allele mining, tilling and functional genomics

2. ASSIGNMENT
ON
Use mutagens in genomics, TILLING
& Allele mining
Department of Plant Breeding and Genetics
College of Agriculture
Swami Keshwanand Rajasthan Agricultural University,
Bikaner – 334006
Submitted to: Submitted by:
Dr. A.K. Sharma Sanjay Kumar
Sanadya Professor M.Sc. final
year (PBG)

3. GENOMICS
1. Structural Genomics :
•The structural genomics deals with DNA sequencing, sequence
assembly, sequence organisation and management. Basically it is the
starting stage of genome analysis i.e. construction of genetic, physical or
sequence maps of high resolution of the organism.
•Structural genomics seeks to describe the 3-dimensional structure of
every protein encoded by a given genome.
2. Functional Genomics :
•Based on the information of structural genomics the next step is to
reconstruct genome sequences and to find out the function that the
genes do.
•Functional genomics attempts to answer questions about the function
of DNA at the levels of genes, RNA transcripts, and protein products.

4. Use mutagens in genomics
• Mutations provide genetic variation for plant breeding.
• Mutations are changes in the DNA sequence of a cell's genome caused
by radiation, viruses, transposons, mutagenic chemicals that occur
during meiosis or DNA replication.
• Mutations are naturally occurring, or can be induced.
For identification of gene function there are two methods used
commonly.
– Forward genetics (Classical genetics)
A traditional approach to the study of gene function that begins
with a phenotype (a mutant organism) and proceeds to a gene that
encodes the phenotype.

5. – Reverse genetics
A molecular approach that begins with a genotype ( a DNA
sequence) and proceeds to the phenotype by altering the sequence or
by inhibiting its expression.
• It is possible due to the advancement in the molecular genetics
Forward Genetics Reverse Genetics
Known phenotype Unknown phenotype
Unknown sequence Known sequence

6. Tools for forward and reverse genetics
1. Transposon tagging
• Transposons, also known as jumping genes,
were first discovered by Barbara McClintock in the
1940’s when she analyzed the mosaic pigmentation
patterns of maize kernels.
• Transposable elements or transposons are DNA
elements that have the ability to move from one
chromosome site to another.
• DNA transposons flanked by short inverted
repeats move by excising from one chromosome site to
another
• Retrotransposons move via RNA intermediates.

8. 2. T-DNA tagging
• T-DNA is a segment of DNA from Agrobacterium tumefaciens tumor
inducing (Ti) plasmid that is moved into the plant upon infection.
• Agrobacterium tumefaciens has traditionally been used in some plant
species for transformation of foreign DNA into the genome (i.e.
RoundUp Ready gene) generating transgenic plants.
• A marker selection gene (i.e. antibiotic resistance) is inserted between
the borders of T-DNA so that transformed cells can be selected.
Advantages:
•Effective interruption of genes
•Low copy number (1.5)
•Random insertion in the genome
Disadvantages:
•Time consuming for transformation
•Somatic variation caused by tissue culture process (i.e. high percentage
of untagged mutations)
•Not available in many species

9. 3. Radiation & Chemical mutagenesis
• Ionizing radiation (i.e. fast neutron, gamma ray)
are used to generate random mutations (breaks) in
DNA segments. For example, fast neutron breaks
the chromosomes leading to loss of DNA
sequences (i.e. large deletions).
• Chemicals (e.g. carcinogens) can also cause
mutations in DNA sequences. Etheylmethane
sulfonate (EMS) induces point mutations in DNA.
EMS alkylates primarily guanine leading to
mispairing: alkylated G pairs to T instead of C. The
resulting mutations are mainly transitions (GC ::
AT).

10. 4. homologous recombination
• Recombination is the exchange of genetic information between DNA
molecules; when the exchange is between homologous DNA molecules
it is called homologous recombination.
• Works in bacteria, yeast, mice and other mammals.

