Erin J. Gilchrist

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa

Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large‐scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000‐bp region examined. Overall nucleotide diversity, measured as Total, was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.

TILLING is an effective reverse genetics technique for Caenorhabditis elegans

BackgroundTILLING (T argeting I nduced L ocal L esions i n G enomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified.ResultsTo determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms.ConclusionWe conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.

Reverse genetics techniques: engineering loss and gain of gene function in plants

Genetic analysis represents a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo. Genome sequencing projects have identified large numbers of plant genes for which no role has yet been defined. To address this problem a number of techniques have been developed, over the last 15 years, to enable researchers to identify plants with mutations in genes of known sequence. These reverse genetic approaches include RNAi and related technologies and screening of populations mutagenised by insertion (PCR), deletion (PCR) and point mutation (TILLING), each with its own strengths and weaknesses. The development of next-generation sequencing techniques now allows such screening to be done by sequencing. In the future, it is likely that the genomes of thousands of plants from mutagenised populations will be sequenced allowing for the identification of plants with mutations in specific genes to be done in silico.

Forward and Reverse Genetics of Rapid-Cycling Brassica oleracea

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today’s B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes.

A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.

Gene Identification: Reverse Genetics

The number of sequenced genes whose function remains unknown continues to climb with the continuing decrease in the cost of genome sequencing. Comparative genetics and bioinformatics have been invaluable in investigating the function of the genes that have been sequenced, but the elucidation of gene function in planta remains a huge challenge. Many gene functions have been defined through the use of forward genetics, where a phenotype is identified and used to clone the gene responsible. However, in most instances, genes of known sequence are not associated with a phenotype. This is particularly true in non-model species where forward genetics can be more challenging due to genetic redundancy. Reverse genetics is a powerful tool that can be used to identify the phenotype that results from disruption of a specific sequenced gene, even with no prior knowledge of its function. Several approaches have been developed in plants that have led to the production of resources including collections of T-DNA insertion mutants, RNAi-generated mutants, and populations carrying point mutations that can be detected by TILLING, direct sequencing or high resolution melting analysis (Table 4.1). These reverse genetics resources allow for the identification of mutations in candidate genes and subsequent phenotypic analysis of these mutants. In addition, new advances in technology and reduction in technical costs may soon make it practical to use whole genome sequencing or gene targeting on a routine basis to identify or generate mutations in specific genes in a variety of different plant species. This chapter will present the current status and promising prospects for the future of reverse genetics in plants.