A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat

Background  

Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.     

Discovery of chemically induced mutations in rice by TILLING

Background  

Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.     

Discovery of induced point mutations in maize genes by TILLING

Background

Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.

Results

We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.

Conclusions

Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.     

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes.

Results

We screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes.

Conclusions

Our TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1348-0) contains supplementary material, which is available to authorized users.     

The use of two-dimensional electrophoresis to detect mutations induced in mouse spermatogonia by ethylnitrosourea

Two-dimensional electrophoresis should, in theory, be a suitable method for the measurement of induced mutation rates in the germ cells of mice. Not only can the polypeptide products of a large number of genes be resolved on a single gel but the detection of mutations which lead to proteins with altered electrophoretic properties (but not necessarily altered function) is possible. Our attempts to apply two-dimensional electrophoresis to the detection of mutation in vivo have involved three stages: (i) the rapid production of gels of high resolution and reproducibility; (ii) the identification of eight interstrain protein variants and demonstration of their simple genetic basis; and (iii) a pilot experiment using the powerful germ-cell mutagen ethylnitrosourea. It was found that although interstrain protein variants could be detected and shown to be inherited in a codominant manner, induced variants were rarely detected even on high quality gels. Only 2 variants were detected among 67 offspring of male mice treated with 150 mg/kg ethylnitrosourea. This represented a mutation rate of 0.88 X 10(-4) mutations per locus per gamete.     

Discovery of rare mutations in populations: TILLING by sequencing

Discovery of rare mutations in populations requires methods, such as TILLING (for Targeting Induced Local Lesions in Genomes), for processing and analyzing many individuals in parallel. Previous TILLING protocols employed enzymatic or physical discrimination of heteroduplexed from homoduplexed target DNA. Using mutant populations of rice (Oryza sativa) and wheat (Triticum durum), we developed a method based on Illumina sequencing of target genes amplified from multidimensionally pooled templates representing 768 individuals per experiment. Parallel processing of sequencing libraries was aided by unique tracer sequences and barcodes allowing flexibility in the number and pooling arrangement of targeted genes, species, and pooling scheme. Sequencing reads were processed and aligned to the reference to identify possible single-nucleotide changes, which were then evaluated for frequency, sequencing quality, intersection pattern in pools, and statistical relevance to produce a Bayesian score with an associated confidence threshold. Discovery was robust both in rice and wheat using either bidimensional or tridimensional pooling schemes. The method compared favorably with other molecular and computational approaches, providing high sensitivity and specificity.     

TILLING in extremis

Targeting induced local lesions in genomes (TILLING), initially a functional genomics tool in model plants, has been extended to many plant species and become of paramount importance to reverse genetics in crops species. Because it is readily applicable to most plants, it remains a dominant non-transgenic method for obtaining mutations in known genes. The process has seen many technological changes over the last 10 years; a major recent change has been the application of next-generation sequencing (NGS) to the process, which permits multiplexing of gene targets and genomes. NGS will ultimately lead to TILLING becoming an in silico procedure. We review here the history and technology in brief, but focus more importantly on recent developments in polyploids, vegetatively propagated crops and the future of TILLING for plant breeding.     

New sources of soybean seed meal and oil composition traits identified through TILLING

Background  

Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.     

A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis

Background  

Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.     

An ensemble approach to accurately detect somatic mutations using SomaticSeq

SomaticSeq is an accurate somatic mutation detection pipeline implementing a stochastic boosting algorithm to produce highly accurate somatic mutation calls for both single nucleotide variants and small insertions and deletions. The workflow currently incorporates five state-of-the-art somatic mutation callers, and extracts over 70 individual genomic and sequencing features for each candidate site. A training set is provided to an adaptively boosted decision tree learner to create a classifier for predicting mutation statuses. We validate our results with both synthetic and real data. We report that SomaticSeq is able to achieve better overall accuracy than any individual tool incorporated.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0758-2) contains supplementary material, which is available to authorized users.     

