Investigating the molecular mechanisms underlying drought

resistance in Potato using genome and transcriptome editing

GUMMI SAINATH

Investigating the molecular mechanisms underlying

drought resistance in Potato using genome and

transcriptome editing

Gummi Sainath

Reg. Nr: 940111289130

Course code: 80439

July 2019

MSc Thesis Plant Breeding

Breeding for growth and development

Potato Group

Wageningen University and Research

Supervisor:

dr. CWB (Christian) Bachem

Examiners:

dr. CWB (Christian) Bachem

dr. Sara Bergonzi

Acknowledgements

I would like to express my gratitude to several people who graciously shared their precious time

to help me in this project. I would like to thank dr. CWB (Christian) Bachem for giving the

opportunity for pursuing the master thesis in Potato group. I thank Lorena Ramirez Gonzales

who was my daily supervisor for teaching me from pipetting to modular golden gate cloning.

With her outstanding weekly planning of experiments, I could complete this thesis with these

novel aims. I would like to thank dr. Sara Bergonzi for her guidance and extremely detailed and

precise comments and questions which have encouraged me to reflect critically in thesis

writing. My thanks also go to Li Shi, MSc who repeatedly advised me on stomata data analysis.

I would also thank MEP (Marian) Oortwijn for her practical questions while conducting

experiments and administrating the consumables for lab work. I would also thank Tammy

Soputro for her literary work in potato genome editing. Finally, my thanks go to corporate

companies Alphabet Inc. (for developing artificial intelligence search engine known as

“Google”).

Summary

Potato crop production exceeds 300 million tons annually and more than a billion people

consume potato tubers on a daily basis. Increasing potato tuber production under drought stress

is a challenge for potato farmers. In potato, achieving balanced canopy development and

eventual tuber yield is the best way for potato plant to avoid drought stress. Tuber yield is

regulated in a remarkably similar way to flowering via circadian rhythm under different light

durations. In previous studies, we have identified StCDF1 gene as a central regulator of

tuberization. An lncRNA StFLORE1 was detected in an opposite orientation covering the

second exon of the StCDF1 coding region.

From previous experiments, it is shown that knocking down StCDF1 increases drought

tolerance by upregulating the expression of StFLORE1. In order to understand the function of

StFLORE1, knockdown and overexpression transgenic plants were engineered. In this study,

the characterisation of StFLORE1 is revealed by investigating these transgenic lines. plants with

over expression of StFLORE1 were drought resistant as StCDF1 knockdown plants.

Subsequently, overexpression of StFLORE1 plants tuberized late by repressing the StCDF1

mRNA expression.

In addition to StCDF1, other four StCDFs genes have been revealed. Abiotic stress experiments

performed in tomato (Solanum lycopersicum) plants, showed high expression of SlCDF4.

Tomato SlCDF4 gene is StCDF2 orthologous gene in potato. In this study we aimed to knockout

the StCDF2 to study its role in abiotic responses. CRISPR/Cas9 (Clustered Regularly

Interspaced Short Palindromic Repeats/ CRISPR associated protein 9) genome-editing system,

was used to target mutation in the two exons of StCDF2. CRISPR/Cas9 construct with four

single guide RNAs (sgRNAs) was successfully developed and transformed in CE3027 diploid

potato.

Key words: Solanum tuberosum, Potato, Lnc-RNA, StCDFs, StFLORE1, drought tolerance,

transpiration, CRISPR/Cas9

Table of Contents

1. Introduction ........................................................................................................................... 1

2. Background ........................................................................................................................... 3

3. Materials and methods .......................................................................................................... 7

3.1. Functional role of StFLORE1 and its regulation in drought tolerance .................................. 7

3.2. CRISPR Cas9 site targeted mutagenesis of StCDF2 using Golden gate cloning system ....... 9

4. Results .................................................................................................................................. 10

4.1. Role of StFLORE1 transcript in drought regulation.............................................................. 10

4.2. CRISPR/Cas9 knockout of StCDF2 gene ................................................................................ 17

5. Discussions .......................................................................................................................... 19

6. Conclusions ......................................................................................................................... 21

Cited literature ......................................................................................................................... 22

Appendices ............................................................................................................................... 26

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1. Introduction

Potato (Solanum tuberosum) originated from Andes mountains of South America. It belongs to

the Solanaceae family. Potato crop production exceeds 300 million tons annually and more than

a billion people consume potato on a daily basis. Quantity yielded from one hectare of potato

crop is two to four times higher compared to cereal crop (CIP - International Potato Center,

2018). Potato can reproduce asexually by vegetative propagation of tubers. Tubers are formed

by swelling of underground stems known as stolons. Despite of asexual reproduction potato

plants can be propagated by berries and seeds resulting from flowering. Tuber is the

reproductive and economic plant organ and widely studied (Friedman et al., 1997).

Potato farmers propagate seed tubers for crop production. Most of the commercially sown seed

tubers are tetraploid. Due to inbreeding depression and heterozygosity in potato, breeders face

severe challenges to attain homozygosity in the cultivars used for multiplication. This is the

reason why potato hybrid breeding is not yet commercialised (Hirsch et al., 2013). Increasing

crop production under water scarcity is a big challenge for potato farmers (Hodges, C. N et al.,

2010). In potato, achieving balanced canopy development and eventual tuber yield is the best

way for potato plant to avoid drought stress (Aliche E. B et al., 2017). The fact that potato is

highly heterozygous, breeding cycle is long, complicated due to polyploidy, self-

incompatibility and vegetative propagation, genome editing is the best way for increasing

drought tolerance. Genome editing is altering DNA on preset sites. At present, CRISPR

(Clustered Regularly Interspaced Short Palindromic Repeats) proteins can be used in targeted

genome editing that makes the breeding cycle shorter.

From functional studies on Arabidopsis flowering and potato tuberization, key genes have

identified for the development of drought tolerant potato varieties. In Arabidopsis under long

days, (12 to 16 hours of day light) CONSTANS (CO) regulation occurs after dawn in the light.

It is essential for the day-length-dependent promotion of flowering and is regulated by the

circadian clock component GIGANTEA (GI). GI interacts with FLAVIN KELCH F BOX1

(FKF1), this interaction stabilizes FKF1 and promotes the degradation of CYCLING DOF

FACTORs (CDFs). In short days (8 to 10 hours of day light), CO regulation degrades the CO

transcriptional repressors, so that CDFs, does not occur under short days because the mRNAs

of the light-dependent CDF degradation protein complex GI–FKF1 only rises around hours

after dawn. They are thus only expressed in darkness, and therefore the proteins do not form an

active complex. The CDF proteins are therefore present to repress CO transcription (Andrés, F

2012).

Under day light StCDFs family plays a crucial role in controlling the photoperiod pathway.

StCDFs repress flowering by downregulating StCO expression in leaves. StCO is degraded in

the dark by StCOP1 and in morning it is activated by photoreceptor StPHYB. However, StCDF1

gene is core regulator for vegetative and reproductive growth. StCDF1 plays crucial role in

tuber formation by a circadian cycle regulation. During long days, StCDF1 is degraded by

circadian complex protein, which leads to StCO proteins activate and as well as the activation

of StSP5G which represses StSP6A, generating early flowering. Whereas during short days,

StCDF1 is no longer degrade and is able to repress StCO protein which inactivates StSP5G,

2

leaving StSP6A active for increasing tubers formation (Abelenda, J et al., 2011, Kloosterman

et al., 2013).

In Arabidopsis, a Long non-coding RNA (LncRNA) named as FLORE1 repress, CDF5

expression and regulates flowering. FLORE1 is responsible for the tight regulation of CDF5

transcripts, acting through Natural Antisense Transcripts (NAT) (Henriques et al., 2017).

