First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.     

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.     

TILLMore, a resource for the discovery of chemically induced mutants in barley

A sodium azide-mutagenized population of barley (cv. 'Morex') was developed and utilized to identify mutants at target genes using the 'targeting induced local lesions in genomes' (TILLING) procedure. Screening for mutations at four agronomically important genes (HvCO1, Rpg1, eIF4E and NR) identified a total of 22 new mutant alleles, equivalent to the extrapolated rate of one mutation every 374 kb. All mutations except one were G/C to A/T transitions and several (approximately 68%) implied a change in protein amino acid sequence and therefore a possible effect on phenotype. The high rate of mutation detected through TILLING is in keeping with the high frequency (32.7%) of variant phenotypes observed amongst the M(3) families. Our results indicate the feasibility of using this resource for both reverse and forward genetics approaches to investigate gene function in barley and related crops.     

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.     

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor?

Background  

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor^? software, aimed at simultaneous detection of mutations in three homoeologous genes.     

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.     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.     

TILLING技术及其应用

定向诱导基因组局部突变(targetinginducedlocallesionsingenomes,TILLING)可快速、有效地鉴定和定向筛选突变,是一种全新的反向遗传学技术。现对TILLING的技术流程、核心与特点,及其在突变研究、反向遗传学及功能基因组学、SNP检测、资源创新与分析以及作物遗传改良等方面的应用进行了综述。     

Engineering melon plants with improved fruit shelf life using the TILLING approach

Background

Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening.

Methodology/Principal Findings

To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect.

Conclusions/Significance

We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.     

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes（TILLING）, which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2（or M3） plants.     

MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativa japonica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M₂ mutant population was calculated as 7.4 × 10⁻⁶ per nucleotide representing one mutation in every 135 kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1 kb of gene region when using 1,000 M₂ mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A mutation screening platform for rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) and the detection of sinapine biosynthesis mutants

We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M₂ plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80–90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.     

A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING

We report the use of TILLING (targeting induced local lesions in genomes), a reverse genetic, nontransgenic method, to improve a quality trait in a polyploid crop plant. Waxy starches, composed mostly of amylopectin, have unique physiochemical properties. Wheat with only one or two functional waxy genes (granule-bound starch synthase I, or GBSSI) produces starch with intermediate levels of amylopectin. We have identified 246 alleles of the waxy genes by TILLING each homoeolog in 1,920 allohexaploid and allotetraploid wheat individuals. These alleles encode waxy enzymes ranging in activity from near wild type to null, and they represent more genetic diversity than had been described in the preceding 25 years. A line of bread wheat containing homozygous mutations in two waxy homoeologs created through TILLING and a preexisting deletion of the third waxy homoeolog displays a near-null waxy phenotype. This approach to creating and identifying genetic variation shows potential as a tool for crop improvement.     

TILLING,high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.     

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.     

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.     

A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

Background  

Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.     

Optimizing TILLING populations for reverse genetics in Medicago truncatula

Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project ( http://www.eugrainlegumes.org ), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula . Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase     

Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.     

TILLING by Sequencing (TbyS) for targeted genome mutagenesis in crops

TILLING (Targeting Induced Local Lesions in Genomes) by Sequencing (TbyS) refers to the application of high-throughput sequencing technologies to mutagenised TILLING populations as a tool for functional genomics. TbyS can be used to identify and characterise induced variation in genes (controlling traits of interest) within large mutant populations, and is a powerful approach for the study and harnessing of genetic variation in crop breeding programmes. The extension of existing TILLING platforms by TbyS will accelerate crop functional genomics studies, in concert with the rapid increase in genome editing capabilities and the number and quality of sequenced crop plant genomes. In this mini-review, we provide an overview of the growth of TbyS and its potential applications to crop molecular breeding.