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With the development of rapid and inexpensive sequence technologies, the efficiency and accuracy in sequencing have interpreted the genomic information of many plant species and the emphasis on genomics has been changing from the study of complete sequenced genomes to the functional genomics. To understand the function of gene/genes in species of concern many approaches like RNAi, gene knockout, site-directed mutagenesis, transposon tagging have been applied for many years. All these approaches depend upon the use of transgenic material which is not always possible in many commercially important crops, impeding to find out gene function, the improvement in existing and development of improved cultivars. A nontransgenic technique called Targeting Induced Local Lesions IN Genomes (TILLING) was established to determine an allelic sequence of induced point mutations in genes of concern. TILLING allocates the rapid and cost-effective detection of induced point mutations in populations of physical/chemically mutagenized individuals. In this technique previous DNA sequence information were used to identify the induced mutations created by the use of endonuclease. The technique can be applied not only to model organisms but also to economically important plants. It provides a powerful approach for gene discovery, DNA polymorphism assessment, plant improvement and can also be used as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants. In this review the application of TILLING in plant studies is thrashed out.
Key words: mutagenesis, reverse genetics, functional genomics, dHPLC,
Crop improvement has a long history as key agronomic traits have been selected over thousands of years during the domestication of crops. More recently, this progress has been accelerated as the green revolution has brought about great increases in crop yields [1, 23]. With the advent of genomics in the last 25 years, opportunities for crop improvement have continued to grow and may help to meet future challenges of food production and land sustainability. Novel DNA sequence information allows the development of additional molecular markers for breeding as well as providing targets for transgenic alteration of gene expression and introduction of new traits. Completion of the genome sequence projects of Arabidopsis thaliana and rice (Oryza sativa L.) [4, 22] has brought plant science research into a new era of genomics. The amount of sequence information stored in public database has increased which resulted in a very urgent demand to develop genome-scale reverse genetic strategies that are automated, broadly applicable, and capable of creating a wide range of mutant alleles that is needed for functional analysis . The forward genetics can hardly meet the demand of high-throughput and large-scale survey of gene functions as most of the phenotypes are obscure. TILLING (Targeting induced local lesions in genomes), a newly developed general reverse-genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically/chemically mutagenized individuals. This makes TILLING an attractive strategy for a wide range of applications from basic functional genomic study to practical crop breeding.
TILLING was first explored in the late 1990's by the efforts of Claire McCallum and his collaborators (Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute), who was experimenting on Arabidopsis . He used T-DNA lines and antisense RNA as reverse genetic approaches to illustrate the function of two chromomethylase genes, but was impotent to successfully apply these methodologies to describe the CMT2. The TILLING (Targeting Induced Local Lesions in Genomes) approach was developed by pooling chemically mutagenized plants together, creating heteroduplexes among the pooled DNA, intensify the region of concern and using dHPLC (denaturing high performance liquid chromatography) to identify the mutants by chromatographic variations . A less expensive and faster modification of the TILLING protocol was published later, which employs a mismatch- specific celery nuclease, CEL1, combined to the LI-COR gel analyzer system suited for this application [2, 32]. In 2001, the standard proposal was developed, and the practical software was explored, and the TILLING technique has become the routine method to detect mutations and satisfactory results have been obtained . Since from its origin, TILLING has been automated and exploited in many plant taxa. As a reverse genetic high throughput method, it purposes to detect SNPs (single nucleotide polymorphisms) and/or INDELS (insertions/deletions) in gene/genes of interest created from a mismatch in a mutagenized populace.
Outline of TILLING technique
To create a induced population with the use of physical/chemical mutagens is the first pre-requisite for TILLING approach (Fig. 1) and most of plant species are compatible with TILLING due to their self-fertilized nature and the self-seeds produced by these plants can be stored for long periods of time [7, 30]. In plants, seeds are treated with mutagens and raised to harvest M1 plants, which are consequently self-fertilized to raise the M2 population. To extract DNA and leaf tissues from M2 plants are collected. The extracted DNA is used in mutational screening . To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction . The M3 seeds can be produced by selfing the M2 progeny and can be well-preserved for long term storage. Ethyle methan sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective (Table 1). EMS produces transitional mutations (G/C, A/T) by alkalyting G residues which pairs with T instead of the conservative base pairing with C . It is a constructive approach for users to attempt a range of chemical mutagen to assess the lethalicity and sterility on germinal tissue before creating large mutant populations. When the population has been primed, the genomic DNA targets must be selected. CODDLE (http://www.proweb.org) is the web based programme, and by putting the genomic, cDNA or protein sequences, it allow the researchers to evaluate the possible gene function in the induced mutant population . Optimal PCR primers were designed for a functional domain target. In the next step DNA was collected from the population and the concentration was normalized. Once samples have been standardized, they were pooled together. It is essential to confirm that all DNA samples are the same concentration so that no biasing of samples occurs.
