosses to familiarize students with the collection and interpretation of classical genetic data (MacIntyre 1974; Pye 1980). The difficulty with contrived genetic unknowns carrying well-mapped genetic mutations is that many students become frustrated that their hard work evaluating the crosses over a period of several months is devoted to a fundamentally trivial exercise, as the recombination map locations of the genes are already established in the scientific literature and have been recalculated thousands of times by generations of genetics students.We have expanded upon the structured inquiry approach to genetics to include novel research experiences for the students in our undergraduate genetics laboratories. They conduct mapping crosses with Drosophila strains carrying P-element transposon insertions in genes without documented recombination map positions. The stock centers maintain very large collections of P-element transposon stocks with known insertion sites on the cytological and genome maps (Spradling et al. 1999). However, in spite of the cytology to recombination map equivalence table available in FlyBase (2009), very few of the transposon inserts have been formally placed on the recombination map. By using the eye color phenotypes associated with many transposon inserts as visible markers in genetic crosses (Marcus 2003), it is straightforward to calculate recombination map positions for the interrupted loci. The stock collections contain many stocks with identical transposons inserted at different chromosomal locations, providing a large number of unique, but equivalent genetic unknowns that can be used for recombination mapping exercises. At the same time, this approach provides students with the opportunity to map genes that have never been mapped before, allowing them to make small but useful contributions to the field of Drosophila genetics.     

Genetic Analysis of 63 Mutations Affecting Maize Kernel Development Isolated from Mutator Stocks

Sixty-three mutations affecting development of the maize kernel were isolated from active Robertson's Mutator (Mu) stocks. At least 14 previously undescribed maize gene loci were defined by mutations in this collection. Genetic mapping located 53 of these defective kernel (dek) mutations to particular chromosome arms, and more precise map determinations were made for 21 of the mutations. Genetic analyses identified 20 instances of allelism between one of the novel mutations and a previously described dek mutation, or between new dek mutations identified in this study; phenotypic variability was observed in three of the allelic series. Viability testing of homozygous mutant kernels identified numerous dek mutations with various pleiotropic effects on seedling and plant development. The mutations described here presumably arose by insertion of a Mu transposon within a dek gene; thus, many of the affected loci are expected to be accessible to molecular cloning via transposon-tagging.     

影响果蝇心脏发育的基因突变

最近的研究表明,果蝇与脊椎动物及人的心脏早期发育具有极为相似的基因控制机理,果蝇已成为研究人体心脏早期发育基因控制的理想模式动物。利用化学诱变剂甲磺酸乙酯大规模地诱变影响果蝇心脏发育的基因,利用心脏特异性抗体染色进行筛选,获得了112个有心脏突变表型的致死系,其中32个致死系的心脏畸变表型有别于目前已知心脏发育基因的突变表型。细胞遗传学定位研究表明在多线染色体的13个带纹区的某些隐性致死突变基因是目前未知的,其功能可能与发育有关的基因。     

Steady-state transposon mutagenesis in inbred maize

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.     

Saturating the genetic map of Arabidopsis thaliana with embryonic mutations

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo-defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo-genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty-six of these genes are tagged with T-DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo-defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.     

玉米雄穗颜色QTL分析

雄穗是玉米的重要生殖器官,不同品种间玉米的雄穗外观差异明显。对玉米雄穗的颜色进行遗传分析和QTL定位,筛选与雄穗颜色紧密连锁的分子标记,可以作为玉米的品种保护和品种鉴别的有用工具。同时,紫色雄穗中花色苷类色素含量较高,与玉米雄穗的抗虫性密切相关。本研究利用一个黑玉米自交系SDM为共同父本,分别与白玉米自交系木6和黄玉米自交系Mo17杂交,构建2个相关F2∶3群体,分别命名为MuS(木6×SDM)和MoS(Mo17×SDM),在云南和重庆两个不同的环境中种植,对玉米花药颜色(COAn)和花药护颖颜色(COCa)2个性状进行QTL定位。结果表明:玉米花药和花药护颖的颜色均为数量性状,受主效基因和微效基因共同控制。2个群体在2个环境中共检测到7个与花药颜色相关的QTL,位于第2、3、6和10染色体上,其中位于第10染色体标记区间umc1196a-IDP8526内的QTL在重庆和云南同时表达,对表型的贡献率分别为23.17%和19.98%;2个群体在2个环境中共检测到9个与花药护颖颜色相关的QTL,位于第3、6、9和10染色体上,其中3个QTL为环境钝感QTL(在2个环境中均表达,且至少在1个环境中贡献率大于10%),分别位于第6染色体标记区间umc1979-umc1796、mmc0523-umc2006内和第10染色体标记区间umc1196a-umc2043内,对表型的贡献率为10.69%～59.30%。2个群体检测到的主效QTL的位置和效应高度一致,且控制花药颜色和花药护颖颜色2个性状的主效QTL有连锁分布的现象,主要表现在bins 6.04处的标记mmc0523和bins 10.04处的标记IDP8526附近。位于第6和第10染色体上的在不同环境和遗传背景下稳定的QTL可以作为进一步精细定位的靶位点,也可以为玉米雄穗颜色的分子标记辅助选择提供有价值的参考。     