11. 5. RNA interference (RNAi)
• RNA interference (RNAi) is a biological process in which RNA
molecules inhibit gene expression, typically by causing the
destruction of specific mRNA Molecules.
• Previously, it was known by other names, including co-
suppression, posttranscriptional gene silencing (PTGS), and
quelling.
• Double stranded RNA (dsRNA) can lead to specific post
transcriptional gene silencing (PTGS).
• This mechanism is part of a natural response of the host that
most likely evolved to control virus.
• Sometimes RNAi does not completely eliminate expression of
the target gene, but only reduces it.
• In these cases, it is referred to as a “knock down” instead of a
“knock out”.

12. •During RNAi, long dsRNA is cut or "diced" into
small fragments ~21 nucleotides long by an
enzyme called "Dicer".
•These small fragments, referred to as small
interfering RNAs (siRNA), bind to proteins from a
special family: the Argonaute proteins.
•After binding to an Argonaute protein, one
strand of the dsRNA is removed, leaving the
remaining strand available to bind to messenger
RNA target sequences.
• RNA-induced silencing complex, or RISC, is a
multiprotein complex, specifically a
ribonucleoprotein, which incorporates one
strand of a single-stranded RNA (ssRNA)
fragment, such as microRNA (miRNA), or double-
stranded small interfering RNA (siRNA).
•Once bound, the Argonaute protein can either
cleave the messenger RNA, destroying it, or
recruit accessory factors to regulate the target
sequence in other ways.

13. 6. Genome editing
• Several methods have been developed to target mutations to
a specific location in the genome.
• These can be used to knock-out target genes, make specific
point mutations in a target gene, or even insert new genes or
DNA sequences at a specific target site in the genome.
E.g.
– Zinc-finger nucleases (ZFNs).
– Transcription activator-like effector nucleases (TALENs).
– Clustered regularly interspaced short palindromic repeat
(CRISPR) and CRISPR associated (Cas) nucleases.

14. 7. Site-directed mutagenesis and transgenics
• Can make a specific nucleotide changes at an
exact site in gene of interest or in DNA.
• Requires: Gene of interest cloned into
plasmid, host with null background (knockout).
• Mutagenesis is performed in vitro on the
cloned gene in a plasmid replicated in bacteria.
• Then the mutated gene is inserted into the
host genome in a transposable element vector.

15. 8. Virus-induced gene silencing (VIGS)
• is a virus vector technology that exploits this RNA-mediated defence
mechanism.
• After the infection the replication of viral RNA proceeds and
produces a dsRNA replication intermediate which in turn produce
siRNA in the infected cell that correspond to parts of the viral vector
genome, including any non-viral insert.
• The siRNA in the infected cell guide an RNase complex by base
pairing and specifically knock out the expression of single-stranded
(ss) target RNA recognized by the dsRNAs.
• Thus, if the insert is from a host gene, the siRNA/Rnase Complex
would target the RNase complex to the respective host mRNA and the
symptoms would appear in the infected plant as the loss of the
function of the target protein.

17. What is TILLING ?
(Targeting Induced local lesions in Genomes)
• TILLING is a general reverse genetic technique that combines chemical
mutagenesis with PCR based screening to identify point mutations in regions of
interest. (McCallum et.al, 2000).
• TILLING is a powerful technology that employed heteroduplex analysis to
detect which organism in a population carry single nucleotide mutation in
specific genes.
• 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.
Reverse genetics is-
AGCTC AATC AGATAATC
TCGAGTTAGTCTATTAG

18. 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.

19. Discovery of TILLING
>TILLING first began in the late 1990’s by McCallum who
worked on characterizing the function of two chromomethylase
gene in Arabidopsis.
>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.

20. The TILLING Methodology
Development of mutagenized population
•EMS mutagenesis
•Development of M2 population
DNA preparation and pooling of individuals
Mutation Discovery
■PCR amplification of a region of interest
■Mismatched cleavage
■Detection of Heteroduplexesas extra peak or band (Acrylamide gel)
■ Identification of the mutant individual
■ Sequencing of Mutant PCR product.

21. Heteroduplexesas
A heteroduplex is a double-stranded (duplex) molecule of nucleic
acid originated through the genetic recombination of single
complementary strands derived from different sources, such as from
different homologous chromosomes or even from different
organisms.

