mismatch_cleavage_by_cel-1_1__final

Mismatch Cleavage by CEL-I Endonuclease: A Tool for Rapid Detection 1

of Homozygous and Heterozygous Mutants

Mismatch Cleavage by CEL-I Endonuclease:

A Tool for Rapid Detection of Homozygous

and Heterozygous Mutants

Sulabha Sharma1, Kamal Tyagi2, M Lakshmi Narasu3, Y Sreelakshmi4 and Rameshwar Sharma5

In recent years, mutants have served as useful starting material to unravel the mechanisms

governing many biological processes, including both plants and animals. Mainly, there are

three ways of inducing mutations; by using physical agents (radiations UV, X-rays, -rays),

chemical agents (Ethyl Methane Sulphonate (EMS), Nitrous Oxide (NO), acridine, etc.) and

biological agents (transposon and T-DNA). The mutation induced by these agents lead to

isolation of most commonly recessive or uncommonly dominant mutants. The isolation and

screening of a mutant are tedious and cumbersome job and even after isolation, ascertaining

the homozygous nature of the plant is equally difficult, especially for a dominant mutant.

The commonly known methods for screening of homo/heterozygosity of the mutant are

either highly expensive as sequencing of the gene or involve very tedious/time-consuming

genetic segregation analysis. Here, we present a rapid tool for identifying the homo/

heterozygous nature of the plants by simple digestion of PCR-amplified genomic DNA with

CEL-I endonuclease and resolving the cleaved product on conventional agarose gels.

This method is quite robust and provides a great help to researchers in developing countries

where instrumentation access is limited.

Keywords: Homozygosity, Heterozygosity, Homoduplex, Heteroduplex, CEL-I Endonuclease, Mutant,

Agarose

Introduction

Plant mutants have been used in genetic studies and breeding for decades, yet huge

number of mutants remains to be characterized at the molecular level. In recent years,

the information of genome sequence has become available for several plant species but

the functions of many of the genes are not known. The analysis of well-characterized

mutants using saturated mutant populations, coupled with recent methods for the detection

1 Sulabha Sharma (Researcher), Department of Plant Sciences, School of Life Sciences, Hyderabad Central University, Hyderabad

500046, India. E-mail: m a i l 2 s u l a b h a @ g m a i l . c o m

2 Kamal Tyagi, Department of Plant Sciences, School of Life Sciences, Hyderabad Central University, Hyderabad

500046, India. E-mail: [email protected]

3 Professor. M. Lakshmi Narasu ( Director), Institute of Science and Technology, Department of Biotechnology, Jahawarlal Nehru Technological

University, Hyderabad, India. E-mail: [email protected]

4 Dr. Y. Sreelakshmi (Associate Professor), Department of Plant Sciences, School of Life Sciences, Hyderabad Central University, Hyderabad

500046, India. E-mail: [email protected]

5 Professor. Rameshwar Sharma (Professor), Department of Plant Sciences, School of Life Sciences, Hyderabad Central University, Hyderabad

500046, India; and is the corresponding author. E-mail: [email protected]

© 2011 IUP. All Rights Reserved.

mailto:[email protected]

and generation of mutants are bridging the gap between plant genes and their functions.

There are basically two ways to analyze function of a mutated gene: reverse and forward

genetics (Caldwell et al., 2004; Jose and Joseph, 2006; and Christian et al., 2009). In reverse

genetic approaches, one starts with a (sequenced) gene of interest, selects a mutation in

that gene, and then tries to identify a phenotypic change associated with mutation.

In forward genetic approaches (Caldwell et al., 2004), one begins with a prediction of

specific effect of a mutation for physiological and morphological processes and then

isolates mutants with the predicted phenotype followed by mapping and isolation of the

genetic sequence that determines the above phenotype. Thus, contrary to reverse genetics