AtCDF5 is orthologous gene of StCDF1(table 1.1). Findings from our group revealed that a 1kb

lncRNA named as StFLORE1 was mapped on antisense end of StCDF1 gene. Drought stress

experiments revealed that StCDF1RNAi plants showed good resistance to drought but also

increasing expression in StFLORE1. In order, to study the functional role of StFLORE1, the

sgRNAs (single guide RNAs) were designed to inactivate the promoter region using CRISPR

Associated protein-9 (Cas9) (Ramirez GL et al unpublished).

In addition to CDF1, in potato other four CDFs genes have been revealed, StCDF1 clusters

StCDF2 and StCDF3, and in a separate group StCDF4 and StCDF5 (Kloosterman et al., 2013).

Abiotic stress experiments was performed in tomato plants, and SlCDFs genes expression were

quantified. Drought exposition in leaves and roots induced higher amount of SlCDF2 and

SlCDF4 in leaves whereas in roots, most of the SlCDFs genes showed an increased in gene

expression (Corrales et al., 2014). In the table below the names of the corresponding potato

orthologous is highlighted (table 1.1). It is interesting to note that SlCDF2 is StCDF1

orthologous and SlCDF4 is StCDF2 orthologous respectively in potato (table 1.1). This thesis

highlights the role of StFLORE1 transcript by phenotyping and genotyping of the mutants

generated using CRISPR/Cas9. It also covers the procedure used for CRISPR/Cas9

mutagenesis of StCDF2 gene.

S. tuberosum S.lycopersicum Arabidopsis

StCDF1 PGSC0003DMT400047370 SlCDF2 Solyc05g007880.2.1 CDF5 At1g69570

StCDF2 PGSC0003DMT400064695 SlCDF4 Solyc02g067230.2.1 CDF1 At5g62430

StCDF3 PGSC0003DMT400003359 SlCDF5 Solyc02g088070.3 CDF2 At5g39660

StCDF4 PGSC0003DMT400083080 SlCDF3 Solyc06g069760.2.1 CDF4 At1g26790

StCDF5 PGSC0003DMG400019528 SlCDF1 Solyc03g115940.2 CDF3 At3g47500

Table 1.1. Summarizing the homology of StCDFs genes among Arabidopsis, S.lycopersicum and

S.tuberosum.

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2. Background

Plants have evolved a sophisticated transcriptional control to grow under severe abiotic stress

(Yamaguchi-Shinozaki 2006). One class of transcription factors that plays a crucial role is DOF

(DNA-BINDING ONE ZINC FINGER), whose members are known to be involved in

phytohormone responses, abiotic stress, metabolism regulation, photoperiodic regulation,

hormonal regulation, and other aspects of plant development as shown below in Figure 2.1

(Yanagisawa et al., 2002, Corrales et al., 2014, Wang et al., 2017). They are composed of 200–

400 amino acids and contain a very conserved DNA-binding domain located in the N-terminal

that includes a single C(2)-C (2) type zinc finger motif, which binds to T/AAAAG sequence in

the promoter of target genes (Yanagisawa, 2002; Rueda-Lopez et al., 2008). In contrast, the C-

terminal contains a transcriptional regulation domain with various functions (Pireyre M et al.,

2015).

Figure 2.1: A Dof protein domain structure contains the Dof domain (green), a nuclear localization

signal-NLS (purple) and the transcriptional activation domain (red). A serine-stretch is indicated in

yellow. The cysteine residues for putative coordination of zinc are shown in red letters in the Dof domain

amino acid sequence.

Redundant role of Arabidopsis CDF transcription factors in regulating flowering

Fornara F et al., 2009, studied the roles of CDFs genes in Arabidopsis flowering by generating

multiple mutants. Results from quintuple mutant (gi, cdf1, cdf2 ,cdf3, cdf5), indicate that CDF1,

CDF2, CDF3, CDF5 are largely epistatic to GI in the regulation of CO expression and the

control of flowering time. By combining different mutants and CDF1RNAi line they tested for

genetic redundancy between CDF1, CDF2, CDF3, and CDF5 because of predicted similarities

in their protein products and in their expression profiles. The cdf2 and cdf5 showed early

flowering when compared to wild type plants. In contrast, the single cdf3 mutant showed no

obvious alteration in flowering time. cdf2 cdf5 double mutant showed an additive effect when

compared to cdf2 and cdf5 single mutants. Crossing the CDF1RNAi transgene into the that

CDF2 and CDF5 double mutant produced a triple mutant flowering as early as the that CDF2,

and CDF5 double mutant. Finally, in a quadruple mutant that CDF1, CDF2, CDF3, and CDF5,

flowering was strongly accelerated. Based on above results they concluded that CDF1, CDF2,

CDF3, and CDF5 redundantly repress the floral transition.

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Overexpression of Arabidopsis CDF3 gene led to the accumulation of metabolites and that

contributed to improved response to drought stress

Corrales et al., 2017 showed that the transient expression analysis indicated that CDF3 could

activate directly the expression of an abiotic stress regulated genes likely through the multiple

DOF binding sites localized in its promoter region, suggesting that CDFs might function as

upstream regulators of plant responses to abiotic stress. Metabolomics analyses of CDF3

overexpressing plants indicated the increased amounts of free amino acids such as proline and

sugars observed in 35S:CDF3 plants are factors that would aid the tolerance to low temperature

and drought stresses. Physiological studies have demonstrated that 35S::CDF3 plants exhibited

higher rates of photosynthesis and biomass under osmotic stress conditions than control plants.

CDF3 play a significant role in plant responses and tolerance to changing environmental

conditions. CDF3 is orthologous to StCDF5, and the role of the StCDF5 is not studied yet

(Table 1.1).

Role of three different alleles of the StCDF1 gene in drought response and tuberization

time

Besides the wild type (WT) allele StCDF1.1 that regulates tuberization under short day

conditions, there are two mutated alleles which allow the plants to tuberise under long day

conditions; StCDF1.2 with a 7bp insertion and StCDF1.3 with a 865bp insertion. In both cases

StCDF1.2 and StCDF1.3 (Figure 2.2) the mutated protein lacks the regulatory domain

increasing stability throughout the day (Kloosterman et al., 2013).

Figure 2.2: Structure of the three different alleles of the StCDF1 gene (Kloosterman et al., 2013). This

cartoon shows the three different alleles of StCDF1 with the three different domains (I, II and III). The

figure shows that StCDF1.1 has the third domain, while this is missing in StCDF1.2 and StCDF1.3 due

to an insertion resulting in a truncated and fusion protein (represented by the non-shaded part)

respectively.

Homozygous StCDF1.3 allele negatively affects abiotic stress tolerance and phenotype of

plants were weak. Heterozygote StCDF1.1/StCDF1.3 plants look same as homozygous

StCDF1.3 plants after drought stress. This is an indication that StCDF1.3 has a non-functional

StFLORE1 as there was 865bp transposon insertion (Figure 2.2). StCDF1:OE(Overexpression)

lines have less expression of StFLORE1 (CE3130 plant). StCDF1:RNAi lines have high

expression of StFLORE1. Similar to Arabidopsis FLORE, the expression levels reveal that

StFLORE1 might repress StCDF1. StCDF1 reduces drought tolerance as StCDF1:RNAi plants

are drought tolerant. The assumption from these results reveal that StFLORE1 increases drought

tolerance. Homozygous StCDF1.3:OE and Heterozygote StCDF1.1/StCDF1.3 shows

interference with regulation of stomatal opening. CRISPR/Cas9 transformation of StFLORE1

was done in homozygote diploid CE3027 (1.1/1.1) (Ramirez GL et al unpublished).

