“Tilling and Eco-tilling A Reverse Genetic approaches for Crop Improvement”

 

  Nucleotide sequence variation is a major determining factor of heritable phenotypic difference and has been exploited by humans for crop improvement since the start of domestication. Variation can either be natural, from divergent populations, or induced through treatment with mutagens. An important goal of modern crop science is to use nucleotide sequence variation to improve crops. The modern crop scientist has a large amount of available nucleotide sequence information to identify genes of potential agronomic importance. Using reverse genetic approaches, specific genes can be disrupted, and hypotheses regarding gene function directly tested in vivo (Till et al., 2007).

TILLING first began in the late 1990’s from the effort of a, Claire McCallum and collaborators. Who worked on characterizing the function of two chromomethylase genes in Arabidopsis (Henikoff et al., 2004).

TILLING (Targeting Induced Local Lesions in Genomes) which is a reverse genetic high throughput approach, aims to identify SNPs (single nucleotide polymorphisms) and INDELS (insertions / deletions) in a gene or genes of interest from a mutagenized population.

The main three steps in TILLING are development of a mutagenized population, DNA Extraction, Pooling and Mutation Discovery (Till et al., 2007). To induce mutation Ethyl Methane Sulfonate is used. EMS typically produces transition mutations (G/C: A/T) because it alkylates Guanine residues and the alkylated Guanine residue pairs with Thymine instead of the conventional base pairing with Cytocine.

In maize TILLING identified 319 mutations in 62 genes, which has greatly assisted functional genomics studies in maize (Weil and Monde 2007).

In barley Two genes (Hin-a and HvFor1) were examined and 10 variants were identified, six of which were missense mutations. Phenotyping the M₃ individuals demonstrated that 20% had visible phenotypes (Caldwell et al., 2004).

Eco-TILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations. Many species are not amenable to chemical mutagenesis; therefore, Eco-TILLING can aid in the discovery of natural variants and their putative gene function (Gilchrist et al., 2005). Another valuable application of Eco-TILLING is mining for variation in resistance genes to help speed up the process of identifying alleles that could provide immunity to various diseases. In Hordeum vulgare (barley) allelic variation was examined and identified by using Eco-TILLING in mlo and Mla resistance genes These genes are involved in defending the plant from the fungal pathogen that causes powdery mildew (Mejlhede  et  al.,  2006).

TILLING and Eco-TILLING is a non-transgenic, high throughput reverse genetic approach for SNP (single nucleotide polymorphism) detection. It also identifies the approximate location within a few base pairs of the induced mutation and helps to know the function of genes. TILLING is so sensitive enough to detect homozygous mutations as well as heterozygous mutations. But the disadvantage is initial large expenses for the purchase of automated sequencers (LI-COR DNA Analyzer) commonly used for cleaved fragment detection.

REFERENCES

CALDWELL, D. G., MCCALLUM, N., SHAW, P., MUEHLBAUER, G. J., MARSHALL, D. F. AND WAUGH, R., 2004, A structured mutant population for forward and reverse genetics in barley (Hordeum vulgare L.) Plant J., 40:143–150.

GILCHRIST, E. J. AND HAUGHN, G. W., 2005, TILLING without a plough: A new method with applications for reverse genetics. Curr. Opin. Plant Biol., 8:211–215. 

HENIKOFF, S. TILL, B. J AND COMAI, L., (2004), Traditional mutagenesis meets functional genomics. Plant Physiol., 135: 630–636. 

MEJLHEDE, N., KYJOVSKA, Z., BACKES, G., BURHENNE, K., RASMUSSEN, S. K. AND JAHOOR, A., 2006, Eco-TILLING for the identification of allelic variation in the powdery mildew resistance genes mlo and Mla of barley. Plant Breed., 125:461–467.

TILL, B. J., COOPER, J., TAI TH., COLOWIT, P., GREENE, E. A., HENIKOFF, S. AND COMAI, L., 2007, Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol.,  7: 19. 

WEIL, C. F. AND MONDE, R. A., 2007, Getting to the point - Mutations in maize. Crop Sci., 47:60–67.