Exploring the role of the two GIGANTEA genes in the life cycle length and tuberisation of the potato cultivar Désirée

Exploring the role of the two GIGANTEA genes in the life cycle length and tuberisation of the potato cultivar Désirée | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the role of the two GIGANTEA genes in the life cycle length and tuberisation of the potato cultivar Désirée Flóra Karsai-Rektenwald, Khongorzul Odgerel, Zoltán Gábor Tóth, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8542741/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Earliness of tuberisation is an important agronomic trait. It was demonstrated earlier that GIGANTEA (GI), a plant-specific nuclear protein that regulates multiple processes, is indirectly involved in tuberisation in a diploid potato. Commercial potatoes, including the cultivar Désirée, are tetraploids and carry two copies of GI genes, designated GI.04 and GI.12 . The aim of our study was to explore the role of the two GI genes in Désirée in relation to tuberisation. Results To obtain information on GI.04 and GI.12 functions in Désirée, mutations were introduced into the two genes individually and simultaneously using the CRISPR/Cas9 system. Two different segments of the genes were targeted by gRNAs. PCR was used for mutant identification. Three mutants from each mutagenesis were selected, and the mutations were localised at the DNA sequence level. The phenotype and tuberisation of the plants were tested by growing the plants in pots in a greenhouse. The individual mutations affecting all four copies of the genes, in general, reduced plant size. Plants of one GI.04 mutant line and two GI.12 mutant lines with truncated proteins and deletions in the 816–869 and 834–863 amino acid (a.a.) regions, respectively, were shorter and remained green for a longer time than Désirée. GI.04 and GI.12 mutants with truncation or deletion in the 567–632 a.a. and 618–694 a.a. regions, respectively, differ in phenotype; one GI.04 mutant had longer, whereas all three GI.12 mutants and the double mutants had shorter life cycles. However, only one of the GI.12 mutants and one of the double mutants tuberised earlier than Désirée. The tuber yield of the double mutant with the shortest life time was lower than that of Désirée. Conclusions Both GI genes of Désirée influence the development and life cycle length of plants. The influence of GI.12 is more pronounced than the influence of GI.04. In conjunction with the shortened lifetime, the onset of tuberisation occurs earlier. CRISPR/Cas9 Mutation mapping Plant development Solanum tuberosum Tuber skin colour Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Earliness of tuberisation is an important agronomic trait. Early potato varieties withstand various biotic and abiotic stresses and complete their life cycle before stress becomes a serious constraint and, therefore, are more profitable for growers. Tuberisation is a complex developmental process influenced by environmental factors. The molecular mechanisms that control tuber formation have been investigated in detail over the past few decades in S. tuberosum ssp. andigena and reported that the stolon-to-tuber transition is governed by mobile RNAs and proteins, phytohormones, a plethora of small RNAs and their targets. In the wild andigena subspecies, tuberisation is strictly dependent on short days (SDs). This control is mediated by the potato SELF-PRUNING 6A (SP6A) protein, which acts as the main phloem-transported tuberigen signal. SP6A is negatively controlled by CONSTANS (CO), whereas CO transcription is increased through proteolytic degradation of CYCLING DOF FACTOR 1 (CDF1), which is mediated by the FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1/GIGANTEA (FKF1/GI) complex [ 1 ]. Thus, GI is indirectly involved in light-dependent tuberisation. GI encodes a plant-specific protein with pleiotropic functions, including flowering, photoperiodic response, phytochrome B signalling, circadian clock regulation, and carbohydrate metabolism. GI interacts with hormonal signalling pathways, balances stress responses, promotes growth and optimises plant productivity [ 2 ]. In Arabidopsis , GI is comprised of 1173 amino acids. It has a nuclear localisation signal (NLS) and predominantly resides in the nucleus [ 3 ]. However, it is also localised in the cytosol, where it stabilises ZEITLUPE (ZTL), an F-box protein that binds to GI through its light-oxygen voltage-sensing (LOV) blue-light-absorbing domain [ 4 ]. In addition to ZTL, two other LOV domain containing blue light receptors, LKP2 and FKF1, also bind to the middle region of GI (residues 563–789) to prevent the degradation of TOC1 and PRR5, thereby contributing to the correct oscillation of the plant circadian clock, whereas GI binding to FKF1 facilitates the degradation of CDF flowering repressors [ 5 ]. The E3 ubiquitin-ligase COP1 also interacts with GI through the clock-associated protein ELF3, which acts as a substrate adaptor protein to accelerate GI destabilisation and degradation [ 6 ]. Although the function of GI in tuberisation has been demonstrated in S. tuberosum ssp. andigena , the current commercial potato varieties from Europe and America have genetic closeness to ssp. tuberosum , compared with the tubers of ssp. andigena [ 7 ]. The introduction of potato into temperate latitudes was accompanied by alterations in its photoperiodic requirements, i.e., tuberisation became day-length independent [ 8 ]. Thus, even though the role of the GI in tuberisation is well demonstrated in the SD-tuberising ssp. andigena , its role in tuberisation in tetraploid commercial cultivars, such as cv. Désirée, is still