(Jansen et al., 1997; Sessions et al., 2002; Jose and Joseph, 2006; and Christian et al., 2009),

forward genetics starts with phenotyping of mutants and later identifies the gene

responsible for the altered phenotype and both the approaches are valuable and

complementary. Once corres ponding gene linked to a mutation is identified, the

introgression of the gene in a new cultivar for breeding requires at least two backcrosses

for removal of background mutations and elimination of genotype of donor plant. Moreover,

the essential confirmation that a phenotype of interest results from a given mutation

though can be achieved via complementation testing, which is also used for determining

allelism of recessive mutations, such confirmation is not possible in case of dominant

mutants. In addition, once the mutant is isolated the next requirement is to maintain the

purity of the mutant, i.e., homozygous nature, which is slightly difficult in case of dominant

mutant (Jack et al., 1997), where the mutant shows same phenotype in both homozygous

and heterozygous forms. Likewise, if the population of different mutants were grown

together, there are always chances of cross pollination from neighboring plants and

heterozygous progeny may result. Tomato crop grows well under warm climate, but

cannot be grown under temperate climate. As a consequence in temperate climate

tomatoes are grown densely in green houses (Liu et al., 2004; and Menda et al., 2004).

In such growth conditions, close proximity of plants also increases the chances of cross

pollination leading to heterozygous population.

In mutation breeding experiments, it is essential that the plant materials be genetically

pure and uniform for the traits to be examined and that pollination be rigidly controlled

both prior to and during the experiment to prevent outcrossing. The identification of

homozygous wild type, heterozygous and homozygous mutant plants, can be done by

normal genetic crossing experiments. However, it involves considerable time and money

for genetically screening of mutant. Moreover, in crops, it is difficult to distinguish between

homozygous dominant or heterozygous dominant mutations. In procedures, that assist

in the identification of mutations include growing progeny of suspected mutants and

observing whether segregation occurs or back crossing the suspected mutant to the

parent followed by selfing or sibling of the progeny of a true recessive mutant.

Apart from genetic screening, another technique for the confirmation of mutation

identity is sequencing of the mutated gene. But the cost of sequencing is not affordable

to every laboratory or company, particularly, when a large plant population of at least 100

2 The IUP Journal of Genetics & Evolution, Vol. IV, No. 2, 2011

to 200 plants need to be analyzed for homozygosity or heterozygosity of mutation.

Therefore, there is an enormous potential for simple and fast methods, which can allow

one to distinguish homo/heterozygosity of mutants efficiently using in house facility.

Recently, use of mismatch specific endonuclease CEL-I (Oleykowski et al., 1998; Till et al.,

2004a; and Yeung et al., 2005) has been extensively applied for TILLING, a reverse

genetic method for mutant isolation. Currently, the use of above endonuclease enzymes

is being made in numerous approaches like TILLING ECOTILLING, SNP detection, etc.

(Peterson et al., 1997; McCallum et al., 2000; Colbert et al., 2001; Henikoff and Comai,

2003; Perry et al., 2003; Till et al., 2004b; Gilchrist and Haughn, 2005; Altmann et al., 2007;

and Raghavan et al., 2007). Here we describe the use of CEL-I endonuclease enzyme

(Oleykowski et al.1998; and Till et al., 2004a) for detection of homo/heterozygous

mutants in a pool of mixed population. This method can be easily carried out in a

breeding lab with simple PCR machine and conventional gel electrophoresis apparatus.

The above tool can detect both homozygous and heterozygous mutants in a cost-

effective manner. This method is also quite convenient for detecting dominant

mutations (Wilson et al., 1990) where it is difficult to differentiate between homozygous

and heterozygous mutants. Moreover, this method can be applied to any organism right

from bacteria to higher eukaryotes for any gene, whose gene sequence is known.

Materials and Methods

Plant Material and Growth Conditions

The tomato mutant population and wild type were grown in green houses, net houses,

growth chambers in confined areas to avoid adverse climatic conditions and even protect

crop from damage caused by birds and animals. The plants used for screening of

homozygous and heterozygous mutation from a pool of mixed population were Solanum

lycopersicon. Mill cv Ailsa Craig (wild type), Money maker, (wild type), fri (far red insensitive

mutant) (Galina et al., 1998) obtained from Prof. M Korneeff and Prof. R E Kendrick,

University of Wageningen, Netherlands in 1992 and multiplied in green houses at

University of Hyderabad.