A targeted screen to detect recessive mutations that have quantitative effects

A key problem in mutagenesis research is developing methods that are sufficiently sensitive to detect a wide range of abnormal phenotypes. Major variants may be easy to identify, but it can be difficult to detect mutations that have subtle effects, particularly on a complex genetic background. This paper describes a targeted mutagenesis protocol with enough sensitivity to detect recessive mutations that have modest quantitative effects. The procedure relies on consomic inbred strains of mice—strains in which one homologous pair of chromosomes of an inbred strain has been replaced with the corresponding pair from a donor strain. Mice that carry the desired donor chromosome—the target of the screen—are mutagenized and bred back to the original recipient strain. The first-generation progeny (G1) that are heterozygous only for the donor chromosome are also bred back to the recipient strain. G2 animals that inherit nonrecombinant donor chromosomes are identified by genotyping. These animals may be backcrossed repeatedly to the recipient strain to dilute off-target mutations, but ultimately, nonrecombinant G2 animals are bred to each other. Their G3 progeny are genotyped at markers spaced at 5- to 10-cM intervals to identify mating pairs that are homozygous for shared segments of the mutagenized donor chromosome. Entire litters of G4 progeny that are homozygous for defined intervals are screened. By comparing phenotypes within and among litters of nearly isogenic G4 animals, mutations can be verified and simultaneously mapped with a precision of 5–10 cM. This method has the potential to consistently detect mutations that have effects on trait means of well under one standard deviation. Received: 29 December 1998 / Accepted: 17 March 1999     

A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.     

A method to detect ras point mutations in small subpopulations of cells.

Biochemical assays for ras mutations are capable of detecting a mutant allele only if it is present in at least 5% of cells tested. Further, ras mutation assays which utilize the polymerase chain reaction (PCR) are unable to distinguish a ras mutation in a small population of cells from mutations resulting from Taq DNA polymerase base misincorporation. We used a standard restriction fragment length polymorphism assay of PCR-amplified c-Ki-ras to detect codon 12 mutations in tumor cells and found a cumulative error frequency for Taq DNA polymerase of one codon 12 mutation per 2 X 10(4) molecules of total amplification product. The Taq polymerase-induced mutations were found to be multiple base transitions and represented a constant proportion of the amplification product at each step of the PCR. The ability to detect the in vitro generated mutation was dependent on the number of thermal cycles and the sensitivity of the detection assay. With these considerations in mind, we developed a two-step RFLP assay in which the thermal cycle number was kept low and molecules containing mutations at codon 12 were selectively amplified in the second step. We were able to detect a ras mutation occurring in 1 per 1000 cells (a two log improvement over standard RFLP methods) without detecting mutations resulting from Taq DNA polymerase infidelity.     

Polymerase chain reaction detect polymorphisms and trait association in soybean

Five sets of synthetic oligonucleotide (20-to 24-mers containing no intenal repeats) primers of known gene sequences [yellow lupin nodule specific (hydroxyl) proline-rich protein, pearl millet alcohol dehydrogenase, Pisum sativum heat shock proteins, Drosophila homeobox, and tRNA] were used to differentiate 73 soybean accessions, including 56 Glycine max (L.) Merr. and 17 G. soja Zucc. & Sieb. The amplified genetic markers revealed polymorphic bands for most genotypes studied. The χ²-analyses of the results showed that several fragments produced with these gene primers were associated non-randomly with resistance to Phytophothora, maturity, seed size, flower colour, seed coat colour, seed hilum colour, growth type, and leaf shape. These markers appear to be valuable for differentiation of G. max and G. soja species and genotypes within these species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population

Background  

Sorghum [Sorghum bicolor (L.) Moench] is ranked as the fifth most important grain crop and serves as a major food staple and fodder resource for much of the world, especially in arid and semi-arid regions. The recent surge in sorghum research is driven by its tolerance to drought/heat stresses and its strong potential as a bioenergy feedstock. Completion of the sorghum genome sequence has opened new avenues for sorghum functional genomics. However, the availability of genetic resources, specifically mutant lines, is limited. Chemical mutagenesis of sorghum germplasm, followed by screening for mutants altered in important agronomic traits, represents a rapid and effective means of addressing this limitation. Induced mutations in novel genes of interest can be efficiently assessed using the technique known as Targeting Induced Local Lesion IN Genomes (TILLING).