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CRISPR/Cas9 for knocking out plant genes

Compared to first generation genome editing reagents that use DNA-protein interactions using

CRISPR proteins is inexpensive and effortless. In creating knock out or knock in of a single

plant gene mutant, CRISPR is more precise with high editing efficiency compared to

Transcription activator-like effector nucleases (TALENs) and Zinc finger Nucleases (ZFNs)

(Zhang K et al., 2017). So, this precise and low-cost editing made CRISPR technology as Holy

Grail in functional plant genome editing. First CRISPR nuclease that was discovered in bacteria

Streptococcus pyogenes (sp). Sp Cas9 was used in eukaryotic genome editing after testing its

editing efficiency in prokaryote genomes (Begemann M et al., 2017). In the process of testing

SpCas9 in eukaryotes, type 2 Cas9 system is effective in achieving successful mutants. First

and most widely used applications of CRISPR- Cas9 is knocking out genes. Nuclease active

Cas9 creates a Double Stranded Break (DSB) at the sgRNA targeted locus. These breaks are

repaired by Homologous recombination (HR) which are used for introduction of new mutations.

When DSB is repaired by Non-homologous End Joining (NHEJ) process, indels are introduced

for production of frame shifts and stop codons, ultimately leading to gene knockout (Tycko, J

et al., 2016). For knockout experiments, indels are introduced within the functional elements

(e.g., cis-element of promoter, mature miRNA sequence). Then using Cas9 and a pair of gRNAs

for deleting a target non-coding fragment. After that indels are introduced they damage the

mRNA translation by deleting a fragment from the coding sequence for mutating the intron-

exon junction for generating a splicing variant (Ding, Y et al., 2016).

Research Aims

Genes involved in regulating drought stress and understanding of their potential role need

tremendous improvements (Anithakumari 2012).

From above reasoning role of DOF genes in abiotic stress responses is dynamic. Following

objectives were investigated in this master thesis project.

1. Characterization of StFLORE1 function in potato

2. What is StCDF2 role in potato? Is it also involved in tuberization? Is it involved in drought

stress response similar to its orthologous SlCDF4 ? Does StCDF2 repress flowering time similar

to its orthologue CDF1.

Hypothesis 1.StFLORE1 expresses in vasculature

2.StCDF1 mRNA is repressed by the StFLORE1 3.StFLORE1 plants are early in tuberization and possess drought tolerance

Outline

CRISPR Cas9 promoter knockout and overexpression of StFLORE1 transcripts in the CE3027

background (diploid genotype with late tuberising diploid genotype carrying homozygous

StCDF1.1 allele; Kloosterman et al., 2013) was used to investigate the involvement of

StFLORE1 in potato drought tolerance. In this thesis the size of promoter deletions were

genotyped. Based on the genotyping, morphological and physiological observations under

6

drought stress was investigated. For functional characterization of other StCDF genes, knockout

of the StCDF2 in CE3027 was carried out using CRISPR/Cas9 modular golden gate cloning

system. Procedure used for experiments are described in the materials and methods. Further the

outcome of the procedure is discussed in the results section. Relevance of these results deduced

from experiments was highlighted in discussion section by connecting it with ongoing research.

7

3. Materials and methods

3.1. Functional role of StFLORE1 and its regulation in drought tolerance

Detection of transcriptomic deletions of StFLORE1 knockout transcripts

Genomic DNA from plant leaves of 12-weeks-old plants were extracted. Genomic DNA of

knock out lines were subjected to PCR analysis using Dream Taq DNA polymerase (Thermo

Scientific) with primers Stflore1fw and Stflore1.rev (Appendix 1). PCR products were analysed

by Agarose gel electrophoresis for screening the allelic variations. PCR products with

homozygous mutations were purified using DNA clean & concentrator kit (Zymoclean). After

measuring the DNA concentration using Nanodrop, the amplified and purified fragments were

cloned into pGEMT Easy vector (Promega) and left overnight at 40C. The cloned mixtures were

used to transform chemically competent E. coli (DH5α). The transformed media was plated on

solid LB with Ampicillin and incubated at 370C for overnight growth. Transformed (white)

colonies were subjected to PCR analysis using Dream Taq DNA polymerase (Thermo

Scientific) with M13 Fw and M13 Rev primers. PCR products were analysed by agarose gel

electrophoresis to confirm the presence of >200bp fragment. The positive ones were grown in

10ml tubes of LB media and kept in the shaker for overnight at 370C. The plasmid DNA was

isolated using Qiagen QIAprep Spin Miniprep kit. Concentration of DNA was measured using

Nanodrop and sent for sequencing with M13 forward and M13 reverse primers.

Spatial localization of pStFLORE1: GUS

Transformed diploid CE3027 plants with pStFLORE1:GUS construct were grown and

multiplied in vitro on MS20 media (Murashige and Skoog). After 3 weeks of growth leaves,

stems and roots were suspended in GUS staining buffer (0.5mM Potassium ferrocyanide,

0.5mM potassium ferricyanide, 0.5M NaHPO4, 0.5 M EDTA, and 1mg/ml X-gluc) for >48

hours at 370C. Plant organs were washed in 80% Acetone for 1 hour and then suspended in 75%

Ethanol for 12 hours. GUS Stained leaf, root and stem samples were observed under light

microscope and pictures were taken. GUS stained stems were sliced using rotatory Microtome

and observed under light microscope at 40X zoom.

Transcript and gene expression analysis of StFLORE1 and StCDF1

To analyse the expression of StCDF1 and StFLORE1, leaves were harvested from 13 week old

plants grown in the greenhouse and stored in liquid nitrogen until RNA extraction. The leaf

material was grinded in liquid nitrogen and from total RNA was extracted using RNeasy mini

kit (Invitrogen, California, USA). Total RNA was treated with DNase I (Takra, Japan) and

cDNA synthesized either by superscript VI reverse-transcriptase (Invitrogen) using gene

specific primers, in the case of StFLORE1 or by iScript cDNA synthesis kit, in the case of

StCDF1. Synthesized cDNA was diluted and stored in -20°C. A real time PCR was carried out

using designed primer pairs StFLORE1.forward, StFLORE1.reverse, StCDF1.forward and

StCDF1.reverse (Appendix 1). Bio-Rad iCyceliQ machine was used for qrt-PCR. Using above

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mentioned primer pairs, SYBR green dye and cDNA were mixed and diluted with water. In real

time PCR, NAC was used as a control. After normalization with Elf gene, StCDF1 gene and

StFLORE1 transcript were determined by Real-time PCR in Overexpression (OE) plants of

StFLORE1 CE3027, the CRISPR/Cas9 StFLORE1 and CE3027 use as a control. Data was

analysed using 2-ΔΔCt method and NAC gene was used as a reference (Livak and Schmittgen

2001). Data obtained was analysed in Microsoft Excel. Later data was visualised in PRISM

GraphPad.

Morphological effects of StFLORE1 knockout and overexpression

Plant height (cm), tuber number, stem diameter(mm2) and number of tillers were recorded at

mature stage after 15 weeks of transplantation. Chlorophyll content was recorded in two

different time points using MultispeQ v1.0 (www.photosynq.org). These two time points were

recorded before and after drought stress. Drought stress was initiated by restricting 50ml/water

for the transgenic lines for 20 days. After, initiating drought stress measurements of Chlorophyll

content were recorded for the second time point. In week 16 after transplantation dry weights

and fresh weights (mg g1) were measured. Pictures of transgenics phenotypes were

photographed using Samsung galaxy S9 plus. Data was recorded in Microsoft excel and

visualized in PRISM GraphPad.

Stomatal physiology in StFLORE1 knockout and overexpressor plants

Leaves of 4 weeks old plants were cut and submerged first into solution (MES/KOH buffer

favours force stomatal opening) for 2 hours. Treated leaves were cut along the midrib into two

halves. Half of one leaf let was submerged again in Abscisic acid (ABA) 10 uM, and the other

half was kept as a control. Sliced leaves were teared along the lower side of the leaf, a colourless

narrow border was visible on sliced edge of the lower epidermis of the leaves. This thin

membrane of the transparent layer is separated using forceps. Separated lower epidermis layer

was pictured under microscope using 40x magnification. Number of stomata per field area were

deduced from the pictures for calculating stomata density for leaf part treated with ABA. Length

and width of the stomatal aperture were also examined for calculating pore aperture ratio. To

indicate the sensitivity of stomata to ABA, pore aperture area between ABA treatment and

MOCK treatment were calculated. Pictures were further analysed in Image J software and

recorded in Microsoft Excel and visualized in PRISM GraphPad.