Plant Genomic DNA Isolation

Extraction of DNA was carried out, as described earlier (Sreelakshmi et al., 2010) with

slight modifications. The leaf tissue (200-300 mg) was homogenized with three steel

balls in microcentrifuge tube in Mini-Bead Beater for 2 min in the presence of 1,000 L

preheated (65 °C) extraction buffer (0.1 M Tris-HCl, pH 7.5, 0.05 M EDTA, pH 8.0; 1.25%

( w / v ) SDS ) c o n t a in in g 0 .2 M - me rc a pt o et h a n o l a n d 3 0 mg o f i n s o l u b l e

polyvinylpolypyrrolidone (PVPP). The tubes were incubated at 65 °C for 30 min for cell

disruption. The residual RNA was degraded by adding 1 L from 10 mg/mL stock and

incubated at 37 °C in a water bath for 30 min. The protein precipitation was done by

the addition of 600 L of 6 M ammonium acetate and incubating the samples at 4 °C for

15 min. The precipitated proteins along with other cellular debris were pelleted by

centrifugation at 13,000 rpm for 10 min and the clear supernatant (800 L) containing



2

DNA in aqueous phase was transferred into a fresh microcentrifuge tube. The nucleic

acid was precipitated by adding chilled 700 L of isopropanol and precipitation was

maximized by incubating the sample at –20 °C for 30 min. The DNA was pelleted by

centrifugation at 13,000 rpm for 10 min, and tubes were dec anted and residual

isopropanol was replaced and salts were removed by washing the pellet with 70% (v/v)

ethanol and finally pellets were air-dried and dissolved in 200 L TE (10 mM Tris, pH

7.5, 1 mM EDTA pH 8.0). The estimation of the yield of isolated DNA samples was done

spectrophotometrically by Nanodrop-1,000 and by electrophoresis on 1% (w/v) agarose

gel by comparative fluorescence quantification of ethidium bromide stained bands using

quantified standard DNA.

Primer Design and Amplification

Fo r wa rd ( 5'G A AG GATG ATG GC A GGA A A AT GC3 ') and rev ers e primers

(5'CACTCAGAAACACCAGCCA A ATTG3') were designed to amplif y a 982 bp region of

plant photoreceptor protein, phytochrome A defective mutant (far red insensitive) from

fri mutant of tomato. The mutant, fri has transition of A to T at the 3' end of the

in tron between exons 1 and 2, in the PhyA gen e of lengt h 6623 bp (AJ0 01913).

Upon digestion with CEL-I enzyme, it generated two fragments of 710 bp and 272 bp.

Amplification reaction was set up in a volume of 10 L with 25-30 ng of DNA having

fri mutant DNA in each of the samples. The reaction consisted of 5 L of template, IX

PCR buffer (10 mM Tris, 5 mM KCl, 1.5 mM MgCl , 0.1% (w/v) gelatin, 0.005% (v/v)

Tween-20, 0.005% (v/v) Np-40, pH 8.8), 0.2 mM dNTPs, 0.5 L Taq polymerase

(in-house isolated) and 5 pmoles of primers. The cycling conditions for amplification

were 94 °C-5 min, 35 cycles of 94 °C-20 s, 55 °C-30 s, 72 °C-30 s followed by elongation

at 72 °C for 8 min.

Heteroduplex Formation

The heteroduplex (Ruano and Kidd, 1992) was prepared by adding equal amount of WT

and fri PCR products and treating at 98 °C for 8 min; 80 °C for 20 s; 60 cycles of

80 °C for 7 s –0.3°/cycle (Colbert et al., 2001). The PCR product was digested as described

by Till et al. (2004a) using CEL-I endonuclease (Yeung et al., 2005). After mixing on ice,

the microcentrifuge tubes were incubated at 45 °C for 15 min and the digestion was

stopped by addition of 2 L of 75 mM EDTA.

Mismatch Cleavage and Mutant Detection

CEL-I was isolated from celery as previously described. The mismatch cleavage reaction

was performed in a total volume of 45 L containing 10 pi PCR product, IX CEL-I digestion

buffer (10 mM HEPES buffer pH 7.0, 10 mM KC1, 10 mM MgCl2, 0.002% (v/v) Triton X-100

and 10 g/mL BSA) and CEL-I enzyme at 1:300 dilution (1 L/300 L CEL-I digestion buffer).