Novel integrative genomics strategies to identify genes for complex traits

Forward genetics is a common approach to dissecting complex traits like common human diseases. The ultimate aim of this approach was the identification of genes that are causal for disease or other phenotypes of interest. However, the forward genetics approach is by definition restricted to the identification of genes that have incurred mutations over the course of evolution or that incurred mutations as a result of chemical mutagenesis, and that as a result lead to disease or to variations in other phenotypes of interest. Genes that harbour no such mutations, but that play key roles in parts of the biological network that lead to disease, are systematically missed by this class of approaches. Recently, a class of novel integrative genomics approaches has been devised to elucidate the complexity of common human diseases by intersecting genotypic, molecular profiling, and clinical data in segregating populations. These novel approaches take a more holistic view of biological systems and leverage the vast network of gene–gene interactions, in combination with DNA variation data, to establish causal relationships among molecular profiling traits and Fbetween molecular profiling and disease (or other classic phenotypes). A number of novel genes for disease phenotypes have been identified as a result of these approaches, highlighting the utility of integrating orthogonal sources of data to get at the underlying causes of disease.     

Dissecting complex phenotypes using the genomics of twins

Genetics in the post-genomic period is shifting from structural to functional genetics or genomics. Meanwhile, the use of twins is largely expanding from traditional heritability estimation for disease phenotypes to the study of both diseases and various molecular phenotypes, such as the regulatory phenotypes in functional genomics concerning gene expression and regulation, by engaging both classical twin design and marker-based gene mapping techniques in genetic epidemiology. New research designs have been proposed for making novel uses of twins in studying the molecular basis in the epigenetics of human diseases. Besides, twins not only serve as ideal samples for disease gene mapping using conventional genetic markers but also represent an excellent model for associating DNA copy number variations, a structural genetic marker, with human diseases. It is believed that, with the rapid development in biotechniques and new advances in bioinformatics, the unique samples of twins will make new contributions to our understanding of the nature and nurture in complex disease development and in human health. This paper aims at summarizing the new uses of twins in current genetic studies and suggesting novel proposes together with useful design and analytical strategies.     

Candidate gene identification of existing or induced mutations with pipelines applicable to large genomes

Bulked segregant analysis (BSA) is used to identify existing or induced variants that are linked to phenotypes. Although it is widely used in Arabidopsis and rice, it remains challenging for crops with large genomes, such as maize. Moreover, analysis of huge data sets can present a bottleneck linking phenotypes to their molecular basis, especially for geneticists without programming experience. Here, we identified two genes of maize defective kernel mutants with newly developed analysis pipelines that require no programing skills and should be applicable to any large genome. In the 1970s, Neuffer and Sheridan generated a chemically induced defective kernel (dek) mutant collection with the potential to uncover critical genes for seed development. To locate such mutations, the dek phenotypes were introgressed into two inbred lines to take advantage of maize haplotype variations and their sequenced genomes. We generated two pipelines that take fastq files derived from next‐generation (nextGen) paired‐end DNA and cDNA sequencing as input, call on several well established and freely available genomic analysis tools to call SNPs and INDELs, and generate lists of the most likely causal mutations together with variant index plots to locate the mutation to a specific sequence position on a chromosome. The pipelines were validated with a known strawberry mutation before cloning the dek mutants, thereby enabling phenotypic analysis of large genomes by next‐generation sequencing.     

Linkage mapping of two mutations that reduce phytic acid content of barley grain

 This study describes the inheritance and linkage map positions of two low phytic acid barley (Hordeum vulgare) mutations, lpa1-1 and lpa2-1, that dramatically reduce grain phytic acid content and increase inorganic seed phosphorus (P). Wide-cross, F₂ mapping populations were constructed by mating six-rowed varieties, ‘Steptoe’ and/or ‘Morex’, with two-rowed ‘Harrington’lpa donor lines homozygous for either lpa1-1 or lpa2-1. The barley lpa1-1 mutation showed normal inheritance patterns, whereas a deficiency of homozygous lpa2-1/lpa2-1 F₂ plants was observed. We identified a codominant, STS-PCR marker (aMSU21) that cosegregated with lpa1-1 in a population of 41 F₂ plants. The aMSU21 marker was then mapped to a locus on barley chromosome 2H, using a North American Barley Genome Mapping Project (NABGMP) doubled haploid population (‘Harrington’בMorex’). We determined that lpa2-1 is located within a recombination interval of approximately 30 cM between two AFLP markers that were subsequently mapped to barley chromosome 7H by integration with the same NABGMP population. Recent comparative mapping studies indicate conserved genetic map orders of several homologous molecular marker loci in maize and the Triticeae species that also show corresponding linkage to the biochemically similar lpa2 mutations of maize and barley. This observation suggests that barley and maize lpa2 mutations may affect orthologous genes. No such evidence for correspondence of the phenotypically similar lpa1 mutations of barley and maize has been revealed. Received: 22 September 1997 / Accepted: 2 December 1997