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Applying genotyping (TILLING) and phenotyping analyses to elucidate gene function in a chemically induced sorghum mutant population
Zhanguo Xin, Ming Li Wang, Noelle A Barkley, Gloria Burow, Cleve Franks, Gary Pederson, John Burke
BMC Plant Biology , 2008, DOI: 10.1186/1471-2229-8-103
Abstract: A sorghum mutant population consisting of 1,600 lines was generated from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). Numerous phenotypes with altered morphological and agronomic traits were observed from M2 and M3 lines in the field. A subset of 768 mutant lines was analyzed by TILLING using four target genes. A total of five mutations were identified resulting in a calculated mutation density of 1/526 kb. Two of the mutations identified by TILLING and verified by sequencing were detected in the gene encoding caffeic acid O-methyltransferase (COMT) in two independent mutant lines. The two mutant lines segregated for the expected brown midrib (bmr) phenotype, a trait associated with altered lignin content and increased digestibility.TILLING as a reverse genetic approach has been successfully applied to sorghum. The diversity of the mutant phenotypes observed in the field, and the density of induced mutations calculated from TILLING indicate that this mutant population represents a useful resource for members of the sorghum research community. Moreover, TILLING has been demonstrated to be applicable for sorghum functional genomics by evaluating a small subset of the EMS-induced mutant lines.Sorghum (2n = 2x = 20, 7.35 × 108 bp for 1C nucleus) is a C4 crop that displays excellent tolerance to both drought and high temperature stresses [1]. Sorghum has the highest water use efficiency among major crop plants and is unusually tolerant to low soil fertility, traits essential for survival and productivity in arid and semi-arid areas with limited irrigation capability. Worldwide, sorghum is the 5th most important grain crop, providing food and fodder for the inhabitants of drought-susceptible regions http://www.fao.org webcite. Recently, sorghum has been demonstrated as a viable bioenergy feedstock [2]. Compared with other bioenergy grain crops, sorghum is particularly advantageous because it can be grown profitably on marginal
Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)  [PDF]
Liang Chen, Linzhou Huang, Donghong Min, Andy Phillips, Shiqiang Wang, Pippa J. Madgwick, Martin A. J. Parry, Yin-Gang Hu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041570
Abstract: Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M2 mutants in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with altered morphological and agronomic traits were observed from the M2 and M3 lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.
NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications
Yellamaraju Sreelakshmi, Soni Gupta, Reddaiah Bodanapu, Vineeta Chauhan, Mickey Hanjabam, Sherinmol Thomas, Vijee Mohan, Sulabha Sharma, Rajeswari Srinivasan, Rameshwar Sharma
Plant Methods , 2010, DOI: 10.1186/1746-4811-6-3
Abstract: The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato.NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations.Several strategies for crop improvement for increased yield, better agronomic or novel traits, improved resistance to diseases and pests, to meet demand for biofuels and secondary metabolites for pharmaceutical or industrial purposes have been targeted by breeders and plant biologists. These strategies range from conventional selection processes, mutagenesis and breeding, to biotechnological approac
Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics  [PDF]
Adnane Boualem, Sebastien Fleurier, Christelle Troadec, Pascal Audigier, Anish P. K. Kumar, Manash Chatterjee, Abdullah A. Alsadon, Monther T. Sadder, Mahmoud A. Wahb-Allah, Abdullah A. Al-Doss, Abdelhafid Bendahmane
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097963
Abstract: Background Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. Principal Findings A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147?Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. Conclusions We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.
A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat
Cristobal Uauy, Francine Paraiso, Pasqualina Colasuonno, Robert K Tran, Helen Tsai, Steve Berardi, Luca Comai, Jorge Dubcovsky
BMC Plant Biology , 2009, DOI: 10.1186/1471-2229-9-115
Abstract: We developed mutant populations of pasta and common wheat and organized them for TILLING. To simplify and decrease costs, we developed a non-denaturing polyacrylamide gel set-up that uses ethidium bromide to detect fragments generated by crude celery juice extract digestion of heteroduplexes. This detection method had similar sensitivity as traditional LI-COR screens, suggesting that it represents a valid alternative. We developed genome-specific primers to circumvent the presence of multiple homoeologous copies of our target genes. Each mutant library was characterized by TILLING multiple genes, revealing high mutation densities in both the hexaploid (~1/38 kb) and tetraploid (~1/51 kb) populations for 50% GC targets. These mutation frequencies predict that screening 1,536 lines for an effective target region of 1.3 kb with 50% GC content will result in ~52 hexaploid and ~39 tetraploid mutant alleles. This implies a high probability of obtaining knock-out alleles (P = 0.91 for hexaploid, P = 0.84 for tetraploid), in addition to multiple missense mutations. In total, we identified over 275 novel alleles in eleven targeted gene/genome combinations in hexaploid and tetraploid wheat and have validated the presence of a subset of them in our seed stock.We have generated reverse genetics TILLING resources for pasta and bread wheat and achieved a high mutation density in both populations. We also developed a modified screening method that will lower barriers to adopt this promising technology. We hope that the use of this reverse genetics resource will enable more researchers to pursue wheat functional genomics and provide novel allelic diversity for wheat improvement.Wheat is an important food crop that is grown worldwide and provides approximately 20% of the calories consumed by mankind [1]. In spite of its economic importance, the ability to modify and understand gene function in wheat is still not fully developed due to several limitations. The large size of the wheat
TILLING in the two-rowed barley cultivar 'Barke' reveals preferred sites of functional diversity in the gene HvHox1
Sven Gottwald, Petra Bauer, Takao Komatsuda, Udda Lundqvist, Nils Stein
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-258