The reaction was incubated at 45 °C for 15 min and then terminated by adding 2 L EDTA

(75 mM EDTA, pH 8.0). Later, the digestion mixture was run on 2% agarose gel, and the gel

was visually assessed for mutations.



Results and Discussion

Low Cost Genomic DNA Isolation

A cost-effective, efficient and high throughput method for genomic DNA isolation from

plants was used (Lin and Kuo, 1998; Kang and Yang, 2004; and Sreelakshmi et al., 2010)

that was specifically designed for high throughput DNA isolation from large number of

plants for TILLING. The basis of this methodology lies on good yield and quality of isolated

DNA along with use of inexpensive chemicals, and minimum time required for complete

DNA isolation (Sreelakshmi et al., 2010). In the present protocol, we modified few steps

as DNA extraction was carried out in microcentrifuge tubes/Eppendorf tubes leading to

the higher yield. One of the advantages of higher yield was that the same DNA after

confirming the homozygous nature of the mutant plant can also be used for other

techniques, such as genotyping or mapping. Figure 1 shows that genomic DNA obtained is

of good quality (260/280 ratio of 1.9) and quantity (up to 10 g/100 mg tissue). Principle of

homozygous mutation detection method. The genomic DNA from three plants was isolated:

homozygous wild type, homozygous mutant and putative heterozygous mutant. The DNA

was subjected to PCR amplification and amplified DNA was denatured and reannealed forming

homo/heteroduplex, while in case of homozygous plants, there is no chance of mismatch,

leading to homoduplex formation. However, in case of heterozygous plants, where both

DNA strands are noncomplementary to each other, and nucleotide bases on reannealing

form a heteroduplex. The CEL-I endonuclease enzyme cleaves at the point of mismatch,

leading to cleavage of PCR fragment into two pieces. The size of cleaved fragments adds up

to main product which can be easily resolved on normal agarose gels.

Figure 1: Genomic DNA

Wild Type Homozygous Mutant Heterozygous Mutant A C C T G T

1) PCR Amplification with Gene Specific Primer, Fragment Carries the Point Mutation

A C C

T G T

2) Denature and Re-Annealing

A C C

T G T

3) Digestion with Cel-I Endonuclease

A C C

(Cut by Cel-I)

T G (Cut by Cel-I)T

4) Fragments were Separated and Resolved on 1.5% Agarose Gel

A C C T G T

C(Cut by Cel-I)

PCR Quality and Quantity

To take advantage of agarose gel detection, special attention should be given towards

the quality and quantity of PCR products. Initially, it is of great importance to amplify

specific and single PCR product from a PCR reaction since the presence of nonspecific

products may lead to heteroduplex formation. The nonspecific amplification would easily

result in the detection of varied CEL-I cleaved products (Ruano and Kidd, 1992; and

Oleykowski et al., 1998). Though here we are aware about the site of mutation in the

gene and the size of expected cleaved products, still complications can easily be avoided

by the single and specific PCR product. Secondly, the yield of PCR product should be

higher since the sensitivity of detection on agarose gel is little low. The PCR products

less than 10 ng/L are not normally detected on conventional agarose gel. A good yield of

PCR product is required to obtain better resolution of both the cleaved products on the

regular agarose gels.

Large-Scale Population Screening for Homozygous and Heterozygous

Mutants in Mixed Crop

In the current era of genomics, plant biologists are striving to understand the function

of all the plant genes. The function of a particular gene can be best determined when

the gene of interest is mutated and the resulting mutant is analyzed for differences

compared to its wild type. The isolation of mutants either by forward genetics or

reverse genetics (Caldwell et al., 2004; and Christian et al., 2009) is equally tedious and

time-consuming. However, maintain ing homozygo sit y of mutants in subsequen t

generations is equally difficult, more so for dominant mutants (Wilson et al., 1990) and

also for those crops where cross pollination is feasible. Therefore, having a tool to

identif y homo/heterozygosity is of great advantage. Here, we adopted a use of CEL-I