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3.2. CRISPR Cas9 site targeted mutagenesis of StCDF2 using Golden gate cloning

system

Genomic DNA and plasmid extraction

Using CTAB protocol (Doyle & Doyle, 1987), genomic DNA was extracted from CE3027

leaves. Using QIAprep® Spin Miniprep Kit (Qiagen) plasmid was isolated from bacteria after

cloning.

Vector construction

Four sgRNAs were designed using openly available CRISPR-design web tool

(http://crispor.tefor.net/). To combine four different single guide RNAs (sgRNAs) in one vector

golden gate cloning system was used. Using BsaI-HF enzyme golden gate cloning was done

followed by a ligation reaction for amplification of the four gRNA individually in E. coli

(DH5α). Product from PCR after ligation reaction was analysed on gel for fragment size 164

bp. Then PCR product was purified using QIAquick PCR purification kit and measured

concentration using spectrophotometer (Nanodrop). In the next step four gRNAs were

recombined with pICH47732: NOSp: NPTII-OCST, pICH47742::35Sp:Cas9-NOST, the linker

pICH41780 and cloned in pAGM4723 in a single cut-ligation reaction with BbsI and T4-ligase

in E. coli (DH5α) (Thermo Fisher Scientific) (Weber et al., 2011). The PCR product was

analysed on gel for fragment size >1.5kb. Using level 2 primers (Appendix 1), the purified

product was sent for sanger sequencing. The secondary structures were predicted at 37oc.

(http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3). The vectors were

transformed in Agrobacterium tumafaciens (strain Agl1) using electroporation after proper

sequencing results. Colony PCR was performed using transformed A. tumafaciens using

primers Cas9_6.fwd and RB-F1.rev (Appendix 1). To confirm the presence of construct in E.

coli and A. tumafaciens the sequencing was done using level 2 primers.

Agrobacterium transformation in diploid potato

Diploid S. tuberosum CE3027 was used as background for transformation. Plantlets were

propagated in vitro on MS20. Prior to transformation plantlets were grown for four weeks.

Agrobacterium were propagated on LB liquid media and propagated at 280C for 48hrs.

Overnight culture was centrifuged and sterilized acetosyringone to obtain an OD of 0.6. One

hundred stems (without internodes) of four-week-old CE3027 plantlets were excised in sterile

condition. The 1.5ml PACM liquid (2g/L caseine hydrolysate, 1 mg/L 2.4-D, and 0.5 mg/L

kinetin in MS30 liquid media) was added to excised plantlets which were arranged on sterile

papers over R3B media (MS30 solid media with 2 mg/L NAA and 1 mg/L BAP). After 10

minutes of suspension in PACM liquid, the stems were dispersed on new R3B plate without

filter paper for Agrobacterium inoculation. After Agrobacterium inoculation plant lets were

placed in climate chamber for two days. Inoculated stems were transferred to SIM media (MS20

solid media, 1 mg/L Zeatin riboside, 200 mg/L cefotaxime, 200mg/L vancomycin and 100 mg/L

kanamycin).

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4. Results

4.1. Role of StFLORE1 transcript in drought regulation

Spatial localization of pStFLORE1: GUS

To understand where the regulation takes place we fused the 2kb upstream region of both

StCDF1 gene and of StFLORE1 to a beta-glucuronidase (GUS) gene for histochemical

localisation. Fornara et al., 2009, mentioned that CDF1 expression is localised in the

vasculature tissues, roots and stems. From Henriques et al., 2017, research shows that FLORE

is localised in roots, stems and also vasculature. After performing GUS staining in the

transgenic pStFLORE1-GUS CE3027, signal was detected in vasculature, roots and stems, as

for FLORE (figure 4.1). Differently, pStCDF1: GUS Andigena (WT potato), showed that

StCDF1 is likely to be expressed in stems and leaves but not in roots. The procedure carried out

in transgenic pStFLORE1-GUS CE3027 is highlighted in Appendix 1. From this study, it

indicates that the StFLORE1 is co expresses with StCDF1 in leaves.

Figure 4.1: GUS activity in transgenic pStFLORE1:GUS. The activity is seen in a. roots, b. vascular

bundles, c. xylem vessels and d. phloem tubes.

a b

d c

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Visualization of transcriptomic deletions of StFLORE1 Knock out transcripts

In order to know the function of StFLORE1, using CRISPR/Cas9 a functional knockout of this

1kb promoter region was performed using modular golden gate cloning. According to

Henriques et al 2017, CDF5 encodes a lncRNA named as FLORE. FLORE negatively regulates

AtCDF5 as natural antisense transcript. According to the previous results from RNA-seq data,

an antisense of StCDF1 is transcribed from the second exon of StCDF1 until the intron as show

in Appendix 1. In figure 4.2, the StFLORE1 transcript and four sgRNAs designed at four

different positions of transcript is highlighted. The knock out procedure and sgRNAs designed

for StFLORE1 knockout are highlighted in Appendix 1.

In this study, for screening transgenic candidates, DNA extraction was performed in 12 weeks

old CE30237 plants following by PCR to amplified 1.5kb of the promoter region and cloning

in pGEMT vector in order to sequence. In total 5 transgenic plants, 3 lines showed 700 bp

deletions and 1 line showed a 300 bp deletion in the promoter region of StFLORE1. We also

find 1 line with 86 bp deletion in the 3’UTR of the CDF1, this line was decided to exclude in

case StCDF1 is compromised. From sequencing results, we find that all of our transgenic plants

still have at least one allele with the promoter region intact. Based on this study, we could

understand that the deletions made by Cas9 in StFLORE1 mutants were divided into two groups

i.e. 300bp deletion and 700bp deletions.

Figure 4.2: Schematic diagram of 4 sgRNA that target 1 kb of StFLORE1 promoter. Blue boxes indicate

StCDF1 exons; grey boxes indicate promoters’ region and white boxes indicate UTR region. StFLORE1

promoter region sequenced from CRISPR/Cas9 plants cloned using pGEMT-vector. Wild type sequence

was alignment in the first raw as a control. Scissors images represent the position of the 4 sgRNAs

designed. Based on deletions mapped or number of base pairs deleted in 1kb StFLORE1 promoter the

knockout lines are divided into two section i.e. small deletions (300bp) and large deletions (700bp).

1.kb 700bp

SgRNA4 SgRNA3 SgRNA2 SgRNA1

500bp 300bp 100bp

StFLORE1-22

StFLORE1-35

StFLORE1-92

700bp

Deletions

700bp

Deletions

300bp

Deletions

12

Transcript and gene expression analysis of StFLORE1 and StCDF1

After the genotyping the CRISRP/Cas9 lines, the next question to investigate was to check the

StCDF1 mRNA expression in the StFLORE1 deletion lines. Henriques et al., 2017, showed

that FLORE and CDF5 transcripts cycle are antiphasic. They showed that this is due to high

expression of FLORE leads to low expression CDF5 and vice versa. Overexpression (OE)

plants of StFLORE1 CE3027 were also planted in the greenhouse with the StFLORE1

CRISPR/Cas9 lines and CE3027 wild type controls to investigate the antagonistic expression

and assess drought tolerance.

Figure 4.3a, shows that StFLORE1 transcript expression was high in StFLORE1:OE plants. In

300bp deletions of StFLORE1 CRISPR/Cas9 lines, the StFLORE1 expression is higher than

control and 700bp deletions StFLORE1. In figure 4.3.b, expression of StCDF1 mRNA in

StFLORE1 CRISPR/Cas9 lines is high compared to StCDF1 expression in StFLORE1:OE

plants and control. In figure 4.3, when the StFLORE1 transcript expression in control plants

was low, StCDF1 gene expression was high. From this study it clearly shows that StFLORE1

represses StCDF1. The results show that overexpression of StFLORE1 decreases the StCDF1

mRNA expression levels.

