EcoTILLING is a method for identifying natural mutations and polymorphisms in populations using TILLING techniques. It allows for the detection of point mutations and small insertions/deletions in DNA in a high-throughput but low-cost manner using gel electrophoresis. EcoTILLING has been used to identify allelic variants in genes related to powdery mildew resistance (mlo and Mla) in barley. It has also been applied to identify natural variation in 196 Arabidopsis ecotypes and 41 cottonwood trees. The method shows potential for discovering alleles relevant to salt tolerance in rice varieties and resistance to potato virus Y in pepper varieties.

1. ECOTILLING
Introduction :
Eco TILLING:
Eco TILLING is a method that uses TILLING techniques to look for natural mutations in
individuals, usually for population genetics analysis. and Eco TILLING which uses an inexpensive
method to identify fragments.
Eco TILLING is a high-throughput method to detect and discover new point mutations and small
insertions/deletions in DNA . The method is gel based and thereby low cost. Eco TILLING is a
variant of TILLING which is a high-throughput, non-transgenic strategy for providing allelic series
of mutations, including knock-outs. It permits the identification of mutations in target genes
without the implementation of genetically modified organisms that cause public concern in Europe.
The method is therefore of great interest in commercial agriculture.
EcoTILLING for the identification of allelic variation in the powdery mildew resistance
genes mlo and Mla of barley:
The wild-type mlo gene encodes a protein which is probably involved in regulating a cell
wall repair process. Its malfunc-tion results in excessive papilla growth and a high level of
resistance to powdery mildew. A number of mlo mutants have been reported, of these at least 32
are of proven independent mutational origin and most of them have been sequenced. They have
been assigned the resistance gene symbols mlo1to mlo32. Of these, most have been induced by
chemical mutagens, five have been induced with radiation and one (mlo11) is naturally occurring
in Ethiopian landraces t al. The known inactivated mlo alleles are either characterized by a single-
point mutation or by deletion of a few nucleotides. Currently, the only way to identify alleles in
the mlo gene is either by pedigree or by sequencing of the gene.
The Mla locus is of great interest because of the diversity of resistant phenotypes that are
conferred by different Mla resistance specificities. These phenotypes can range from near
immunity, associated with a rapid hypersensitive response and early growth arrest of the powdery
mildew, to a late response allowing the development of some fungal mycelium. The Mla region
contains multiple classes of genes associated with plant defence responses and genetic variants of
the Mla locus are found in cultivars world wide.At least 20 very effective alleles of the Mla locus
have been identified in a wild barley collected in Israel. Currently, Mla alleles are mostly

2. determined by resistance tests with a set of powdery mildew isolates. However, the isolates
collected in Europe do not possess the corresponding virulence genes to detect the alleles of the
Mla locus from wild barley. Therefore,isolates sampled from wild barley in Israel have to be
employed for this purpose.In this study it has been demonstrated that point mutations in the mlo
gene and the natural variation at the Mla locus of barley can easily be detected by EcoTILLING.
EcoTILLING of Arabidopsis thaliana
The enzymatic mismatch cleavage method used for TILLING should be applicable to any
heteroduplexed DNA target regardless of the source of the nucleotide polymorphism. Therefore,
the same methods should be applicable to the discovery of natural nucleotide variation in
populations. However, if cleavage of mismatches were complete, then only the closest
polymorphism to the labelled end would be detectable, and this method would be unsuitable for
polymorphism discovery. Fortunately, cleavage is only partial at any site, and TILLING
methodology has been used to score multiple mismatches within single end-labelled
heteroduplexes. To determine if the method could be used to accurately catalogue natural diversity,
196 Arabidopsis ecotypes were screened for nucleotide variation in 5 gene target regions (Comai
et al. 2004). To uncover homozygous polymorphisms that are thought to TILLING AND
ECOTILLING FOR CROP IMPROVEMENT 347 predominate in self-fertile species, an equal
amount of DNA from the sequenced Columbia accession was added to each test sample so that
heteroduplexes could be created. To unambiguously assign haplotypes, individual accessions were
not pooled together. Fifty-five distinct haplotypes were discovered. In addition to SNPs, small
insertions/deletions and a satellite repeat number polymorphism were identified. The study
indicated a low false positive and false negative rate of discovery when compared to sequencing.
Based on this work, the modification of TILLING for the discovery and genotyping of natural
nucleotide polymorphisms was termed EcoTILLING.
Use of EcoTILLING to identify natural allelic variants of rice candidate genes involved in
salinity tolerance
Rice is a salt-sensitive species with enormous genetic variation for salt tolerance hidden in
its germplasm pool.The Eco TILLING technique allows us to assign haplotypes,thus reducing the
number of accessions to be sequenced , becoming a cost effective, time-saving and high-

