J. Antoni Rafalski

[55] – Genetic Analysis Using Random Amplified Polymorphic DNA Markers

Publisher Summary The most commonly used DNA markers in genetic mapping, genetic diagnostics, molecular taxonomy, and evolutionary studies are restriction fragment length polymorphisms (RFLPs). This chapter presents the detailed experimental protocols for random amplified polymorphic DNA (RAPD) assays and applications, emphasizing their use for genetic analysis in plants. To perform a RAPD assay, a single oligonucleotide of an arbitrary DNA sequence is mixed with genomic DNA in the presence of a thermostable DNA polymerase and a suitable buffer, and then is subjected to temperature cycling conditions typical of the polymerase chain reaction. At an appropriate annealing temperature during the thermal cycle, the single primer binds to sites on opposite strands of the genomic DNA that are within an amplifiable distance of each other, and a discrete DNA segment is produced. The presence or absence of this specific product, although amplified with an arbitrary primer, will be diagnostic for the oligonucleotide-binding sites on the genomic DNA. The chapter additionally presents the statistical aspects of genetic mapping with RAPD markers.

Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines

The science of mapping genetic traits, including those of agronomic interest, is well established and many genetic marker systems are available. However, the application of genetic diagnostics in plant breeding is in its infancy. The sample throughput and cost requirements are very different from those of medical DNA diagnostics. It will be necessary to automate the DNA isolation process, DNA amplification-based allele identification and data handling. Here, we discuss recent progress in the development of molecular technology for plant breeding.

Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize

Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an ≈2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize–sorghum–rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution.

4 – Generating and Using DNA Markers in Plants

This chapter presents description and comparison of different DNA marker systems. DNA markers are being applied to a wide variety of problems central to plant genome analysis. Each marker system is characterized by a unique combination of advantages and disadvantages and the choice of a marker system is dictated to a significant extent by the application. Factors to consider in choosing a marker system include the amount of available plant material, the quality of the DNA, and the availability of public collections of DNA markers for the species being examined. Restriction fragment length polymorphism (RFLP) markers are generally codominant, allowing detection and characterization of multiple alleles at a given RELP locus among individuals in a population. Several types of polymorphism can be detected, including single base substitutions, insertions, and deletions. One clear disadvantage to using RFLP markers is the large amount of high-quality genomic DNA required from each individual. It is suggested that once the mapping parents have been chosen and low-copy RFLP probes obtained, those probes that detect useful polymorphisms in the segregating population must be identified. The polymerase chain reaction analysis and identification of polymorphisms are also elaborated.

Novel genetic mapping tools in plants: SNPs and LD-based approaches

Abstract Use of DNA-based genetic markers [1] has forever changed the practice of genetics. In the 20 years since that discovery, many different types of DNA-based genetic markers have been used for the construction of genetic maps, for the analysis of genetic diversity, trait mapping, as well as for applied diagnostic purposes. A bewildering array of acronyms, such as RFLP, SSR, AFLP, RAPD, AP-PCR, DAF, SAMPL, and many others describes these methodologies [2] . AFLPs and SSRs have become especially popular due to the formers high multiplex ratio and the latters high degree of informativeness [3] . Also, arbitrary primer-based methods, such as RAPD, found their applications because of their simplicity. All of these methods constitute indirect approaches towards assessing DNA sequence differences: single nucleotide polymorphisms (SNPs, [4] ) and insertions/deletions (indels). A direct analysis of sequence difference between many individuals at a large number of loci has now become practical. Dramatic advances in sequencing technology have resulted in the determination of complete DNA sequences of many organisms including most notably human, and, from a plant scientists’ perspective, Arabidopsis [5] . The next important objective is to determine sequence diversity of genic and regulatory regions in these and other species. This would allow the understanding of the relationship between phenotypic diversity and genetic diversity. We discuss here the development and applications of SNP genetic markers in corn and other crop plants, and the contribution of these studies towards the understanding of the organization of genetic diversity in plants. We also discuss linkage disequilibrium-based trait mapping approaches.

Structure of wheat gamma-gliadin genes

We have cloned and sequenced two linked members of the wheat (Triticum aestivum) gliadin multigene family. One gene encodes a gamma-gliadin which is 292 amino acids (aa) long. S1 mapping indicates that this gene is transcriptionally active. The second gene, which is only marginally active by S1 mapping, is closely related to the first gene. Moreover, it is probably incapable of encoding a full-length gamma-gliadin due to the presence of two premature in-frame stop codons. Neither gene contains introns. Nucleotide sequences of the two genes show the presence of characteristic repeats which have a derived aa consensus sequence Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln. A comparison of the promoter regions of these gamma-gliadin genes with those of the alpha-gliadin genes shows the presence of a highly conserved region that could be involved in tissue specific and developmental regulation.

Combining expression and comparative evolutionary analysis. The COBRA gene family

Plant cell shape is achieved through a combination of oriented cell division and cell expansion and is defined by the cell wall. One of the genes identified to influence cell expansion in the Arabidopsis (Arabidopsis thaliana) root is the COBRA (COB) gene that belongs to a multigene family. Three members of the AtCOB gene family have been shown to play a role in specific types of cell expansion or cell wall biosynthesis. Functional orthologs of one of these genes have been identified in maize (Zea mays) and rice (Oryza sativa; Schindelman et al., 2001; Li et al., 2003; Brown et al., 2005; Persson et al., 2005; Ching et al., 2006; Jones et al., 2006). We present the maize counterpart of the COB gene family and the COB gene superfamily phylogeny. Most of the genes belong to a family with two main clades as previously identified by analysis of the Arabidopsis family alone. Within these clades, however, clear differences between monocot and eudicot family members exist, and these are analyzed in the context of Type I and Type II cell walls in eudicots and monocots. In addition to changes at the sequence level, gene regulation of this family in a eudicot, Arabidopsis, and a monocot, maize, is also characterized. Gene expression is analyzed in a multivariate approach, using data from a number of sources, including massively parallel signature sequencing libraries, transcriptional reporter fusions, and microarray data. This analysis has revealed that the expression of Arabidopsis and maize COB gene family members is highly developmentally and spatially regulated at the tissue and cell type-specific level, that gene superfamily members show overlapping and unique expression patterns, and that only a subset of gene superfamily members act in response to environmental stimuli. Regulation of expression of the Arabidopsis COB gene family members has highly diversified in comparison to that of the maize COB gene superfamily members. We also identify BRITTLE STALK 2-LIKE 3 as a putative ortholog of AtCOB.

Genetic Analysis with RAPD Markers

For many years the principles of genetics have been applied to crop variety improvement with great success. Several crop species, notably corn, wheat, and tomato, have been used as model genetic systems because of their central importance to food production. Until recently, virtually all progress in both breeding and model genetic systems has relied on a phenotypic assay of genotype. Since the efficiency of a selection scheme or genetic analysis based on phenotype is a function of the heritability of the trait, factors like the environment, multigenic and quantitative inheritance, or partial and complete dominance often confound the expression of a genetic trait (33). Many of the complications of a phenotype-based assay can be mitigated through direct identification of genotype with a DNA-based diagnostic assay (5,7). For this reason, DNA-based genetic markers are being integrated into several genetic systems, and are expected to play an important role in the future of plant breeding.

A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. colis transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

A Customized Gene Expression Microarray Reveals that the Brittle Stem Phenotype fs2 of Barley is Attributable to a Retroelement in the HvCesA4 Cellulose Synthase Gene

The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.