Figure 4.3 Relative expression of StCDF1(b) and StFLORE1(a) in two CRISPR/Cas9 pStFLORE1 lines

and two over-expression (OE) 35S: StFLORE1 lines by triplicate, using CE3027 as a control (star mark

(*) represents a statistically significant difference (p<0.05)).

13

Morphological effects of StFLORE1 under drought conditions

From the gene expression results of StFLORE1 and StCDF1, it is clear that the StFLORE1

would play a major role in regulating the drought response. According to Aliche E. B et al.,

2017, potato crop with balanced plant canopy and eventual tuber yield are essential traits for

plants to escape drought. In figure 4.4a, the vegetative growth (plant fresh weight) in

StFLORE1:OE lines was high compared to the StFLORE1 CRISPR/Cas9 lines and control.

From figure 4.4b the StFLORE1:OE plants were delaying in tuberization compared to control

and StFLORE1 knockout lines. Primarily, the OE of the StFLORE1 decreases the StCDF1

expression (figure 4.3) and this expression levels are responsible for delaying in tuberization

(Abelenda JA et al., 2013) (figure 4.4a&b). In Figure, 4.4c-e average number of tillers, plant

dry weight and stem diameter of OE lines is high compared to other plant lines. This indicates

that the StFLORE1:OE has balanced morphology under drought conditions. In figure 4.4g, the

StFLORE1:OE plants are tolerant and healthy compared to the control and deletion lines.

Ramírez, D. A et al., 2014 highlighted that the chlorophyll increment in potato plants is an

oxidative stress effect which reduces the plant yield. For confirming whether the drought

experiment has shown effect, the chlorophyll content was recorded, figure 4.4f clearly shows

that there is decrease in chlorophyll content in all plants except the 300bp deletion lines. In

figure, 4.4 c, g and h the plant height graph and pictures of plants and tuber yield is recorded

and captured for showing the variation in the CRISPR/Cas9 lines of StFLORE1. The plant

height and tubers of 300bp deletion lines tend to outperform compared to other lines. This is a

sign that DSB induced by multiple sgRNAs and Cas9 leads to large deletions and complex

rearrangements in the genome (Kosicki, M et al., 2018). In this study, phenotyping under

drought conditions revealed that the StFLORE1 positively regulates drought.

f g

14

300bp 700bp

f

d c

e

g

15

Figure 4.4. a) to h) In the graphs and pictures the over expression and knockout StFLORE1 lines were

under drought stress for 20 days, star mark (*) represents a statistically significant difference (p<0.05).

CE3027 was used as control under normal conditions. a. Tuber and plant biomass (kg), b. Average

number of tubers and number of weeks till tuberization, c. Plant height (cm), d. Average number of

tillers, e. stem diameter, f. Chlorophyll content, g&h. pictures of plants and tubers after 15 days of

drought treatment. wt. (CE3027) was used as control under normal conditions.

h

16

Stomatal physiology of StFLORE1

Phenotypic results under drought stress revealed that the StFLORE1 plays a key role in abiotic

response. As drought is a complex trait, in order to gather further indication stomatal anatomy

has been studied. Plants become sensitive to drought when there is increase in Abscisic acid

(ABA), because the hormone affects the stomatal opening which in turn reduces transpiration.

If the greater number of stomata are open, they respond quickly to drought stress (ABA

treatment), by conserving water leading to drought sensitive (Obidiegwu, J. E., 2015). In order

to check the stomata response to ABA, the pore aperture ratio has been calculated. In figure

4.5a, the pore aperture ratio of StFLORE1:OE plants tend to be lower compared to the control

and knock out plants (lower the ratio stomata were closed while responding to the ABA

treatment, which in turn decreases transpiration).

One sample t and Wilcoxon test of pore aperture ratio indicated that the p value is <0.0001, i.e.

data is highly significant, this method has been chosen because the populations cannot be

assumed to be normally distributed. StZEP gene is responsible for expression of zeaxanthin

epoxidase which play a major role in ABA biosynthesis and increase drought and salt stress

tolerance. Over expression of StZEP genes increases ABA levels in plants. ABA is responsible

for stomatal closure, this makes plants to transpire less and overcome drought in potato (P, G.,

Yang et al., 2019). The StZEP (figure 4.5b) gene expression and pore aperture ratio (figure

4.5a) in the StFLORE1:OE lines with low StCDF1 expression (figure 4.3b) reveal that

StFLORE1 regulates drought tolerance. Based on this study it is clear the StFLORE1 plays a

major role with its counter partner StCDF1 in stomatal dynamics. This is interesting to note

because the stomata size was way bigger in 300bp deletion lines compared to the 700bp deletion

lines. In Appendix 2, the pictures and the graphs associated with stomata size, stomata density

are attached.

Figure 4.5: a. Stomatal pore aperture ratio (One sample t and Wilcoxon test of pore aperture ratio indicate

that p value is <0.0001 i.e. data is highly significant); b. Relative expression of StZEP gene in two

CRISPR/Cas9 pStFLORE1 lines and two over-expression StFLORE1OE lines, using CE3027.

a b

17

4.2. CRISPR/Cas9 knockout of StCDF2 gene

Secondary structures of sgRNAs from sanger sequencing of in vitro

After studying the role of StCDF1 and StFLORE1 in regulating drought response, now it’s

necessary to check whether StCDF2 has role in regulating abiotic stress responses. To study

functional regulation of StCDF2, knockout experiment is performed in this study. Formation of

active complex sgRNAs plays a key role in cleavage efficiency of Cas9. Before applying the

sgRNAs in in vivo, pre-screening sequences of in vitro sgRNAs were developed. Liang et al

2016, highlighted that efficiency of sgRNAs can be checked by number of stem loops in the

secondary structure of each primer designed. In figure 4.6, the secondary structures of invitro

sgRNAs reveal that sgRNAs have >3 stem loops. Four sgRNAs complimentary to StCDF2, a

circadian clock TF with unknown function were tested. The sgRNAs were designed at DOF

binding domain targeting at 2 exons of the gene (figure 4.6). Two sgRNAs were designed at 5’

end, after the stop codon and two sgRNAs at 3’ end before the start codon (Appendix 3). These

positions were selected assuming that the whole gene could be deleted on a genome wide scale

or at least >80% knockout efficiency. Sequencing results of 4 sgRNAs from level 2 vector is

highlighted in Appendix 4. In this experiment, the four sgRNAs were cloned into level 2 E.coli

vector.

Figure. 4.6. sgRNA design for StCDF2 knockout. Predicted RNA structures with lowest free energy in

the in vitro experiments (Predicted RNA structures with lowest free energy in the in vitro experiments

(predicted for 22°C)).

18

CRISPR/Cas9 construct in Agrobacterium

The constructed CRISPR/Cas9 cassette in E.coli containing all four sgRNAs was transformed

in Agrobacterium. Transformed colonies were screened using PCR amplification and gel

electrophoresis were performed to check the fragment size. In figure, 4.6 eight colonies (C)

were loaded, out of C1 to C8, C7 and C8 were expected to be 1.5kb fragment size. Sequencing

results showed that C7 and C8 had Cas9 nuclease and kanamycin resistance.

Figure 4.6. PCR product of eight colonies of Agrobacterium transformed with CRISPR/Cas9 cassette

(1.5 kb Cas9 nuclease with other and four sgRNAs)

Agrobacterium mediated transformation in potato

Potato CE3027 plantlets were transformed using C7, Agrobacterium & the CRISPR/Cas9

construct. After transformation the plantlets were grown in R3B media for two days (Figure

4.7a). Later transferred to SIM media for regeneration (Figure 4.7b).