Generally for diploid organisms, a pool of DNA comprising up to eight individual samples can be effective in mutation detection . Thus, depending on ploidy level, heterozygosity, and the extent of certainly arising SNPs, best pooling for a species of concern should be determined practically. Once the pooled DNA is arranged into 96 well microtiter plates, the targeting forward and reverse primers are differentially 5' end labeled with IRD700 and IRD800 dye labels respectively for fluorescent detection at ~700 nm and ~800 nm (Fig. 1). Next, heteroduplexes and homoduplexes are produced from the PCR products of pooled samples (comprising of mutants and the wild form) by heating (denaturing) and cooling (annealing). The endonuclease enzyme CEL I is used and a short heating is vital for the enzymatic reaction to progress. CEL I, extracted from celery, not only recognize gaps in the heteroduplex, but it also cleaves DNA on the 3' side of the mismatch . After the enzyme incubation period, digested fragments were recognized on a denaturing polyacrylamide gel attached to a LI-COR 4300 DNA analysis system (Fig. 1). Pools holding an induced mutation will contain a mixture of homo- and heteroduplexes. Therefore, when fragments are separated a full length product (detected in both 700 and 800 channels) and two cleaved fragments (one IRD700 labeled, one IRD800 labeled) will be measurable. The amount of the sliced fragments should be equal the full length PCR product. The size of the cleaved fragments can be evaluated by comparison to a size standard, and therefore, the estimated position of the mutation will be recognized and further confirmed by sequencing. The PARSESNP (http://www.proweb.org/parsesnp/) can be used to identified and display the positions of the polymorphisms in a gene/genes in a graphical layout .
Boons of TILLING
TILLING is a non-transgenic, reverse genetic approach and contrasting to other SNP detection methods, provides the imprecise position within a few base pairs of the induced mutation [7, 9]. As the chemical mutagen creates a range of numerous mutations throughout the genome such as nonsense, splice site, and missense, and these can possibly affect the protein structure and the subsequent phenotype. Therefore, through mutagenesis one can acquire partial loss or complete loss of function, which can provide valuable insight into the true role of a gene  in a species of interest.
The high mutation-detecting efficiency of TILLING is credited to its high-throughput screening capacity. The densities of traditional chemical mutagenesis could be estimated. For example, EMS, one of the highly stable alkylation that is commonly used to induce point mutation in DNA produces primarily C to T changes resulting in C/G to T/A transition mutations in Arabidopsis. Ninety-nine percent of mutations from alkylation of guanine induced by EMS are reported as G/C-to-A/T transitions . From these consequences, the most appropriate fragment is selected in a specific gene of interest. Because of the ability of chemical mutagenesis to induce high density of mutations in multiple locus, genome wide saturated mutagenesis can be achieved using a relatively small mutant population. According to the general estimation made by the Arabidopsis TILLING Project (ATP), approximately 7 mutations per 1 Mb could be identified after screening the mutant Arabidopsis plant lines. On the basis of the above estimation, a total of 10000 mutant plants will achieve satisfied mutant densities . The use of chemical mutagenesis, coupled with LI-COR analyzer and TILLING procedure to locate induced mutations is useful in the interpretation of gene function in plants without the development of transgenic material. It is found be highly sensitive to identify induced mutations and naturally occurring SNPs , as well as the detection of heterozygotes in a population. This has been proved in the original work by McCallum and her colleagues . Seven different PCR fragments that ranged from 345 to 970bp in size were examined for a total of 2Mb of DNA sequence screened by dHPLC to detect mutations in CMT2 and CMT3 among 835 M2 plants in Arabidopsis. Thirteen chromatographic alterations were detected and confirmed to be mutations by amplification and sequencing; no PCR errors were found, indicating an error rate of <10−6 . Combination of Cel 1, double- end fluorescent dyes labeling and LI-COR system as an alteration to dHPLC maintained and secured the high sensitivity of the modified high-throughput TILLING.