Abstract: In barley, we generated a new TILLING (Targeting Local Lesions IN Genomes) resource comprising 10,279 M2 mutants in the two-rowed malting cultivar 'Barke,' which has been used in the generation of other genomic resources in barley (~150,000 ESTs, DH mapping population). The value of this new resource was tested using selected candidate genes. An average frequency of approximately one mutation per 0.5 Mb was determined by screening ten fragments of six different genes. The ethyl methanesulphonate (EMS)mutagenesis efficiency was studied by recording and relating the mutagenesis-dependent effects found in the three mutant generations (M1-M3). A detailed analysis was performed for the homeodomain-leucine-zipper (HD-ZIP) gene HvHox1. Thirty-one mutations were identified by screening a 1,270-bp fragment in 7,348 M2 lines. Three of the newly identified mutants exhibited either a six-rowed or an intermedium-spike phenotype, and one mutant displayed a significantly altered spikelet morphology compared to that of the 'Barke' wild type. Our results indicate a bias in the frequency of independent functional mutations at specific base pair (bp) positions within the gene HvHox1.A new TILLING population was developed as a resource for high-throughput gene discovery in an alternative barley germplasm. Pilot screening demonstrated a similar or even slightly higher mutation frequency when compared to previously published barley TILLING populations that should allow for the identification of diverse allelic variation. Partial phenotypic evaluation of the M2 and M3 generations has revealed the presence of a wide spectrum of morphological diversity that highlights the great potential of this resource for use in forward genetic screens. Altogether, our study shows the efficiency of screening and the applicability of the new TILLING population for genetic studies in the barley crop model system.Barley has a long history as a model plant in mutation research and breeding [1]. One year afte
Towards a TILLING platform for functional genomics in Piel de Sapo melons
Mireia González, Meihong Xu, Cristina Esteras, Cristina Roig, Antonio J Monforte, Christelle Troadec, Marta Pujol, Fernando Nuez, Abdelhafid Bendahmane, Jordi Garcia-Mas, Belén Picó
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-289
Abstract: A new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1 mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point mutations were found for the seven gene targets, six of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the Phytoene desaturase gene, which is involved in carotenoid biosynthesis.The TILLING approach was successful at providing new mutations in the genetic background of Piel de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic and breeding studies.Melon (Cucumis melo L.) is an important vegetable crop. Genetic and genomic information for this crop is increasing significantly due to several national and international projects [1]. A broad range of genomic tools are available today [2-7]. An effort is also in progress, through a Spanish initiative, to obtain the whole genome sequence of this crop [8]. These tools are generating a lot of information about genes involved in various biological processes, such as plant resistance and fruit quality [9,10]. However, the tools necessary for reverse genetic studies to conduct the functio
A rich TILLING resource for studying gene function in Brassica rapa
Pauline Stephenson, David Baker, Thomas Girin, Amandine Perez, Stephen Amoah, Graham J King, Lars ?stergaard
BMC Plant Biology , 2010, DOI: 10.1186/1471-2229-10-62
Abstract: Separated by only 10-20 million years, Brassica species and Arabidopsis thaliana are closely related, and it is expected that knowledge obtained relating to Arabidopsis growth and development can be translated into Brassicas for crop improvement. Moreover, certain aspects of plant development are sufficiently different between Brassica and Arabidopsis to warrant studies to be carried out directly in the crop species. However, mutating individual genes in the amphidiploid Brassicas such as B. napus and B. juncea may, on the other hand, not give rise to expected phenotypes as the genomes of these species can contain up to six orthologues per single-copy Arabidopsis gene. In order to elucidate and possibly exploit the function of redundant genes for oilseed rape crop improvement, it may therefore be more efficient to study the effects in one of the diploid Brassica species such as B. rapa. Moreover, the ongoing sequencing of the B. rapa genome makes this species a highly attractive model for Brassica research and genetic resource development.Seeds from the diploid Brassica A genome species, B. rapa were treated with ethyl methane sulfonate (EMS) to produce a TILLING (Targeting Induced Local Lesions In Genomes) population for reverse genetics studies. We used the B. rapa genotype, R-o-18, which has a similar developmental ontogeny to an oilseed rape crop. Hence this resource is expected to be well suited for studying traits with relevance to yield and quality of oilseed rape. DNA was isolated from a total of 9,216 M2 plants and pooled to form the basis of the TILLING platform. Analysis of six genes revealed a high level of mutations with a density of about one per 60 kb. This analysis also demonstrated that screening a 1 kb amplicon in just one third of the population (3072 M2 plants) will provide an average of 68 mutations and a 97% probability of obtaining a stop-codon mutation resulting in a truncated protein. We furthermore calculated that each plant contains on ave
sunTILL: a TILLING resource for gene function analysis in sunflower
Wilma Sabetta, Vittorio Alba, Antonio Blanco, Cinzia Montemurro
Plant Methods , 2011, DOI: 10.1186/1746-4811-7-20
Abstract: To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M2 progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M2 lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.Cultivated sunflower (Helianthus annuus L.) is a globally important oilseed crop subjected to intensive molecular genetic and genomic studies during recent decades [1]. Sunflower belongs to the genus Helianthus, which is native to temperate areas of North America and includes 12 annual and 37 perennial species [2,3]. The world production of sunflower is estimated to be 23.4 million tons [4]; seed production in the world increased by 24% (or 5 million metric tons) between 1993 and 2003 [5]. After maize, it is the second largest hybrid crop, and the fifth largest among the oilseed crops, after soybean, rapeseed, cottonseed, and gro
A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing  [PDF]
Erin J. Gilchrist, Christine H. D. Sidebottom, Chu Shin Koh, Tanya MacInnes, Andrew G. Sharpe, George W. Haughn
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0084303
Abstract: 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.
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