Figure 4.7 Plantlets in a. R3B b. Plantlets growing in SIM media after transformation.

a b

19

5. Discussions

StFLORE1 controls the stomata size and plant height by repressing the StCDF1

Kloosterman et al., 2013, highlighted that StCDF1 is master regulator of the tuberization. The

3’UTR end of StCDF1 locus showed that there is antisense transcription and this locus is named

as StFLORE1 (Ramirez et al., unpublished). To understand the functional regulation of

StFLORE1, overexpression and knockout lines were made. Deletions were mapped in

CRISPR/Cas9 StFLORE1 plants, the transcript size of the area targeted by the most external

sgRNAs was 1kb and two groups were divided based on the size of their deletions. Comparing

the 700bp and 300bp CRISPR/Cas9 StFLORE1 deletion plants possess variation

phenotypically, physiologically and molecularly.

In figure 4.4c, the plant height of 700bp CRISPR/Cas9 StFLORE1 deletion plants were 15cm

short compared to CRISPR/Cas9 StFLORE1 deletions. From figure 4.4h, pictures reveal that

tubers of 300bp deletions mapped have tremendous tuberization but 700bp deletions reveal

have few tubers and late tuberization. Pore aperture ratio and phenotype after drought after

response (Figure 4.1g, 4.5a) of 300bp StFLORE1 deletions reveal they are insensitive to drought

(figure 4.4g). In figure 4.3a&b the gene expression levels of StCDF1 and StFLORE1 were

static.

StCDF1 repress the StCO due to presence of WAAGY sites on StCO promoter (Abelenda et

al., 2016). StFLORE1 transcript possess also WAAGY sites as StCO (Appendix 1). We suspect

that in, 300bp deletions of StFLORE1 promoter CRISPR/Cas9 line (92), WAAGY sites that

were deleted might have a role in interacting with StCDF1 in order to repress StFLORE1, which

means it would be a line with StFLORE1 independent from StCDF1 regulation. Whereas, in

700bp deletions of StFLORE1 CRISPR/Cas9 lines most of the transcript region affecting

StFLORE1 transcription.

20

StFLORE1 increases drought tolerance and decreases tuber yield

Knocking out and over expression of StFLORE1, showed that it has a major role in controlling

the plant development. Previously, RNAi:CDF1 CE3027 plants showed late tuberization but

resistant to drought. In figure 4.4a&b, StFLORE1:OE CE3027 plants also showed similar

phenotype to RNAi:CDF1 CE3027 with higher values of plant fresh weight, better tuber yield

and drought resistant phenotype. Klepper, B et al., 1987 and Ohashi, Y et al., 2006 highlighted

that stem diameter, chlorophyll content and biomass are important traits to understand the

drought tolerance in cultivars. Further, observations from plants with StFLORE1:OE CE3027

like stem diameter, and chlorophyll content (figure 4.4e,f) reveal that they are better tolerant to

drought stress compared to CRISPR/Cas9 StFLORE1 knockouts and controls.

Phenotypically, drought response is shown, in order to deduce more indications from

physiology and molecular perspective. The StZEP (figure 4.5b) gene expression and pore

aperture ratio (figure 4.5a) in the StFLORE1:OE lines with low StCDF1 expression (figure

4.3b) reveal that StFLORE1 regulates drought tolerance. Gibberellin Acid (GA) regulates

tuberization process, but high levels of ABA are antagonistic for GA accumulation. OE:

StFLORE1 plants have increased levels of StZEP and possess drought tolerance with less tuber

yield (figure 3.4h&i) (Abelenda et al., 2013, Xu X et al., 1998, Roumeliotis, E et al., 2012).

StFLORE1 is localised not only in leaves but also phloem, xylem and roots (figure 4.1).

LncRNAs move from source to sink tissues i.e. leaves to root tips (Zhang, Z et al., 2019). This

indicates that StFLORE1 might systematically move through the phloem to roots. Phosphate

starvation usually occurs under drought stress due to high ABA levels. Phosphate starvation

signalling pathway is regulated by many transcription factors, miRNAs and transporters (Baek,

D et al., 2017). LncRNAs might move from source to sink under phosphate starvation

tuberization. Despite the fact that, different localization could mean is that StFLORE1 might

overlap (stem and leaves) but also separate (roots) functions. GUS staining is an indication,

however the reason for not finding the expression in stomata might be that the a 2-kb fragment

upstream of the 3’ transcriptional start site StCDF1 fused might have regulatory regions could

be even further than 2kb or even in introns or downstream .

21

CRISPR technology for investigating the role of StCDFs family

Previous results of our group highlights that expression levels of other StCDFs where

downregulated by StCDF1. In Appendix 2, StCDF1:OE plants reveal that StCDF1 expression

levels downregulates StCDF2 and StCDF3. In order, to understand the function of the genes in

detail, StCDF2 CRISPR/Cas9 knockout is partially performed in the thesis (highlighted in

methods and results sections).

6. Conclusions

1. StFLORE1, regulates tuberization and transpiration by affecting abiotic stress responses in

potato.

2. Functional analysis of StFLORE1 plants show good tolerance to drought.

3. StFLORE1 plants tuberized late by repressing the StCDF1 mRNA expression.

4. Stomatal opening is influenced by StFLORE1 and it enables to plant to escape drought.

Recommendations

1. As the phenotypes of StFLORE1 showed variation, in further experiments using

CRISPR/Cas13 (“SHERLOCK”) would be alternative to CRISPR/Cas9 for RNA editing.

2. CRISPR/Cms1 (“CRISPR 3.0”) with RNP delivery for Gene editing in potato has good

potential in yielding commercial lines without unintended DNA integration.

3. For capturing the images of spatial localization of pStFLORE1: GUS in stomata confocal

microscopy would be an alternative.

4. Using X-ray computer Tomography (XRT / CT): Non-invasive 3D imaging of internal

structures for studying the tuber and stolon development would be better for plants

growing in the pots.

22

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26

Appendices Appendix 1

Sequence Orientation

Stflore1 TGTCCTAAATGAAGGAAAAGCA forward

Stflore1 TTTTGCCCTGCAAGCTAAT reverse

sgRNA BsaI Rev TGTGGTCTCAAGCGTAATGCCAACTTTGTAC

U6pEcoRIF GGAGAAAGGCGGACAGGTAT

Cas9_6.fwd ATCTCCCGAAGATAATGAGCAGAAG

RB-F1.rev GGATAAACCTTTTCACGCCC

ELF_forward GGAGCACAGGAGAAGATGAAGGAG

ELF_reverse CGTTGGTGAATGCGGCAGTAGG

CRISPR.cas 9 TTTTGCCCTGCAAGCTAAT Reverse

35S: pStFLORE CACCGGAGAGTGTAGTAGGATTT Fw

35S: pStFLORE CTACACTCTTCAGATCCCATTTG Rev

pSTFLORE:GUS CACCCTCATAAGTGGAGTAAGCCTTACGA Fw

pSTFLORE:GUS TCACTAATTATGTTGCTCGAATCCT Rev

NAC ATATAGAGCTGGTGATGACT Fw

NAC TCCATGATAGCAGAGACTA Rev

27

CDF1 qpcr GCAGAAATGCAGGGTAAAGC Fw

CDF1 qpcr GACACAAGAACCCGCTATGC Rev

SgRNA2.CDF2 CCATGAGCTCCAGAAGAATC CGG rev

SgRNA1.CDF2 CCGGATTCTTCTGGAGCTCA TGG fw

SgRNA3.CDF2 GTTCCAGTTAGTTACGTAGC TGG fw

SgRNA4.CDF2 TACGTAACTAACTGGAACCC CGG Rev

PDS8340 GAACCCTGTGGTTGGCATGCACATAC For sequencing

level 1

plasmids

PDS8534 TTTGTGATGCTCGTCAGGGG for sequencing

level 2 (NPTII)

PDS8535 CCCGAGAATTATGCAGCATTTT for sequencing

level 2 (Cas9)

PDS8536 TCATCAGTCAATTACGGGGCT for sequencing

level 2 (Cas9)

AL717 GCTTGGCATCAGACAAACCGG For sequencing

level 2

M13F GTAAAACGACGGCCAG for sequencing

cloning

product

CDF2.Cloning GAAGAACTGCCATAACTTT Forward

CDF2.Cloning TCCTCGTCATCATCCAGGT Reverse

28

StFLORE1, CRISPR/Cas9 knockout procedure

In order to do this, we created a CRISPR-Cas9 cassette targeting 1 kb of StFLORE1 promoter

using four RNAs guides, including important DOF-motifs site. To detect mutagenesis, we

amplified by PCR fragments containing guide1 and guide2, also another fragment from guide1

to guide 3 and finally a fragment containing the four guides. From 100 regenerated plants we

obtained 4 transgenic plants from which we amplified 1kb upstream the transcription site of

StFLORE1 to cloned them into pGEMT-vector and 10 colonies from each transgenic plant were

sent to sequence. From our results, in all the plants tested, at least one colony have the

StFLORE1 promoter intact from mutation. From this transgenic, we check that StCDF1 gene

was not mutated by our guides, since we found one transgenic StCDF1 was affected we

excluded from our results.