One of the noteworthy benefits of TILLING is the saving of time and money as it does not demand resequencing of all the individuals in a population to comb frequent or rare SNPs. For a diploid organism, TILLING is processed by pooling eight individuals of a population in one time and finding variations due to mismatches in a heteroduplex.. The purpose of reverse genetics is to determine the function of a gene with known sequence by phenotypic analysis of cells or organisms in which the function of this gene is diminished. In plants, the most commonly used reverse-genetic approaches are insertional mutagenesis, anti-sense RNA, and double-stranded RNA interference (RNAi). These reverse-genetic approaches are, still, not equally relevant to all organisms. For example, in rice as cereal model plants, there are over 200000 T-DNA insertional populations; however, only few reports have been published about rice gene knockout by T-DNA insertion . Anti-sense RNA and RNAi techniques have been commonly used to reduce expression of genes [5, 59], but RNAi suppression generates only unpredictable outcomes, and the whole procedure is laborious as it requires vector construction, transformation, and transgenic analysis . From to the demands of high-throughput and of larger-scale of mutant detection the promise of using these reverse- genetic technologies is hampered. The TILLING technique is a permutation of the traditional chemical mutagenesis and the double-dye far-red fluorescent detecting technique. This technique requires no complicated manipulations and expensive apparatus. It enables to screen the mutant pools easily for investigating the functions of specific genes, avoiding both the confounding gene separation steps and tedious tissue-culture procedures involved in anti-sense RNA and RNAi.
4. TILLING Applications
4. 1. For gene discovery
The TILLING technique was first utilized in Arabidopsis, a workshop to develop TILLING as a service to the Arabidopsis community, known as the Arabidopsis TILLING Project (ATP), was initiated in 2001. In the first year of public operation, the ATP has detected, sequenced, and delivered over 1000 mutations in more than 100 genes ordered by Arabidopsis researchers . Through the workshop, mutant materials, DNA samples and mutant information were fully shared by all researchers working on Arabidopsis. CODDLE (for Codons Optimized to Detect Deleterious Lesions) was developed as a general tool for polymorphism analysis and for designing of primers for any organism and for detection of any mutagen based on DNA sequences information that is input by users. For TILLING and for polymorphism analysis, there is a need to assess the effect of missense mutations. For example, when specific DNA sequence with the length of 1kb was selected, CODDLE can evaluate whether a missense mutation is likely to have an effect on the encoded protein. This can be quite effective; for example, the conservation- based SIFT program is able to predict, with approximately 75% accuracy, whether an amino-acid change results in damage to a protein . The ATP continuous operation has proved to be a successful case for the application of TILLING in model system and encouraged the broader utilization of the technique to other organisms. Well-developed and tested protocols have been available for both genetic model organisms, such as Arabidopsis [28, 50] and Lotus japonicus , and important crops, such as maize (Zea mayz L.) , wheat (Triticum aestivum L.)  and rice . High-throughput TILLING is also used in maize, an important crop with a large genome but with limited reverse-genetic resources currently available. The pools of DNA samples that were screened for mutations in 1kb segments from 11 genes and 17 independent induced mutations were obtained from a population of 750 pollen-mutagenized maize plants . The result obtained from maize was consistent with that from Arabidopsis, indicating that TILLING is a broadly applicable and efficient reverse- genetic strategy for large genome. Furthermore, the TILLING strategy also succeeded to create and identify genetic variation in wheat, thereby showing a great potential as a tool for genomic research in polyploidy plants.
4. 2. For DNA polymorphism assessment
DNA polymorphism widely exists in various species and plays an important role in biological evolution. The methods currently available for revealing DNA polymorphism encompass DNA sequencing, single-strand conformation polymorphism (SSCP), hybridization, and microarray, and these methods have their own advantages and limitations. Although DNA sequencing is simple and straight-forward, it is rather costly and time-consuming. SSCP provides a high-throughput strategy for polymorphism detection; however, it has low efficiency in detecting novel mutations with a limit of 200 to 300bp length of target DNA sequence. Microarray holds two disadvantages, one is high cost of operation, and the other is the low detecting-frequency of less than 50% [8, 54]. The TILLING can detect DNA variations from single nucleotide polymorphism (SNP), small fragment insertion and deletions to simple sequence repeat (SSR) and can be performed as a high-throughput, low-cost, and high-accuracy approach compared with the other methods mentioned above because only the sequencing of the unique haplotypes is required to determine the exact nucleotide polymorphism at a locus.
