Phenotypic characterization and map based cloning of a novel mutant causing abnormal leaf in arabidopsis thaliana

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY LE VIET TRINH PCHARACTERIZATION AND MAP-BASED CLONING OF A NOVEL MUTANT CAUSING ABNORMAL LEAF IN Arabidopsis Thaliana...

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LE VIET TRINH

PCHARACTERIZATION AND MAP-BASED CLONING OF A NOVEL

MUTANT CAUSING ABNORMAL LEAF IN Arabidopsis Thaliana

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

LE VIET TRINH

PHENOTYPIC CHARACTERIZATION AND MAP-BASED CLONING OF A

NOVEL MUTANT CAUSING ABNORMAL LEAF IN Arabidopsis Thaliana

Supervisors: Professor Soon- Ki Park

Doctor Bang Phuong Pham

Thai Nguyen, 2016

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Student name Viet Trinh Le

Student ID DTN1153150084

Thesis title PHENOTYPIC CHARACTERIZATION AND MAP-BASED

CLONING OF A NOVEL MUTANT CAUSING ABNORMAL

LEAF IN ARABIDOPSIS THALIANA

Supervisor(s) Professor Soon-Ki Park

Dr Bang Phuong Pham Abstract:

This study was carried out to identify the mutant gene causing Leaf Rolled Inside

(LRI) phenotype of mutant line named as AP-44-1 Mutant line obviously showed curly

leaf for all rosette leaves, less leaf number compared to wild type To identify mutant gene, F2 mapping population was generated for map-based cloning using SSLP markers

Based on PCR analysis, the LRI gene was predicted to locate between 32160 and 32580 markers that containing 49 candidate in the region of approximately 163kb At2g32460

gene that was identified by sequencing and compared with previous report (An et al., 2014) is a strong candidate causing abnormal leaf phenotype Based on the Arabidopsis database (TAIR; http://www.arabidopsis.org), At2g32460 is the gene coding for a

member of the R2R3-MYB transcription factor family and was designated MYB101

The expression and genetic complementation of At2g32460 is being carried out to

investigate the responsible of gene to abnormal leaf phenotype

Keyword: Arabidopsis, map-based, cloning, curly leaf, leaf-rolled inside

Number of pages: 36

Date of

submission: 2016/08/29

This thesis would not have been possible unless their support

Lastly, I would like to thank Faculty of Biotechnology and Food Technology

members for their support through my internship

CONTENT

LIST OF FIGURES i

LIST OF TABLES ii

LIST OF ABBREVIATIONS iii

PART I INTRODUCTION 1

PART II MATERIALS AND METHODS 8

1 Plant materials and growth condition 8

2 Methods 9

2.1 DNA extraction 9

2.2 Genetic analysis using SSLP markers for positional cloning of AP-44-1 9

2.2.1 PCR analysis 10

2.2.2 Gel electrophoresis 10

2.3 Phenotypic characterization of a novel mutant causing abnormal leaf 11

2.4 DNA Purification 16

PART III RESULT AND DISCUSSION 18

1 Plant morphological analysis 18

1.1 Morphological phenotypes of AP-44-1 mutants showing defects and sterility 18

1.2 Comparative analysis of growth and biological activity 18

2 Fine mapping of AP-44-1 locus 21

PART IV SUMMARY AND CONCLUSION 29

REFEFENCES 30

Appendix 1 32

Appendix 2 33

i

LIST OF FIGURE

Figure 5 Procedure of a typical map-based cloning experiment 15 Figure 6 Principle of PCR-based mapping using SSLP markers 15

Figure 8 Comparative analysis of the morphological phenotypes of

wild-type, and AP-44-1 plants

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Figure 10 Example of linkage analysis with four markers using

wild-type plants in the mapping population (AP-44-1)

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Figure 11 Example of linkage analysis with four SSLP markers to

narrow down the region of mutant gene using wild-type

plant in the mapping population (AP-44-1)

Figure 14 A schematic of the positional cloning of the AP-44-1 gene

(A) and structure of candidate gene

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ii

LIST OF TABLE

Table

Table 2 Identification of chromosome containing the gene of

interest

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Table 3 Recombinants by PCR analysis of a large mapping

population with flanking markers

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iii

LIST OF ABBREVIATIONS

ADW Autoclaved distilled water

CTAB Cetyltriethy-ammonium bromide

Ler-0 Landsberg erecta

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

IAA Isoamyl Alcohol

LRI Leaf rolled inside

PCR Polymerase Chain Reaction

SSLP Simple Sequnce Length Polymophic

1

PART I INTRODUCTION

Arabidopsis Thaliana is a small flowering plant that is widely used as a model

organism in plant biology Arabidopsis has been used as an ideal model for studying the

plant biology and genetics As a model organism for agricultural biotechnology,

Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production (Boyes et al., 2001) There is ample

reason to believe that Arabidopsis will serve as a resource base for breeders of crop plant

and as a model plant that furthers the knowledge of plant scientists (Hayashi and