22. Step 1: Mutagenesis
The TILLING process starts with mutagenesis to create a large population containing many
1,000’s of random mutations.
For TILLING in plants the customary procedure involves treating seeds with the chemical
mutagen ethylmethanesulphonate (EMS) that causes G/C to A/T changes. Sodium azide typically
causes A/G to T/C.
The treated seeds are sown and the resulting M1 plants grown to produce the next generation
of seeds.
Step 2: Growing the TILLING population
The M1 plants are self-fertilized and the M2 seed harvested and sown.
The M2 germplasm will allow recessive and lethal alleles to be recovered as heterozygotes.
Each M2 plant is given a unique identifier.
Step 3: DNA
DNA is individually extracted from each M2 plant and stored in 96 well plates.
The M3 seeds are harvested and catalogued for future sampling.
TILLING procedure

23. Step 4: Database
The unique plant identifiers allow the DNA and the archived seeds to be cross-referenced and all
data is stored on a web-accessible database.
Step 5: Pooling the DNA and amplification of your favourite gene
To increase throughput the M2 DNA samples are 8x pooled.
Using gene specific primers, PCR is carried out on the library of 8x pooled DNA samples.
Step 6: Heteroduplex – forming mismatched PCR fragments
In the presence of a mutant, the amplification products when heated and cooled will form
mismatched heteroduplexes between the wild type and mutant DNA.
Step 7: CEL I
To enable identification of the point mutations induced by EMS the re-annealed amplification
products are incubated with a plant endonuclease called CEL I, which preferentially cleaves at
sites of heteroduplex mismatches that occur between wild-type and mutant DNA.
Step 8: Cleavage products
The cleavage products are run on a Fragment AnalyzerTM which is an automated fluorescence-
based Capillary Electrophoresis System that enables sensitive and high-resolution separation of
DNA. Wells containing CelI digested fragments are identified as containing a mutant allele within
the pool.
Step 9: Identification
When a mutation is detected in the pooled DNA, PCR products amplified from the individual
DNA samples that make up the pool are sequenced to identify the specific plant carrying the
mutation.

24. Choosing a target sequence
>The optimal length of target sequence that can be TILLED in a single reaction
is 1,500 bp. But most eukaryotic genes are over 2,000 bp and sometimes much
longer.
>Since the objective in TILLING is to identify plants with deleterious mutations
in the target gene, and most mutations in non-coding sequences such as introns,
untranslated regions, and promoters will have no effect on gene function.
>So the target sequence should be chosen to minimize such sequences and
maximize coding regions.

25. TILLING IN PLANTS
Arabidopsis thaliana - In 2003, Greene et al. reported that the
Arabidopsis TILLING Project (ATP), which was set up and introduced as a
public service for the Arabidopsis community, had detected 1,890
mutations in 192 target gene fragments.
■The mutations in Arabidopsis thaliana that have been identified via
TILLING have provided an allelic series of phenotypes and genotypes to
elucidate gene and protein function throughout the genome for Arabidopsis
researchers.
■Lotus japonicus - TILLING was used to investigate induced mutations
occurring in the protein kinase domain of the SYMRK gene, which is
necessary for root symbiosis.
■ Nitrogen fixation and the functional role of sucrose synthase was the
target of another Lotus japonicus TILLING study. Six isoforms of sucrose
synthase were identified.

26. Advantages
■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 without 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.