4.3. Approach for functional genomics
Two principal approaches forward and reverse genetics have been extensively used to determine the function of gene/genes and how genotypes are linked to phenotypes. Conventionally in forward genetics (phenotype to genotype) one starts with a specific known phenotype or biological procedure and the gene sequence is finally inferred through selecting large numbers of mutagenized individuals for phenotypic variants. In forward genetic methodologies for genome wide analysis primarily for gene coding for a particular phenotype needs a lot of time and work . Whereas in reverse genetics (from genotype to phenotype), the gene sequence is known and mutants are identified and screened with structural alterations in the gene of interest . In this approach generally less time is needed than forward genetics and its strategies have been effectively used for functional genomics in many plant species. The ubiquitous availability of sequence data from different databases permits researchers to design swiftly their reverse genetic schemes to decide gene function. Some of the reverse genetic approaches employed in plants comprise homologous recombination, Agrobacterium mediated insertional mutagenesis, transposon tagging, RNAi (RNA interference) or PTGS (post transcriptional gene silencing), and chemical mutagenesis. Among all these TILLING is a more efficient mutation detection method, grosses the advantage of chemical mutagenesis to generate induced mutations in a population.
What is unique for the TILLING approach compared to transgenic approaches is the identification of numerous mutations within a targeted region of the genome. These mutations constitute allelic series that can potentially confer a range of phenotypes from subtle to strong, and allow structure and functional studies. Mutations in the coding regions of genes have the potential to alter plant metabolism in ways other than changing the effective level of a target gene product. For example, a mutation may change the affinity of an enzyme for its substrate, alter regulatory domains within enzymes, or may interfere with proper subunit or other protein-protein interactions. Within a metabolic pathway, such alterations can have large effects. TILLING offers a way to investigate a target gene of interest in potentially any crop of interest without first having knowledge of the gene product, which seems to us the essence of a useful tool for functional genomics. If a transformation system is available for a crop and there are only a few genes of interest in which one would like to have knockouts to help determine gene function, RNAi may be the current method of choice. However, TILLING offers many advantages in cases where transformation is difficult or if the investigation of a continuing series of unknown genes in a specific crop is desired. Once a TILLING library is set up, it becomes a renewable resource for continued analysis of many different gene targets. Thus the reiterative cost and time to analyze many different targets is much less by TILLING than by gene suppression using transgenics.
4.4 More than a knockout
With the possible exception of naturally occurring transposon systems in maize, most methods (transposon, TDNA, antisense, and RNAi) rely on transgenic introduction of foreign DNA. For Arabidopsis, this is not an issue; however, the efficiency of gene transfer and subsequent plant regeneration can become a serious limitation in many crops. TDNA insertions and/or transposon insertions may be the preferred means to obtain a specific gene knockout but are practically limited to the crops for which they are available. RNAi has the advantage of knocking down the expression of multiple related genes with one construct , whereas TILLING, like TDNA insertions and transposons, is unlikely to affect more than one specific member of a multi-gene family in an individual plant. The application of TILLING to crop improvement may also help with another constraint in domesticated species' genomes having limited genetic variation.
During domestication and subsequent selection, much of the genetic variation available in the wild crop progenitors has been lost . Thus, plant breeders have at times used wild relatives or landraces to introduce useful genetic variation. This practice has been successful in wheat for developing disease resistant and higher yielding varieties  and a landrace was also used for the development of the first full waxy line because it carried a rare deletion allele of one of the waxy loci . As an alternative to the use of wild varieties, TILLING can be a means to introduce genetic variation in an elite germplasm without the need to acquire variation from exotic cultivars, thus avoiding introduction of agriculturally undesirable traits. In addition, the issue of bio-piracy makes the use of exotic varieties to improve modern cultivars potentially filled with complications. The identification of caffeine free Arabica coffee by Brazilian scientists in germplasm that came originally from Ethiopia has prompted dispute over ownership .
4.5. For crop breeding
Conventional mutation breeding, either by radiation or by chemical treatment, has had a proven influence on production of many varieties including high-yielding rice, barley, and wheat . Unlike conventional mutation breeding in which the mutation frequency is unknown or estimated only from mutations conveying a visible phenotype, TILLING provides a direct measure of induced mutations. Besides, TILLING allows not only the prompt, parallel selection of numerous genes but also a forecasting of the number of alleles that will be recognized on the basis of the mutation frequency and library size. The efforts done on plants are deliberated as follows.