Nishimura, 2006) Classified in a member of the mustard and cabbage plants, Arabidopsis

has several advantages that make it an excellent experimental model (Hartwell et al., 2004) Not only the smallest genome makes Arabidopsis useful for genetic mapping and sequencing, also it could be easily grown in the laboratory In addition, its small size and rapid life cycle, approximately 6-8 weeks, are also advantageous for research Finally, mutants are easily induced by treating the seeds with various chemical mutagens The surviving seeds are the germinated and mutant progeny are recovered for analysis (Hopkins et al., 2004)

Over 750 natural accessions of Arabidopsis thaliana have been collected from

around the world and are available from the two major seed stock centers, ABRC (Arabidopsis Biological Resource Center) and NASC (Nottingham Arabidopsis Stock Centre) These accessions are quite variable in terms of form and development (e.g leaf shape, hairiness) and physiology (e.g flowering time, disease resistance) Researchers around the world are using these differences in natural accessions to uncover the complex genetic interactions such as those underlying plant responses to environment and evolution of morphological traits While many collections of natural accessions may not meet a strict definition of an ecotype, they are commonly referred to as ecotypes in the scientific literature

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Figure 1 Arabidopsis Thaliana model (TAIR)

Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental

cues (An et al., 2014) Optimum leaf shape and size are very important for photosynthesis

process that directly effect on seeds of yield and quality also Leaf physiological functions are supported by several specialized cell types, such as paired guard cells in the epidermis for gas exchange, mesophyll cells for photosynthesis, and vascular cells for internal fluid and nutrient transport As a fundamental component of the plant body, the continuous vascular network provides not only mechanical strength but also the key role of transport: the vascular tissue xylem transports water and minerals, and phloem translocates dissolved photoassimilates efficiently Leaf morphogenesis corresponds closely with genetic controls and environmental factors and often used to distinguish different plant species (Tsukaya, 2005) Over the past two decades, the isolation of leaf morphological

mutants of Arabidopsis thaliana has been commonly used to further genetic studies of

leaf development (Scarpella et al., 2010)

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The morphology of multicellular organisms is attributable to mechanisms that regulate the shapes, sizes, and numbers of the constituent cells In higher animals, the body plan is basically established at the stage of gastrulation By contrast, in plants, the body plan is not strictly determined and, throughout the life cycle of the plant, new organs are added to the body via meristems located at the apices of the roots and shoots The fundamental unit of each vegetative shoot system can be considered to consist of a leaf,

an internode, and a lateral bud (Kim et al., 1998) Our current goal is the identification of the various genes that control the development of the leaf, a fundamental component of the shoot

Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype The major strength of this approach is the ability to tap into

a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes (Jander et al., 2002) Genetic mapping of a mutation-defined gene is the first step toward isolating and cloning the corresponding normal gene and ultimately identifying its encoded protein Various techniques are used

to produce a genetic map of a chromosome, which indicates the positions of genes relative to one another along the length of the chromosome In a physical map, the number of nucleotides between known genes is indicated (Lodish et al., 2000)

Mapping a novel mutation to a well-defined chromosomal region is an essential step in the genetic analysis of this mutant, and is also (unless the mutant is tagged) a prerequisite for molecular cloning of the corresponding gene Determining the map position of a gene (as identified by its mutant phenotype) consists in testing linkage with a number of previously mapped markers Once linkage with a specific marker is detected, a refined mapping can be achieved by analysing linkage relations to more markers in that region (Giraudat et al., 2006) By comparing a genetic map and corresponding physical map, the actual physical position of any gene can be determined Whereas reverse genetics strategies seek to identify and select mutations in a known sequence, forward genetics requires the cloning of sequences underlying a particular mutant phenotype

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Map-based cloning is tedious, hampering the quick identification of candidate genes With the unprecedented progress in the sequencing of whole genomes, and perhaps even more with the development of saturating marker technologies, map-based cloning can now be performed so efficiently that, at least for some plant model systems, it has become

feasible to identify some candidate genes within a few months (Janny et al., 2003)