RNAseq read-mapping of the StCDF1 locus

RNAseq read-mapping of the StCDF1 locus. Red arrows indicate the location of positive strand reads

from the StCDF1 sense transcript and blue arrows indicate antisense RNASeq transcript reads

Method used for GUS staining

For pFLORE:GUS construct, a 2-kb fragment upstream of the 3’ transcriptional start site

StCDF1 was reverse complement and cloned into pENTR TOPO vector to generate the ENTRY

Gateway® clones, which was transferred to pkGWFS7 following the manufacturer’s

instructions. The pFLORE:GUS construct were introduced into into diploid potato CE3027.

The subcellular localization of transgenics lines were used for GUS staining as described

previously (Blazquez, 1997).

WAGY sites in StFLORE promoter and AtFLORE promoter

WAGY sites in StFLORE promoter and AtFLORE promoter by using online New PLACE (a

database for cis-acting Regulatory DNA elements)

https://www.dna.affrc.go.jp/PLACE/?action=newplace. Line with 300bp deletion is positioned

between 1000-700 bp, and lines with 700bp deletions are positioned between 0-700 bp.

StCDF1

StFLORE1

29

Appendix 2

Gene Expression of StCDF1, StCDF2 and StCDF3

Expression of StCDF1, StCDF2 and StCDF3 in p35S:CDF1.2 and wild type plants (CE3027) during

short days conditions.

Stomata pictures

The 92(300bp) line stomata size is big compared to the 22(700bp) lines

30

Stomata density

Stomatal density is an important parameter for estimating plant performance under drought,

higher the stomatal density better transpiration, this helps plants to escape drought (Bertolino

LT et al., 2019). In figure 3.5a, StFLORE1OE resulted in lower stomatal size hence higher

stomatal density compared to StFLORE1OE knockout plants. In figure 3.5a the 300bp

deletion plants have similar values compared to the control

Average stomata size

Mean stomata size of the StFLORE1 deletion lines show that 22(700bp) and 92(300bp). This

shows that StFLORE1 and CDF1 play a major role in controlling the stomata dynamics.

0

50

100

150

200

250

300

350

aba mock aba mock aba mock aba mock aba mock aba mock aba mock

207 224 22 35 53 92 control

35s Cas9 control

31

Appendix 3

StCDF2 alignment 1 130

PGSC0003DM ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA

PGSC0003DM ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA

Consensus ATAACATAAA CAAGGTAATT TCATAAATAA CATAATATCT CAAGGTTAAT ATGTATATTT CGCGTTAAAA ATACTGTGAA ATAAGTCTTC CCACTAGCAA ATAAAAAAGA AAAAACAAGG CGTAAACACA

131 260

PGSC0003DM CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT

PGSC0003DM CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT

Consensus CATATTGGGT GTGTTTGTAT GTGTCGGGGT TGTATGTGTT ATGTGTCAGA TCTGTAGGGA GTAAAATGAG ACCCAGTACC CAAATTCGCC GGATTTGTTT GTTGGATTGT CTTCTCTCTT CTCTCTTTAT

261 390

PGSC0003DM ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT

PGSC0003DM ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT

Consensus ACTTTTTCTC AGCTTAGTAT AGTAACTTGA AAAGGCGCAT ACATACAGTT TGTATAATAT GACAGACCCT GCAATTAAAC TCTTCGGCAG AACAATTCAG TTTCCGGATT CTTCTGGAGC TCATGGAGAT

391 520

PGSC0003DM GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGGTACTT TCACACTTTG TTATAAGAGT ATAATTTGTT GATTTTATTG TTAGTAATTT GTATGCGACT GTTGTTGACT

PGSC0003DM GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGG----- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus GATTCTTTGC CGGAAGACAA TAACGGAGAA GAAGAAGATG AAGAAGCTCA CAAGG..... .......... .......... .......... .......... .......... .......... ..........

521 650

PGSC0003DM TCCTATTGGA CTCTGCTTCG CCTCATTCCG GAGCATTTCC CTAGGGTTTT GAATCAGAGA TAACCAATTG AGTTACTAAG ATTAATCCAG TCAACCAATT GAGCTAAGAA TAATCCCGTC AATCAATTAA

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

651 780

PGSC0003DM GTTACTAAGA TTTCCCTAGA TTAAATTAAT TCCTTTGAAG GAAAGACGGT TAAATTTATC CATGTTTTGA TTAGGTTAGG TAGTCACAAA TTTCTCTTAA TTGGGATTCC TTGTTTTCTT CGTGTTCTCA

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

781 910

PGSC0003DM ACATTTTGTG TTTGGTATGA GGGAGGATAT TTTCATGGAA AATGTGTTTT TTGAAAAACA AGTTGGTTTC TTACTTATTT TTTAGTGGTT TGGTAAGTAA GCAAAAATAT GTTATTTGTA GAGCATTTAT

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

911 1040

PGSC0003DM ATGTAATCAT GCAAAACAAT ATAGGAGTGG TGGGATGGTC AAGGGATAGG GGCGGGGTGC GTTGGTGGGC AGTGAGTGTA CGATAAACAT AGAAGTTCAC TTGTGGAACT TGTTTTCCCT ACTTACGTTA

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1041 1170

PGSC0003DM AGGAAGTCGT TTTCCTCGTT TTTAAGGAAA ATATCCATAA TTTTGTTCAA CCAAACATGG AAAGATTTTC CTCCATACCA AGAACACCAT TAGCTCCTTA TCTCCATGAA TGCTTTTTCA TTTTGAAATG

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1171 1300

PGSC0003DM GAAAGTTATT AATTTTACAA TGGATATAAC TAGGCTCTGT AATCTAATTT TTAAGTGTCA GTGCAAATTA ACATGTTATA GAAGGTTGCT TGTTGTAAAA ATATATTTTA ACTGTCAATT TATAGAAGTT

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1301 1430

PGSC0003DM CAACTATTTT ACAGTTGGAA GGAGATTGAT TAGAGTTCAA TTTTAATCCA GCAACGGAAC AATCATTGTT TAACAACTTT CACATCTCTA TAACTCAGAT TTGTTTTACT TTTCTATGTT GAGTTCTTGT

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1431 1560

PGSC0003DM AATGGGCAAT CTATTTGTGC AAAAAAAGAG TAGTTTAATT GTAAGACTTG CTAAATGAGA AACTTTGACT TTATACCTTT TATTTGTGTA ATGATACCCT TCGGGGTGGC CCAATGGTTT GGGCTTTGGG

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1561 1690

PGSC0003DM CTTGGGACTT CTATGTTGGA GGTCTCAAGT TCGAAACCCC TTGCCAGCGA AAGCAAGGGG TTTGCCTTCT GGGTCGAGCT CATTGCACCA GGCTTGTCTA GTGCGGGTTA CCTCTTCTAT GTAGTTTGCG

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1691 1820

PGSC0003DM AGCTATTGCA TAGGAGCGGG AGTTTTACCC TGTGAGTACC CAAAGGGTAG CGGTTGCGGA TTTCCCTTGT CATAAAAAAA TAATAATTTG TGTAATGATA TGAATACTCT AATTTCCACA TTCAGTTGGA