The TILLING Project (ATP) had mentioned 1,890 mutations in 192 target gene and it was detected that heterozygote mutations were twice fold more than homozygote mutations . The several mutations in Arabidopsis thaliana that have been recognized via TILLING that have clearly explain the function of gene and protein throughout the genome for Arabidopsis researchers.
Barley is also used as an important cereal crop having a fairly large genome size of ~5,300 Mb, was used for TILLING experiment to find the induced mutations in two genes . Hin-a and HvFor1 genes were studied and 10 mutants were identified. Among these ten mutants six have missense mutations.
Medicago truncatula has been extensively adopted as a model plant for crop legume species of the Vicieae. Regardless the convenience of transformation and regeneration protocols, there are presently inadequate tools accessible in this species for the systematic investigation of gene function. M. truncatula was treated with chemical mutagens to create mutant population that provide a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database for both reverse and forward genetics screens. Fifty-six targets were identified and screened; 546 point mutations were recognized with a mutation frequency of 1/485 kb .
4.5.4. Common Bean
Phaseolus vulgaris is the main food legume used worldwide, making it an important target for innovative methodologies of genetic analysis. BAT 93 was used for TILLING approach and found that 40 mM EMS was an appropriate concentration for the generation of a mutant population. Higher the concentrations of EMS, lower the survival rates less than 10% and lower the concentrations resulted in the generation of fewer mutants .
4.5.5. Field Mustard
B. rapa was used as the first EMS TILLING source in the diploid Brassica species. It has a genome size of 625Mbp. The mutation frequency in this population is ~1 per 60 kb, which makes it the most densely mutated diploid organism .
Lotus japonicus, a model plant has also been emphasized for explaining gene function through TILLING. It is a perennial temperate legume and is used as a model plant for genomic studies due to its short life cycle, a diploid nature (2n = 2x = 12), with a small genome size (472Mb), and is self-fertilized nature . To discover induced mutations in the protein kinase domain of the SYMRK gene, TILLING approach was used and six missense mutations were discovered in the splice acceptor site. TILLING is also targeted in another project in Lotus japonicus to investigate the functional role of sucrose synthase and nitrogen fixation .
Maize having a large genome size was found to be promising for TILLING project and in 2005 a TILLING project was launched at Purude University. In this project 319 mutations in 62 genes were identified . In another study a population of 750 mutagenised plants was used to illustrate the function of 11 genes and six genes having visible mutation were screened. In this investigation among six genes DMT102 gene which is called chromomethylase gene played a vital role in arabidposis for non-CpG DNA methylation and gene silencing was confirmed.
The oat (Avina sativa) having a genome size of 13000Mbp was treated with chemical mutagen EMS to establish a TILLING population. On an average it exhibited hundreds of mutations in every individual gene in the genome . Thus TILLING can be used as an important tool in oat improvement by developing mutants having specific characters.
Pea (Pisum sativum) a member of legume family which fixes nitrogen was used for TILLING experiments. Using this reverse genetic approach 60 mutants were identified from an allelic series of mutations in five genes . In this study specific mutants were screened from the LE gene encoding for 3-hydroxylase and were further characterized to determine the effect on internode length.
TILLING, with conventional mutagenesis is used for targeted screening of known genes. A peanut TILLING population was generated in tetraploid genome and screened for mutations in genes for allergenic proteins Ara h 1, Ara h 2, as well as the oil biosynthesis enzyme FAD2. It is observed that silencing of Ara h 2 by RNA interference has delivered evidence that this protein and its related family member Ara h 6 may be dispensable for peanut seed growth, development, and viability. Therefore, recovery of knockout mutations in the two genes of Ara h 2 should allow elimination of this most severe allergen from peanut seed. Up to now potential knockout mutations in one copy each of Ara h 1, Ara h 2, and Fad2 have been identified in peanut .
Three potato (Solanum tuberosum) cultivars were treated with different doses of gamma radiations to create a platform for TILLING and Ecotilling studies. Three gene-specific primer pairs were used to amplify a sequence of ~1 to 1.5 kb of targeted gene and 15 putative nucleotide polymorphisms per kilobase were found. Among fifteen, nine discovered polymorphisms were distinctive to one of the three tetraploid cultivars tested .