Historically, mapping in Arabidopsis primarily utilised morphological markers such

as mutants with an easily scorable phenotype and a defined map position Typically, the mutant of interest is crossed to another mutant used as phenotypic marker, the resulting F1 double heterozygote is allowed to self, and the segregation of the two phenotypes is analysed in the F2 population The mutation used as marker should of course not interfere with the phenotype of the mutant to be mapped The genetic distance is the number of meiotic recombination events that occur between the two loci in 100 chromosomes To facilitate mapping, tester lines that are recessive for several morphological markers have been constructed and can be ordered from NASC (http://nasc.nott.ac.uk) In other word,

to map a novel mutation that was generated in ecotype A, this mutant is crossed with a wild-type plant of a polymorphic ecotype B, and the F1 progeny is allowed to self The resulting F2 population can then be used to analyse the linkage between the mutation of interest and any DNA marker that distinguishes ecotypes A and B As compared to morphological markers, an additional advantage of molecular markers is that in most cases homozygous and heterozygous individuals can be readily distinguished (Giraudat et al., 2006)

• Map-based cloning in Arabidopsis

In the course of map-based cloning, mutant genes are identified through linkage to a sufficiently small region of the genetic map and subsequent DNA sequencing This

process has become fairly straightforward for Arabidopsis mutations, owing to the

completed genome sequence and the discovery of many thousands of molecular markers Initially, plants with the desired phenotype are identified in populations treated with

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ethylmethanesulfonate (EMS) or other mutagens A typical mutation- mapping project has four phases (Figure 2) First, the mutant line is crossed to another Arabidopsis ecotype, preferentially one with known DNA differences Phenotypic analysis of the F1 and F2 plants is used to determine whether the mutation is dominant or recessive Second, approx

50 homozygous mutant (for recessive mutations) or homozygous wild-type (for dominant mutations) F2 plants are identified based on the phenotype These F2 plants are genotyped with markers on each of the five chromosomes to narrow the position of the mutation about 20cM (1cM~250kb) resolution Additional markers in the 2cMinterval are used to narrow the position as much as possible, ideally to less than 5cM Third, a larger population of 1000 to 2000 plants is generated and genotyped with markers flankig the 5cM or smaller interval containing the mutation The phenotypes are tested for plants that are recombinant in this interval to infer the genotype at the site of the mutation and to determine on which side the mutation the meiotic crossover occurred Additional DNA markers are used to narrow down the position of the crossover and thereby also the site of the mutation Fourth, once the position of the mutation has been narrowed less than 50kbp, the DNA sequences is analyzed to determine the underlying genetic lesion Because the entire Arabidopsis genome sequence is known, it is often possible to pick out candidate genes that might be mutated to cause a given phenotype Therefore, the iterative mapping process can be short- circuited at any stage to sequence likely candidate genes and identify the mutation (Jander., 2006)

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Figure 2: Procedure of Map and Clone Mutation (Jander., 2006)

Left: Phase of map-based cloning project, with possible time link leading to gene identification in 1 year

Right: Schematic of the five pairs of Arabidopsis chromosomes during critical

stages of a sample mapping of a recessive mutation on chromosome 1 in the Col

back-ground, crossed to wild-type Ler

The CURLY LEAF (CLF) gene in Arabidopsis thaliana is required for stable repression of a floral homeotic gene, AGAMOUS in leaves and stems (Kim et al., 1998) The clf mutants had normal roots, hypocotyls, and cotyledons, but the foliage leaves and the stems had reduced dimensions A decrease both in the extent of cell elongation and in the number of cells was evident in the mutant leaves, suggesting that the CLF gene might

be involved in the division and elongation of cells during leaf morphogenesis An analysis

of the development of clf mutant leaves revealed that the period during which cell

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division or cell elongation occurred was of normal duration, while the rates of both cell production and cell elongation were lower than in the wild type Two phases in the elongation of cells were also recognized from this analysis Thus, the CLF gene appears

to affect cell division at an earlier stage and cell elongation throughout the development

of leaf primordial (Kim et al., 1998)

Morphological variation within organisms is integrated and often modular in nature That is to say, the size and shape of traits tend to vary in a coordinated and structured manner across sets of organs or parts of an organism The genetic basis of this morphological integration is largely unknown (Juenger et al., 2005)

In this study, we carried out to identify gene responsible for abnormal leaf phenotype which has the same morphological phenotypes with CLF gene- leaf rolled inside (LRI) by map-based cloning approach using a large mapping population Results

suggested that At2g32460 coding for a member of the R2R3-MYB transcription factor

family is a strong candidate causing leaf rolled inside phenotype Our analysis of the leaves of leaf rolled inside mutant plants has allowed us to identify a gene that appears to regulate both the division and the elongation of cells during leaf development Although this phenotypes were results of ectopically expressed genes, our work do demonstrate the utilities of gain-of-function genetic approaches in uncovering potential regulators of plant development and this genes may be exploited in the future for generating curly leaf traits when desired Development analysis of this mutant may play as new important roles apart from rolling inside leaf, such as slow plant grow and short silique, …

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PART II MATERIALS AND METHODS

