रासायनिक उत्परिवर्तन द्वारा प्लांट ब्रीडिंग में टीलिंग और इको- टिलिंग की भूमिका

TILLING (Targeting Induced Local Lesions in Genomes) is a method in molecular biology that allows directed identification of mutations in a specific gene or allele. TILLING was introduced in (McCallum et al. 2000) by using the model plant Arabidopsis thaliana. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, corn, wheat, rice, soybean, tomato and lettuce.

TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest. TILLING is a powerful technology that employed heteroduplex analysis to detect which organism in a population carry single nucleotide mutation in specific genes.

McCallum utilized reverse genetic approaches such as  T-DNA lines and antisense RNA, but was unable to successfully apply these approaches to characterize CMT2.The approach that was successful turned out to be what is now known as TILLING.

TILLING can also be used to detect naturally occurring SNP in genes among the accession, variety or cultivar. To study the gene function, or to detect genetic marker in population.

TILLING methodology can also be used to uncover natural nucleotide variation linked to important phenotypic characters, a process termed EcoTILLING (Comai et al. 2004). The current status of various plant TILLING and EcoTILLING methods are generally applicable across the plant kingdom.

 DNA sequence     → Protein  → Phenotypes 

To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003).

Why  TILLING ?

• Tool for functional genomics that can help decipher the functions of the thousands of newly identified genes.
• To identify SNPs and/or INS/DELS in a gene of interest from population.
• Genetic mutation is a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo.
• Non transgenic method for reverse genetics.

History of TILLING

TILLING first began in the late 1990’s by a graduate student, Claire McCallum (and collaborators from Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute), who worked on characterizing the function of two chromomethylase genes in Arabidopsis (Henicoff et al., 2004).

Claire McCallum utilized reverse genetic approaches such as T-DNA lines and antisense RNA, but was unable to successfully apply these approaches to characterize CMT2.

The approach that was successful turned out to be what is now known as TILLING (Targeting Induced Local Lesions in Genomes). This was accomplished by pooling chemically induced mutagenized plants together, amplifying the region of interest, creating heteroduplexes among the pooled DNA, and performing dHPLC (denaturing high performance liquid chromatography) to detect the mutants by chromatographic alterations (McCallum et al., 2000).

The TILLING methodology

Development of mutagenized population: 

1. EMS mutagenesis
2. Development of M2 population

DNA preparation and pooling of individuals:

1. Mutation Discovery
2. PCR amplification of a region of interest
3. Mismatched cleavage
4. Detection of Heteroduplexes as extra peak or band (HPLC or Acrylamide gel) Identification of the mutant individual
5. Sequencing of Mutant PCR product.

Basic procedure

TILLING is straight forward for

1. the creation of a large genetically diverse population of plants;
2. the high throughput identification of individual plants whose genotype predicts a phenotype of interest; and
3. the evaluation of these individuals’ phenotypes for the accelerated development of novel cultivars that exhibit traits of interest.

It combines random mutagenesis of seeds with an alkylating agent such as ethyl methanesulfonate (EMS) or other mutagenic agent with the targeted identification of induced alterations in the genes of interest.

Germplasm collection and maintaining genetic stocks

Germplasm repositories can be beneficial resources for research scientists for TILLING and EcoTILLING experiments by providing a source to help fill genetic gaps and / or acquire material needed for a study.

On the other hand, TILLING could be applied in collaboration with gene pool repositories to develop mutant lines that have beneficial characters for breeders, such as improved drought tolerance in plants.

Genetic stocks created by chemical mutagensis may be beneficial not only for linking a genotype to a phenotype, but also to develop germplasm resources for breeding or obtaining new agronomically important traits.

TILLING in the plants

1. Arabidopsis thaliana:

TILLING was first applied to Arabidopsis thaliana (McCallum et al.2000). A mutagenized population was created by treating seed with EMS, using the single seed descent method of plant breeding.

2. Lotus japonicus :

Perry and colleagues adapted the TILLING method for the model legume Lotus japonicus (Perry et al. 2003). Seeds were treated with EMS similar to what was done for Arabidopsis.

3. Zea mays:

As part of a NSF-funded research project to ascertain the suitability of plant populations for TILLING, the STP screened maize populations donated by Clifford Weil and Nathan Springer (Till et al. 2004).

4. Wheat :

The feasibility of TILLING in a polyploid species was shown for wheat by Slade and colleagues (Slade et al. 2005). Starting with seed mutagenized with EMS, they developed TILLING populations in tetraploid and hexaploid wheat.

5. Other Plant Species :

The number of plant species in which TILLING has been successfully applied continues to grow. Caldwell and colleagues used a combination of denaturing HPLC and CEL I to identify mutations in barley (Caldwell et al. 2004). In collaboration with Tom Tai, the STP has developed TILLING for rice (Till et al. 2007). 