In Brassica napus two EMS mutant populations of the semi-winter rapeseed were created to provide a TILLING platform for functional genomics in Brassica napus, and for introduction of novel allelic variation in rapeseed breeding. Forward genetic selection of mutants from the M2 populations caused in identification of a large number of unique phenotypes. In that study existing SNPs were used as positive control to find the distinguishing novel mutations. The method was used to 1344 M2 plants and 19 mutations were identified . Among them three were functionally conceded with reduced seed erucic acid content.
Rice is an important economic and staple food crop providing about 80% of the caloric intakes of three billion people of the world . Its genome has ~50, 000 genes but the function of all genes is empirically not yet determined. To detect the mutations and identify the function of genes, TILLING studies were done on indica rice  using chemical mutages EMS and Az-MNU respectively. In this study among 10 target genes 57 polymoprphism were identified . The use of agarose gel and LI-COR DNA analyzer was also used in rice to find the induced mutations .
Sorghum bicolor (L.) Moench is used as a most important grain crop and fodder resource for most of the arid and semi-arid regions of the world. A sorghum inbred line BTx623 was treated with chemical mutagen EMS to create a mutant population. Out of 1,600 lines, 768 mutant lines was analyzed by TILLING using four target genes and only five mutations were identified resulting in a calculated mutation rate of 1/526 kb .
Soybean (Glycine max) an important economic crop and a rich source of protein (35-50%) is beneficial for human health . It also improves soil quality by fixing nitrogen . Two cultivars viz; Forrest and Williams 82 were used to create four mutagenised populations by treatment with EMS or NMU to identify induced mutations . For seven targeted genes, about 116 mutations were identified through TILLING approach. Most of the mutations discovered were found to be the estimated as G/C to A/T transitions .
Using TILLING approach, a set of new mutants was created in tomato (Solanum lycopersicum) cultivar i.e., Red Setter at 0.7%-1.0% EMS dose. To confirm the Red Setter TILLING platform, induced point mutations were investigated in 7 tomato genes with the mismatch-specific ENDO1 nuclease. 9.5 kb of tomato genome were explored out and 66 nucleotide substitutions were identified. The overall mutation rate was estimated to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatments respectively .
Wheat having polyploidy nature is used as an important staple crop, yielding annually 600 million tons . To make a good in quality partial waxy wheat cultivars are desirable  which is good for noodles and superior flour. It is hexaploid and having a large genome size (17,000 Mbp). Using TILLING approaches in wheat 246 allelic series were identified in the waxy gene homologues. Among this allelic series 84 missense, 3 non-sense and 5 splite site mutations were identified .
Albeit TILLING was primarily designed in Arabidopsis but it has been recognized as an exceptionally flexible approach as compared to many other reverse genetic techniques [table-1]. To find the mutation in hexploid and diploid organism was very difficult but TILLING has proven to be very successful to explain the gene function in such large genome size plants. Besides, the use of physical/chemical mutagens in diploid and polyploid plants yields a series of various allelic mutations and a high density of mutations present throughout the genome (Table 1).
5. Experimental encounters in application of TILLING
There are some scientific challenges in employing TILLING experiments; regarding the creation of a high quality mutant population about one to two years may be predictable to be required for population development [8, 42]. The first step to generating a population is to use different concentration of the chemical mutagen to assess lethality to find an optimal concentration for conducting the experiments . It is challenging because the lethality of species and varieties respond differently to physica/chemical mutagenesis . An ideal population would exploit mutational load i.e, more than 50% survival of mutant population is pre-requsite  and viable. Creating mutant populations in vegetatively propagated plants  also slow down the progress of generating a mutant population. The species that are highly heterozygous may confuse mutation detection for researchers due to natural polymorphisms in the genome, which may deter in finding of rare induced mutations . The production and maintenance of clones of vegetatively propagated plants for future analysis is somewhat problematic . After the creation of mutant population, it is essential that all DNA extracts be equivalent in concentration so that they are all correspondingly characterized in the pools being investigated. Otherwise, unique induced mutations may not be recognized as the amount of mutant DNA decreases in contrast to others in a pool of DNA. Another task, particularly in plants is the selecting of target genes that sometimes exist as a single copy throughout the genome. This leads to a problem when experimenting on polyploid plants that have complex genomes such as wheat or peanut. To overcome this contest primers need to be designed that are precise to single gene of interest, which may entail some extra effort . Another strategy is to sequence the multiple alignments of the homologous target genes to find restriction site differences