1 Plant materials and growth condition

The following plants were use: Arabidopsis Thaliana ecotype Columbia (Col-0); Landsberg erecta (Ler-0) These mutants, which were isolated on an Ler-0 back-ground,

were back-crossed twice to Col wild type to allow analysis of the phenotype in a

comparison with strains with the Ler back-ground The Ler-0 wild type was also analyzed

in order to estimate the effects of the genetic background All the backcrossed mutants were comparable with wild-type Ler-0 All seeds were sown in the soil (soil mixed with vermiculite at the ratio 1:1) and cold-treated at 4ºC in the dark for 3-4 days before

transferring to plant growth room for germination Wild-type and AP-44-1 mutants were

grown under condition of 16 hours light, 23ºC/21ºC of day/night temperatures in a controlled- environment growth room

Figure 3 Arabidopsis growth room

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2 Methods

2.1 DNA extraction

Mature leaves, cauline leaves, flowers, and roots were harvested from flowering

plants of about 3–6 weeks of age DNA was extracted from leaf tissue that collected from 3-week-old seedling using CTAB method following below steps:

1 Collect 0.3g of leaf tissue into 2mL eppendorf tube and adding a bead into

2 Freeze in liquid nitrogen

3 Homogenize the sample in liquid nitrogen using a Tissue Layer machine (QIAGEN,

http://www.quiagen.com)

4 Immediately, add 250µL of extraction buffer (CTAB buffer), vortex briefly and incubate at room temperature for 15-20 minutes

5 Add 250µL of 24:1 solution (chloroform: isoamyl alcohol), mix well

6 Spin for 12 minutes at 12000RPM and transfer aqueous layer to fresh tube (approx 200µL) containing 0,7 transfer volume of Iso-propanol (approx 140µL)

7 Shaking and incubating at room temperature for 5 minutes

8 Mix, then spin for 7 minutes at 12000RPM and discard supernatant

9 Wash DNA pellet with 700µL 70% ethanol, centrifuge at 12000RPM for 5 minutes

10 Pour of ethanol, remove remainder with pipette and leave to dry on clean bench for approx 30 minutes

11 Dissolve pellet in 50µL ADW (autoclaved distilled water)

12 Store DNA solution at -20ºC

A successful DNA extraction will yield a sample with long, non-degraded strands

of DNA which require further preparation according to the sequencing technology to be used

2.2. Genetic analysis using SSLP markers for positional cloning of AP-44-1

To generate mapping population for the positional cloning of 1 locus,

AP-44-1 plants in the Ler-0 background were crossed to wild-type Col-0 to obtain F1 plants and

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self-fertilized Genomic DNA was prepared from leaves of F2 plants according to the

modified Cetyl Trimethyl Ammonium Bromide (CTAB) method (Murray et al., 1980)

PCR-based mapping was conducted using Simple Sequence Length Polymorphic

(SSLP) markers which showed clear polymorphism between Ler-0 and Col-0

Information of existing molecular markers were obtained from public database (TAIR,

http://www.arabidopsis.org), and new markers also designed based on the sequence

polymorphism found between Ler-0 and Col-0 The primer sequences used in the

mapping are shown in Table 1

2.2.1 PCR analysis

Polymerase Chain Reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template DNA polymerase adds nucleotides to the 3’ end of a custom-designed oligonucleotide when it is annealed to a longer template DNA Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains

a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3’ end to generate an extended region of double stranded DNA (Aryal., 2015)

In our work, PCR reactions were conducted in 15µL in 40 cycles A 1µL volume of DNA samples with mixture consisting of 1.5µL e-taq buffer, 0.3µL dNTPs, 0.075µL e-taq polymerase, 0.375µL gene-specific primers and 11.375 autoclaved distilled water was used for PCR amplification at 95ºC for 2 minutes and 40 cycles at 94ºC for 30 seconds, 55ºC for 15 seconds and 72ºC for 15 seconds The final extension was carried out at 72ºC for 5 minutes

2.2.2 Gel electrophoresis

Agarose gel electrophoresis is a method to separate DNA molecules by size This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix

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with an electric field (electrophoresis) Shorter molecules move faster and migrate farther than longer ones The amplified products were loaded into 4% agarose gel with 100 bp ladder marker in 1X TAE buffer (Elpis Bio) and stained with ethidium bromide

2.3 Phenotypic characterization of a novel mutant causing abnormal leaf

Wild-type and AP-44-1 mutants grown in a controlled- environment growth room

after 3 weeks were observed leaf phenotype To have a clear view of the rosette leaves, the inflorescence stems were removed prior to photographing

The second platform consists of an extensive set of measurements from plants grown on soil for a period of ∼2 months When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function

We present here analyses of the development of wild-type plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation

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Table 1: Primer sequences used in this study

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Table 1: Primer sequences used in this study (continued)

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Figure 4.Generation F2 mapping population

Use 689 plants (or 1378 chromosomes) of AP-44-1