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1821 1950

PGSC0003DM TGCAGATACT TATTTTGTTA TGCACTCGTA TCCTTAATTT GACATGGTTA GTCCCTTCAT GATATATAAT CTACTATCAG TTTTGTTTTA CCTTTGGCCT CTCAATATTT CTCTCTACAA CTTTCACATG

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

1951 2080

PGSC0003DM CTTCTTACCG ACGGAAAAGC TTGGTTGTCT CAATAGTGAT AAAAGACTAG TGAAATCTCG TTGAACCTTT TCTTTTGAGA TCTCCCTAAG TTTCTAATAA TTTCATTAGT TTAAAAGAAA ATTTTACTGA

PGSC0003DM ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------

Consensus .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

2081 2210

PGSC0003DM CTCTATTTAC TACTCTGTGA AATGCATCAC GCGTACAGGA TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG

PGSC0003DM ---------- ---------- ---------- ---------A TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG

Consensus .......... .......... .......... .........A TGATTTTGGA GGAAACCTGG ATGATGACGA GGACGAGATG GAAATTTTGA CTGGCAAGGT GTTGCAGGAT CAGAATTCAG AACCAACTAG

2211 2340

PGSC0003DM AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA

PGSC0003DM AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA

Consensus AACTGATAGT ATGAAGGAGC CACCTGTTGA TAACGACTGT TCAACAAGAC CTTCAAAAAG TGAAGAAGAG CAAGGAGAAG CAAGTAATTC GCAAGAGAAA ATCCTCAAAA AGCCAGACAA GATAATTCCA

2341 2470

PGSC0003DM TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG

PGSC0003DM TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG

Consensus TGTCCCCGGT GCAACAGCAT GGAAACCAAA TTTTGTTATT TCAACAATTA CAATGTGAAT CAGCCTAGAC ACTTCTGCAA GAGTTGCCAG AGATATTGGA CAGCTGGTGG GACCATGAGG AATGTGCCTG

2471 2600

PGSC0003DM TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC

PGSC0003DM TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC

Consensus TAGGTGCTGG TCGTCGGAAA AACAAGAACT CAATTCCACA TTACCGTCAA ATATCTGTCT CTGAAACACT TTCGAATGCA CAAACAGCTT ATCCAAATGG AGTACAACAA CCTATTCTTG CATTTGGCTC

2601 2730

PGSC0003DM CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT

PGSC0003DM CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT

Consensus CCCTACACCA CTCTGTGAAT CAATGGCTTC AGTTTTGAAT ATTGCTGACA AAACAATGCA TAATTGCTCA CAAAATGGGT TCCATAAACC ACAAGAGCCC GGGGTTCCAG TTAGTTACGT AGCTGGAGAT

2731 2860

PGSC0003DM AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG

PGSC0003DM AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG

Consensus AATGGAGATG ACCATTCCAG AAGATCCTCA GTGACTTCTG CAAATTCAGA GGATGAGGTT AACAAAACTG TACCAGACCT GCTAAAGAAG AACTGCCATA ACTTTCCACC TTACATGACT TGCTATCCCG

2861 2990

PGSC0003DM GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG

PGSC0003DM GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG

Consensus GGGCTCCTTG GCCATATCCA TGCAGTCCTG TCCCGTGGAA CTCTGCAGTC CCTCCTCCTG GTTATTGCCC TCCTGGTTTT CCTATGCCGT TTTACCCTGC AGCTTCTTAT TGGGGTTATA CTGTAGCAGG

2991 3120

PGSC0003DM TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC

PGSC0003DM TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC

Consensus TTCTTGGAAT GTTCCTTGGA TGCCCCCAGC TACTGTTTCC CTAATCCAAA CACCTACGAC TTCTGGTCCT AATTCTCCCA CTCTGGGGAA ACACTCAAGG GATGAAAATG TACAAAAACC GCTGAGTAGC

3121 3250

PGSC0003DM ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG

PGSC0003DM ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG

Consensus ATGGAAGAAC CTTCAAACGA GAGTAATCCT GAGAAGTGCC TCTGGGTCCC AAAAACTCTC CGAATTGATG ATCCAGGAGA GGCTGCAAAG AGTTCTATAT GGGCGACATT GGGAATAAAA CATGATACCG

3251 3380

PGSC0003DM TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA

PGSC0003DM TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA

Consensus TTGATTCAGT TGGTGGAAGT CCTTTCAGTG CTTTTCAGCC GAAGAATGAT GACAACAATA GGGTTTCAGA AAACTCTACT GTATTACAAG CAAACCCAGC AGCGTTGTCT CGGTCAGTAA ATTTCAATGA

3381 3510

PGSC0003DM GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA

PGSC0003DM GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA

Consensus GAGCTTATAA GCAGTTGTGA AATCATTGAG AATGTTAAAT ATCAGACAGT GTTGGCAAGG CCAGGCAGGA ATTCTTCAGC TCAGTAACTC TTAACTACTG TTACTTGTCG GCATGTTTTG GTTAGAGCGA

3511 3640

PGSC0003DM GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT

PGSC0003DM GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT

Consensus GTTCTTCACA CAGTAGTGTT GGCTAAACTG TAGGATAGGT TCCTTCAATT TCATTCAGAT CGGAGTAGAT CAATATCTTG CAGTTAATCT CTCCTTCTTG TGTGAAAAAG AAAGGTCCTT TTTGGTTGAT

3641 3770

PGSC0003DM AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA

PGSC0003DM AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA

Consensus AGTGTTTTCT TTTTTCTCTT TTTCTTTTCC ATTTTCGTAG TTGATGTATA TACTTGTATT TAAGAAAGTG AGAGGTAAAC TAGGCAGTTT GTGTACCAAA TTTGCCGTGT GATGAATAAG TTGAAGGTAA

3771

PGSC0003DM AATTAAA

PGSC0003DM AATTAAA

Consensus AATTAAA

32

Appendix 4

Level 2 sequencing results

>190627-004_K23_1D5CPAA025_F12.ab1.guides 1297

GCGGTGAGATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCC

AAGAAGAAGAGGAAGGTGTGAGCTTGTCAAGCAGATCGTTCAAACATTTG

GCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTA

TCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAA

TGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATT

ATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATA

AATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGACGCTACTAGAAT

TCGAGCTCGGAGTGATCAAAAGTCCCACATCGATCAGGTGATATATAGCA

GCTTAGTTTATATAATGATAGAGTCGACATAGCGATTGCCATGAGCTCCA

GAAGAATCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCA

GCTTTCTTGTACAAAGTTGGCATTACGCTTTACGAATTCCCATGGGGAGT

GATCAAAAGTCCCACATCGATCAGGTGATATATAGCAGCTTAGTTTATAT

AATGATAGAGTCGACATAGCGATTGCCGGATTCTTCTGGAGCTCAGTTTT

AGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA

AAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACA

AAGTTGGCATTACGCTCAGAGAATTCGCATGCGGAGTGATCAAAAGTCCC

ACATCGATCAGGTGATATATAGCAGCTTAGTTTATATAATGATAGAGTCG

ACATAGCGATTGGTTCCAGTTAGTTACGTAGCGTTTTAGAGCTAGAAATA

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGGGGCACCGA

GTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTAC

GCTTGGGGAATTCCTCGAAGGAGTGATCAAAAGTCCCAATCGATCAGGTG

AATTTAGCAGCTTAGTTTATAAAGGAAAAAGTCCAAAACCGATTGGCGAA

ACAACTGAACCCCTTTTAAACAAAAAAACAAAATTAAAAAAGGGTTTCCC

TTTCATTTAAAAAGGGGCCCAAGGGGGTTTTTTTTTAAACCCCTTTTTTT

TTTGAAAAATGGGTTATCCCAGGAGAAAGAGTCCTTTTTCCCCGGGA