Applications of TILLING

Major areas of applications are

Functional genomics: The identification of numerous mutations in target region of genome. Construction of TILLING library is useful for scientists to search for mutations in gene of interest. TILLING offers a way to search  target GOI in any crop of interest without first having knowledge of gene product.

Genetic engineering: Agricultural interest in producing phenotypic variants without introducing foreign DNA of any type into plants genome. T-DNA/ Transposon insertions are used to obtain specific gene knockouts but practically limited to some crops only. TILLING is in front of transgene, as consists of identification of numerous mutations within a targeted region of whole genome.

Evaluation of genetic diversity of natural populations: Alternative to wild relatives, TILLING is used to introduce useful genetic variation of elite germplasm. Also applicable in a population which has several pre-existing polymorphism for developing SNPs.

Eco TILLING

• The first publication of the EcoTILLING method in which TILLING was modified to mine for spontaneous polymorphisms was in 2004 from work in Arabidopsis thaliana.
• EcoTILLING is similar to TILLING, except that its objective is to identify natural genetic variation as opposed to induced mutations
• Many species are not amenable to chemical mutagenesis; therefore, EcoTILLING can aid in the discovery of spontaneous variants and their putative gene function
• This approach allows one to rapidly screen through many samples with a gene of interest to identify naturally occurring SNPs and / or small INs/DELS.

Eco TILLING for Populus trichocarpa

As with TILLING, EcoTILLING is general, and should be applicable to most species. Much can be learned from studying natural nucleotide diversity; new markers to be generated from EcoTILLING projects, and non-synonymous SNPs may be identified that provide a beneficial phenotype. For species in which mutagenesis is impractical, exploiting natural nucleotide diversity to be invaluable for plant breeding.

Merits of Eco TILLING

• Its applicability to virtually any organism.
• Its facility for high-throughput and its independence of genome size, reproductive system or generation time.
• Since it uses Chemical mutagenesis virtually all genes can be targeted by screening few individuals.
• High degree of mutational saturation can be achieve barring excessive collateral DNA damage.
• Eco- TILLING is useful for association mapping study and linkage disequilibrium analysis.
• Ecotilling can be used not only to determine the extent of variation but also to assay the level of heterozygosity within a gene.

Perspective

• TILLING and EcoTILLING have been proven to be highly effective reverse genetic tools for functional genomic studies in plants and animals. Since the inception of these techniques, many researchers have gained indispensable insight on gene function and have identified natural and induced variants.
• These methods are now well established for many model plant and animal systems regardless of their mating system, genome size, or ploidy level. TILLING is one of the few reverse genetic applications that has not been proven to be applicable in a species specific manner unlike other approaches (i.e.-RNAi or homologous recombination), which potentially makes this application available for all species.
• 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.
• The main limitation for TILLING is that the species is capable of being mutagenized. So, for ethical reasons TILLING should not be employed for analyzing functional genomics in humans.

Conclusion

TILLING and EcoTILLING are high-throughput and cheap methods for the discovery of artificial mutations and spontaneous polymorphisms. The methods are general and have successfully been applied to many crop spcies.

With sequence data and general tools such as TILLING, reverse genetics can be applied to lesser studied species. Now that successes have been found in a variety of important plant species, the next challenge to be use the technology to develop improved crop varieties in plant breeding.

The utility of artificial mutations and spontaneous polymorphisms has already been established for plant breeding and hence the task is mostly one of implementation. Cost effective method than Genetic engineering and has no associated bio-safety issues.

But the problem lies in the fact that the rate of induction of mutation is low, requires skilful labours and moreover a mutagenized organism must be kept alive long enough to screen mutant population in vegetatively propagated plant species.

REFERENCES

1. Borevitz, J. O., Liang, D., Plouffe, D., Chang, H.S., Zhu, T., Weigel, D., Berry, C.C., Winzeler, E. and Chory, J. (2003). Largescale identification of single-feature polymorphisms in complex genomes. Genome Research 13(3): 513-523
2. Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, Waugh R (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40:143–50
3. Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff s (2004) Efficient discovery of DNA polymorphisms in natural populations by EcoTILLING. Plant J 37:778–786
4. Henikoff, S., Till, B.J. and Comai, L. (2004). TILLING: Traditional mutagenesis meets functional genomics. Plant Physiology 135: 630-636.
5. McCallum CM et al. (2000) Targeting induced local lesions in genomes (TILLING) for plant functional genomics. Plant Physiology 123, 439-442
6. Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol 131:866–871
7. Slade AJ, Knauf VC (2005) TILLING moves beyond functional genomics into crop improvement. Transgenic Res 14:109–115
8. Till BJ, Burtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641
9. Till BJ, Cooper J, Tai TH, Colowit, P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19

Authors

Dr. R. S. Parmar  

Assistant Professor and Head, Collage of Agriculture,

Junagadh Agriculture University, Motabhandariya -Amreli(365610)

Email: This email address is being protected from spambots. You need JavaScript enabled to view it.