3. throughput method,ideal to be used in laboratories with limited financial resources.Aiming to find
alleles associated with salinity tolerance,we are currently using the Eco TILLING technique to
detect single nucleotide polymorphisms(SNPs) and small indels across 375 germplasm accessions
representing the diversity available in domesticated rice.We are targeting several genes known to
be involved in salt stress sigal transduction (OsCPK17) or tolerance mechanisms (SalT).So far,we
found a total of 15 and 23 representative SNPs or indels in OsCPK17 and SalT,respectively.These
natural allelic variants are mostly located in 3’-untranslated region, thus opening a new path for
studying their potential contribution to the regulation of gene expression and possible role in salt
tolerance.
Automation of TILLING Gel Analysis
The major difference between TILLING and EcoTILLING is the amount of information
present in gel data from a single run. For TILLING, there are typically 3–5 mutations per 1.5 kb
Arabidopsis gene per 768 pooled individuals screened on a gel. With this amount of information,
manual gel analysis and database entry is practical. For EcoTILLING, over 100 polymorphic bands
could be identified in a single gel run, when screening 96 samples with a 1-kb target fragment.
This large increase in data points created a serious bottleneck and increased the possibility of
human error during manual analysis. To facilitate the gel reading and data entry processes, Zerr
and Henikoff developed the PC/Mac program, GelBuddy (http://www.gelbuddy.org) (Zerr and
Henikoff 2005). GelBuddy provides automated lane identification and molecular weight
calibration. Bands are scored with a click of the mouse, and reports containing lane and molecular
weight information for each band are automatically generated. A fully automated version that
includes band scoring has recently been introduced as part of an effort to apply EcoTILLING to
the high-throughput discovery of rare human SNPs and cancer mutations (Till et al., 2006). 5.3.
EcoTILLING for Populus trichocarpa As with TILLING, EcoTILLING is general, and should be
applicable to most species. Gilchrist and colleagues recently reported a genotyping analysis of
western black cottonwood populations (Populus trichocarpa), using EcoTILLING for SNP
identification (Gilchrist et al. 2006). Sixty-three novel SNPs were identified in 9 target genes, for
41 tree accessions. From these data, the group estimated the degree of linkage disequilibrium,
heterozygosity, and nucleotide diversity. Much can be learned from studying natural nucleotide
diversity; new markers will be generated from EcoTILLING projects, and non-synonymous SNPs
may be identified that provide a beneficial phenotype. For species in which mutagenesis is

4. impractical, exploiting natural nucleotide diversity will be invaluable for crop improvement. 348
TILL ET AL.
Functionality of new alleles identified by EcoTILLING
The main objective of Eco TILLING is to isolate useful new haplotypes or alleles in target genes.
Thus, the molecular variation in eIF4E and eIF(iso)4E genes could be very useful for identifying
new resistance alleles against important viruses in pepper. Among the pepper poly viruses, Potato
virus Y is widespread throughout most of the cultivated areas. PVY can be transmitted by many
species of aphids, but chemical methods are effective enough to control the vector. Nevertheless,
the impact of this virus has increased due to the restriction of phytosanitary treatments. In recent
years, resistance alleles for this virus have not been very important in breeding programs, but now,
with the treatment reduction, the use of resistant varieties is the most effective way to prevent
damage from the virus. Therefore, a screening with PVY-F14K was done to study the response of
the new eIF4E and eIF(iso)4E proteins discovered. The F14K isolate of PVY completed the viral
cycle in some or all accessions of each analysed species, which indicated that resistance is not
species-specific.
The results according to the protein combinations of eIF4E and eIF(iso)4E). From analysis of the
correlation between proteins and disease resistance, it is hypothesised that the eIF(iso)4E proteins
were not involved in the resistance to PVY as most of the accession responses could be explained
by eIF4E proteins. Accessions carrying the proteins eIF4E_C, eIF4E_F, eIF4E_G, eIF4E_J and
eIF4E_P generated symptoms, while accessions with the proteins eIF4E_M, eIF4E_L, eIF4E_R
and one accession with the eIF4E_Q protein (CDP04710) and another with eIF4E_F (CDP00614)
showed systemic infection but were symptomless. Other accessions carrying new eIF4E proteins
found in this study (eIF4E_D, eIF4E_H, eIF4E_K, eIF4E_O and eIF4E_N) showed a resistance
response as the viral infection was not detected throughout the experiment.

5. PVY-F14K symptoms in pepper plants and representative resistant accessions. (A)
Agridulce negative control leaf at 15 DPI. (B) Agridulce leaf showing mosaic and deformation at
15 DPI. (C) Agridulce leaf showing dark green vein-banding between 45-60 DPI. (D) CDP08791
negative control (C. chacoense) at 45 DPI. (E) CDP08791 susceptible plant showing stunted
growth at 45 DPI. (F) CDP02521 negative control (C. chinense) at 60 DPI. (G) CDP02521
resistant plant showing no symptoms at 60 DPI. (H) CDP09688 negative control (C. annuum) at
60 DPI. (I) CDP09688 resistant plant showing no symptoms at 60 DPI.
CONCLUSION:
EcoTILLING are high-throughput and low-cost methods for the discovery of induced
mutations and natural polymorphisms. The methods are general and have successfully been
applied to many plants, including crops. With sequence data and general tools such as TILLING,
reverse genetics can be applied to lesser studied species. Now that successes have been reported
in a variety of important plant species, the next challenge will be to use the technology to develop

6. improved crop varieties. The utility of induced mutations and natural polymorphism has already
been established for crop breeding and so the task is mostly one of implementation