27. T able 1, Year-wise overview of TILLING approach among various crops
Year Organism Genus species Mutagen applied Mutagen dose Estimated
genome
Ploidv Mutation rate
2000 Arabidopsis Arabidopsis thaliana EMS 20mM to 40 mM 125 Mbp 2X 1/300 kb
2001 Arabidopsis Arabidopsis thaliana EMS 20mM to 30mM 125 Mbp 2X 1000 genome
2002 Arabidopsis Arabidopsis thaliana Fast neutrons 60 Gy 125 Mbp 2X 0.7 to 12 kb
2003 Lotus Lotus japonicus EMS 60mL 10mL(v/v) 472Mbp 2X l/154kb
2004 Barley Hordeum vulgare EMS 20mM to 30 mM 5300 Mbp 2X 1 Mb
2004 Maize Zea mays EMS 0.0625% 2500 Mbp 2X 0.93 kb
2005 Rice Oryza sativa MNU lmM 430 Mbp 2X 0.80/Ikb
2006 Field mustard Brassica rapa y radiation 500Gy 500Mbp 2X 1/6190kb
2007 Pea Pisum sativum EMS 4 mM 4300 Mbp 2X 1/669 kb
2007 Rice Oryza sativa EMS 1.5% 430 Mbp 2X 1/294 kb
2007 Rice Oryza sativa Az-MNU 1 to 15 mM 430 Mbp 2X 1/265 kb
2008 Rape seed Brassica napus EMS 0.4% to 1.2% w/v 1150 Mbp 4Y 1/41.5kb
2008 Sorghum Sorghum bicolor EMS 0.1 to 0.6% (v/v) 7.35 xl0sbp 2X 1/526 kb
2008 Soybean Glycine max NMU 2.5 mM 1115 Mbp 2X -1/140 kb
2008 Soybean Glycine max EMS 50 mM 1115 Mbp 2X 1/250 kb
2009 Barley Hordeum vulgare Sodium azide 1.5 mM 5500 Mbp 2X 1/ 2.5 Mb
2009 Clover Medic ago truncatula EMS 0.075% to 0.40% 470 Mbp 2X l/485kb
2009 Common Bean Phaseolus vulgaris EMS 20niM to 60 rnM 625 Mbp 2X 2 to 3/Mb
2009 Peanut Arachis hypogaea EMS/y-radiation (0.5%)/ 20 &30 kr 3.0 x 109 bp 4X 0.5 xlO'7
2009 Potato Solanum tuberosum y radiation 0.5% to 2.0% 850Mbp 4X <1/1810 bp
2009 Wheat Triticum turgidum EMS 0.7% to-1% 12000 Mbp 4X 1/40 kb
2010 Field mustard Brassica rapa EMS 0-1% 500Mbp 2X l/60kb
2010 Oat Avina sativa EMS 0.9% (v/v) 13000 Mbp 6X 1/ 20-40 kb
2010 Tomato Solatium EMS 0.7% to 1.0% 950Mbp 2X l/322kb
fycopersicum
Source: Muhammad Rashid et al, 2011

28. EcoTILLING
• The first publication of the EcoTILLING method in which TILLING
was modified to mine for natural 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 natural 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.

29. Results
>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.
>We screen the pools of DNA sample for mutation in 1-kb
segment from 11 different genes, obtaining 17 independent
induced mutations from a population of 750 pollen mutagenized
maize plants.
>One of the gene targeted was the DMT102 chromomethylase
gene, for which we obtained an allelic series of three missense
mutation that are predicted be strongly deleterious.

30. Allele mining
•Identification and access to allelic variation that affects
the plant phenotype is of the utmost importance for the
utilization of genetic resources, such as in plant variety
development.
•Considering the huge numbers of accessions that are held
collectively by gene banks, genetic resources collections
are deemed to harbor a wealth of undisclosed allelic
variants.
•The challenge is how to unlock this variation. Allele
mining is a research field aimed at identifying allelic
variation of relevant traits within genetic resources
collections.

31. STEPS IN ALLELE MINING

32. IMPORTANCE
• It helps in tracing the evolution of alleles.
• Identification of new haplotypes and development of allele-specific
markers for use in marker-assisted selection.
• This capability will be important for giving rice breeders direct access
to key alleles conferring.
(1) resistance to biotic stresses,
(2) tolerance of abiotic stresses,
(3) greater nutrient use efficiency,
(4) enhanced yield,
(5) improved quality, including human nutrition
• It can also provide insight into molecular basis of novel trait variations
and identify the nucleotide sequence changes associated with superior
alleles. In addition, the rate of evolution of alleles.