endonuclease enzyme (Oleykowski et al., 1998; and Till et al., 2004) for detection of

homozygous and heterozygous mutants in a pool of mixed population. This method is

cost-effective and institutes or small companies with just a PCR facility can deploy this

method (Figure 2). The basic methodo logy is demo nstrated using a phytochro meA

receptor defective mutant in tomato (fri, far red insensitive) (Galina et al., 1998). The

cartoon depicts the methodology in Figure 3. The DNA from wild type and fri mutant

was PCR amplified using the phytochrome A gene specific primers and subjected to

mutation detection using the standard protocol of TILLING (Till et al., 2004). The

mismatch cleavage by CEL-I cuts the heteroduplex DNA fragment of 982 bp size at the

mismatch site, releasing two fragments of 272 bp and 710 bp (Figure 4). The specificity

of the reaction was confirmed since the cut bands were present only in samples where

genomic DNA from wild type and fri were combined to make heteroduplex. In contrast,

samples with either wild type or fri mutant DNA alone, no complementary fragments

were released, on CEL-I digestion. The resultant PCR product of homozygous mutant

releases a band on CEL-I digestion, only when it was mixed with WT PCR products to

make heteroduplex. In all other samples, cleavage of CEL-I endonuclease shows no cut

fragments, as there was no heteroduplex formation (Figure 5).



Hom

ozy

gous

WT

Hom

ozy

gous

frt

Dig

este

d W

T/f

ri

Un

dig

este

d W

T/f

ri

M

Figure 2: Plant Genomic DNA Isolation from a Mixed Population of Homozygous

and Heterozygous Plants

A

B

Note: The protocol used for tomatoplant genomic DNA isolation is highly cost-effective and adapted from

our previous publication.

Source: Sreelakshmi et al. (2010)

Figure 3: Gene-Specific PCR Amplification Using in House Isolated Taq DNA Polymerase

Note: The genomic DNA isolated from homo/heterozygous mixed population was used for PCR amplification.

The PCR amplified bands were resolved on 1% agarose gel. Amplification of the PCR product without

any nonspecific amplification is important for this technique.

Figure 4: Detection of Mutation in PHYA Gene of Fri mutant

by Mismatch Cleavage Using CEL-I

Nonspecific Band

0.98 Kb 0.71 Kb

0.27 Kb

1.0 Kb 0.3 Kb

Note: Homoduplex PCR product is ca. 0.98 kb in both wild type and fri mutant, while digestion of heteroduplex

shows presence of cleaved bands of 0.71 kb and 0.27 kb (adding uptothe size of full length product

0.98 kb); and * indicates nonspecific band.


Figure 5: Methodology for Screening of Homozygous Mutation

from a Pool of Homo/Heterozygous Population

1 Kb 0.9 Kb 0.8 Kb 0.7 Kb 0.6 Kb 0.5 Kb 0.4 Kb

0.3 Kb

0.2 Kb

M 1 2 3 4 5 6 7 8 9 1 0

Note: Lanes 1 and 3 show undigested PCR amplified DNA fragment, homozygous wild type and mutant, lanes

2 and 4 show CEL-I digested homozygous wild type and mutant DNA fragment. Lane 5 shows undigested

heteroduplex of Wild type DNA fragment mixed with mutant DNA fragment and lane 6 shows

heteroduplex made of WT + mutant DNA fragment digested with CEL-I endonuclease. The two extra

bands indicated by arrow shows the cleaved bands after digestion of CEL-I. Lanes 7 and 9 show one

putative homozygous and one heterozygous mutant plants respectively, the genomic DNA of putative

plants was PCR amplified using gene specific primer and then subjected to CEL-I endonuclease treatment,

the presence of two extra bands indicated by an arrow shows that the plant in lane 10 is heterozygous

carrying one copy of wild type and another copy of mutant DNA while lane 8 appears to be a

homozygous mutant with no other extra cleaved bands even after CEL-I treatment. The star* shows

some nonspecific cleavage observed in all the samples.