between the target genes. Proceeding to TILLING, the DNA can be digested which may cleave the annoying target leaving the desired gene unbroken for analysis . Additionally, another potential difficulty in TILLING may be the increased number of SNPs per fragment, the identification, scoring and tracking of cleaved fragments hence becomes more challenging. Single SNPs discovered in a heteroduplex needs a high focused as compared to multiple mismatch sites [11, 36, 47]. Moreover, care should be taken during scoring fragments as large numbers of SNPs are existing in a gene portion. Another point for consideration when designing an Eco- TILLING or TILLING experiment is the selection of the nuclease to digest the mismatches in the heteroduplexes. CEL I that identify and cleave mismatched fragments in a heteroduplex and also contain 5' to 3' exonucleolytic activity [3, 60] can also digest the full length PCR product starting with the 5' fluorescent label. Therefore, care should be taken not to over digest DNA samples to avoid the loss of the fluorescent signal of the PCR products. The last challenge is allocating a particular phenotype to a genotype and supposing the putative function of a gene. Chemical mutagenesis sometimes creates background mutations, which can make phenotype analysis more difficult . This may take several generations of outcrossing or backcrossing [3, 30]. Obviously, to assign a function to a gene will be more challenging if there is any epistasis or pleiotropic effects created from the background mutations 
As a novel reverse-genetics technique, TILLING has been put into practices since its origin. It has been convincingly proved that TILLING is obviously a technique with considerable potential for crop improvement. From 2000 to 2009, 84 articles have been published which are available on www.scholar.google.com as shown graphically. It represents an extension of the use of spontaneous and induced mutants in plant breeding and allows direct identification of beneficial nucleotide and amino-acid changes in genes with known functions and in their use as the genetic markers for selection. The range of alleles that can be developed via TILLING in a short time is matchless and unlikely to be found elsewhere in the pool of germplasm accessible to plant breeders, including landraces and undomesticated relatives. As the TILLING population is a stable source, the results of basic scientific research can be well interpreted into crop improvement as new information about the functions of potential gene targets becomes available. There are at least two instantaneous applications in plant breeding using TILLING as a haplotyping tool for detection of genetic loci that are putatively associated with agronomically important traits. The first application is the identification of allelic variation in genes exhibiting expression correlating or cosegregating with phenotypes. This will link gene expression with DNA variation. Because haplotypic variation caused by SNP or small indels can be detected, it helps overcome the main difficulty of finding DNA variation based on restriction-site polymorphism or linkage to hypervariable markers such as SSR. The second application is the establishment of an allelic series at genetic loci for the traits of interest in germplasm or induced mutants. Allelic series at such loci will provide confirmatory evidence of the relationship between the phenotypes and candidate gene sequences. A large collection of alleles at a locus will provide patterns of association to deduce the functional significance of certain SNPs. It has been suggested that the recent progress in the area of plant molecular biology and plant genomics have the potential to initiate a new Green Revolution. Though, these findings need to be executed in new cultivars to realize that potential. Now, TILLING service centers are accessible for Arabidopsis thaliana, Lotus japonicus, barley (Hordeum vulgare), common bean (Phaseolus vulgaris), field mustard (Brassica rapa ), maize (Zea mays), oat (Avina sativa), pea (Pisum sativum), peanut (Arachis hypogaea L), potato (Solanum tuberosum L), rice (Oryza sativa), rape seed (Brassica napus), Sorghum (Sorghum bicolor L), soybean (Glycine max), Medicago truncatula, , tomato (Solanum lycopersicum), and wheat (Triticum turgidum). Many of these aforesaid species already have widespread genomic information publicly and now the emphasis for these species has shifted from genome to genomics (to empirically find the function of gene/genes). With passage of time, more information on genomic will available for other plant species, and thus focuses on the use of reverse genetics approaches to allocate the putative genes functions. This ambition of geneticists to find and explain the function of coded DNA may ultimately lead to the development of public TILLING services in numerous plant species, which will enable to streamline the procedure of functional genomics for all researchers. TILLING, as a unique approach for genome-wide functional genomics, coupled with other recently developed genomic resources including detailed genetic maps, large numbers of ESTs, deep-coverage large insert libraries with extensive contig assemblies, both targeted and complete genome sequencing and annotation, will allow for the efficient identification of gene (s) controlling phenotypes in both model systems and economically important plants. It can be predicted that more and more direct or indirect benefits will be revealed through continuous applications of TILLING in the near future.