This robust technique can be even applied to screen for homozygous plants from a

population exhibiting doubtful phenotypes. Some times in tomato, wild type or any mutant

plant grown in green houses or open field shows unusual phenotypes, which can be due

to adverse climatic stress, pests, or cross pollination from neighboring plants. So it is

not advisable to use these plants for any analysis before confirming the identity of the

mutant. The only available option is to wait for few generations before we can screen

them genetically by analyzing segregation pattern or obtaining expensive gene sequence

of these plants. Here we used CEL-I endonuclease mismatch cleavage for identifying the

mutant gene identity either homozygous or heterozygous in nature. From our mutant

plant growth facility, we have identified few plants with unusual phenotypes and to

determine the homo/heterozygous nature of the plants the plant genomic DNA was isolated

and used for gene specific amplification and the PCR products were subjected to CEL-I

digestion, and the digested products were resolved on the 1% agarose gel, all the putative

mutants turned out to be heterozygous in nature (Figure 6).

The important application of this method is large-scale screening of plants for detection

of homozygous and heterozygous mutations within a population. A set of 24 to 48 plants

can be screened in one batch. Once the genomic DNA is isolated 48 to 96 samples can be

amplified in a single PCR block in one set. A normal water bath or even PCR block can also

accommodate 48 to 96 samples for CEL-I digestion. The time taken for resolving the

cleaved fragments is important since this step cannot be done in a 96 well agarose gel

plates. In order to obtain better resolution, samples should be run at a voltage less than

50 V/min and a broad well gel casts should be used to cast agarose gel. Still 28 to 30

samples can be resolved in one batch (Figure 7). A population of about 150 to 200 plants

can easily be screened in less than three days.



Figure 6: Identification of Putative Heterozygous Plants Selected

on the Basis of Unusual Phenotype

M 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 M

1 Kb

0.6 Kb

0.2 Kb

1 Kb 0.6 Kb

0.3 Kb

0.1 Kb

Note: The six tomato plants were selected from the large plant population of fri mutant with some unusual

phenotype, which were never observed during normal screening of mutant. DNA was isolated and

subjected to gene specific PCR amplification, lanes 1 to 6 show the amplified PCR product while was

cleaved with CEL-I enzyme, shown in lanes 7 to 12. All putative hetreozygous plants were easily

confirmed by CEL-I digestion, as lanes 7 to 12 showed two extra bands.

Figure 7: Rapid Method of Homo/Heterozygous Mutation Detection

from the Tomato Mutant Crop

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 71 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 M

1 Kb 0.8 Kb

0.6 Kb 0.5 Kb 0.4 Kb

0.2 Kb

Note: A mixed population of homo/heterozygous mutant plants grown in green house with densely packed

growth conditions was screened. Lanes 1, 3, 5, 7 and 9 show the PCR product of control plants out of

which lane 1 wild type homozygous, lane 3 mutant homozygous while lane 5 heteroduplex formed

between wild type and mutant. Lanes 7 and 9 are previously known homozygous and heterozygous

mutants respectively. The DNA fragments in lanes 6, 8 and 10 were treated with CEL-I and presence of

extra bands in lane 6 confirms the plant as heterozygous which matches with the lane 10. Subsequently,

a set of 20 plants were screened similarly. The genomic DNA was isolated and gene specific PCR was

amplified and PCR products were digested with CEL-I endonuclease. Lanes 13, 14, 15, 16, 18, 20, 22,

23, 25 and 29 confirm the presence of hetreozygousplants, while 11, 12 17, 19, 21, 24, 26, 27 and 28

are homozygous mutant plants. The first cleaved band of around~ 800 bp can easily help in identif ying

the homo/heterozygous nature of the plants.

Conclusion

In this work, we report a simplified procedure to detect homozygous and heterozygous

mutant plants from a mixed crop. This novel use of CEL-I endonuclease helps to detect

the mutant population carrying not only recessive mutation but also dominant mutation

where it is extremely difficult to differentiate the homo/heterozygosity on the basis of

their phenotypes. The method is highly robust and does not require sophisticated

instrumentation or expensive dye labeled primers. More importantly, it can be carried

out in a just one day and eliminates the need of genetic crosses to confirm the homo/


heterozygosity of the population This technique will enable more laboratories, particularly

in developing countries to carry out work on mutants without worrying about costs and

facilities ^

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