elusive. Previously, we demonstrated that there are two copies of GI genes in potato homologous to Arabidopsis GI located on chromosomes 4 and 12 ( GI.04 and GI.12 ) with different regulatory elements in their promoter region; however, with 84% identity at the transcriptional level[ 9 ]. The antisense repression of GI.04 by approximately 50% affected the transcription of the genes involved in the circadian clock, flowering, starch synthesis, and stress responses in the leaves of Désirée plants; however, it did not influence tuber formation or yield but did cause a reduction in tuber colour [ 10 ]. To further investigate the function of the GI genes in the potato cv. Désirée, targeted mutations were introduced into GI.04 and GI.12 and simultaneously into both genes via the CRIPR/Cas9 gene editing system. Here, we report that both GI genes influence the development and life cycle length of Désirée plants and, in certain cases, mutations in GI.12 can lead not only to plant size reduction and shortening of the vegetation period but also to earlier onset of tuberisation. Methods Plant material, growing conditions and phenotyping The potato ( Solanum tuberosum L.) cultivar Désirée from Fritz Lange KG (Bad Schwartau, Germany), cultivated in tissue culture at the Max Planck Institute of Molecular Plant Physiology (Golm, Germany), was used as the starting material and propagated in vitro from stem segments in the rooting medium RM (MS without vitamins [ 11 ]) containing 2% (w/v) sucrose and 0.8% agar in 40-ml tubes closed with paper plugs at 24°C under a 16 h/8 h day/night cycle and a light intensity of 75 µmol m − 2 s − 1 . For plant phenotyping, 4-week-old in vitro plantlets were transferred into 18x14 cm pots filled with sterile Tabaksubstrat soil A200 (Stender GmbH, Schermbeck, Germany) and grown further under greenhouse conditions. From the middle of November until the middle of March, the ambient light conditions were supplemented with artificial lighting by sodium lamps to provide a minimum of 12 h light conditions. The temperature regime varied from 18 to 24°C. The soil humidity reached approximately 80%, which was provided by regular watering. Pesticides and fungicides were regularly applied for pest and fungal pathogen control. The morphological characteristics of the plants were observed visually and documented via photos. The earliness of tuberisation was assessed by carefully tipping the plants out of the pots and counting the number of tubers seven weeks after planting. After counting, the plants were replanted into the pots and grown further in the greenhouse. At the end of the vegetation period, the tubers were harvested, weighed, and peeled for anthocyanin measurement. Cloning and sequencing the coding sequences of Désirée GI.04 and GI.12 RNA was isolated from the leaves of in vitro- grown Désirée plants at 4 h after the beginning of the light period using the method of [ 12 ], and cDNA was synthesised with the Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Fisher Scientific, Waltham, MA, USA). The GI.04 and GI.12 coding sequences (CDSs) were obtained with the help of the GI04 attB1–GI04 attB2 and GI12 attB1–GI12 attB2 primer pairs (Table S1 ), respectively, in PCR reactions using the CloneAmp HiFi PCR Premix (Takara Bio Inc., Shiga, Japan) and following the manufacturer’s instructions. The PCR products were introduced into the pDONR™221 vector with Gateway™ cloning (Thermo Fisher Scientific, Waltham, MA, USA) and Sanger sequenced at Eurofins BIOMI Ltd. (Gödöllő, Hungary) in fragments generated with the M13, GI04 640–2070, GI04 2000–3400, GI12 630–2100 and GI12 2030–3440 forward‒reverse primer pairs (Table S1 ). Targeted mutagenesis of GI.04 and GI.12 in Désirée Targeted mutagenesis was carried out using pCBC-DT1T2 as gRNA template and the Cas9 delivery vector pKSE401 [ 13 ]. Gene-specific gRNAs were designed using the CRISPOR tool ( http://crispor.tefor.net ), and the oligos were synthesised by Integrated DNA Technologies (Coralville, Iowa, USA). For the gRNA sequences, see Table S2 . The Agrobacterium tumefaciens LBA4404-mediated leaf transformation was performed according to the protocol published in [ 14 ]. After transformation, the Agrobacterium was eliminated by adding 500 mg l − 1 cefotaxime to the medium. Kanamycin at a concentration of 50 mg l − 1 was used for the regeneration, rooting and propagation of the transgenic plants in vitro . Detection and mapping of the mutations in transgenic Désirée plants Genomic DNA from in vitro -grown plants was isolated using the method of Shure et al. [ 15 ]. Selection of mutated plants was performed by PCR using primer pairs corresponding to gRNAs, and the amplified fragments were detected on agarose gels. The lack of or lower amounts of PCR products than those obtained from Désirée were supported by qPCR using PowerTrack™ SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) in a LightCycler-96 (Roche, Basel, Switzerland) following the manufacturer’s instructions. ACTIN was used as a reference gene [ 16 ]. For the localisation of mutations, gene-specific PCR primers (Table S1 ) surrounding the region restricted by gRNA sequences were designed using the Primer-BLAST tool ( https://www.ncbi.nlm.nih.gov/tools/primer-blast ). The PCR products amplified from the genomic DNA were subsequently cloned using the pGEM-T-Easy (Promega, Madison, WI, USA), CloneJet or TOPO Cloning Kit for Sequencing (Thermo Fisher Scientific, Waltham, MA, USA) and transformed into Escherichia coli DH5α competent cells. Transformed cells were selected on antibiotic-containing LB plates, and plasmid DNA was isolated. For basic molecular techniques, e.g., plasmid DNA isolation, transformation of E. coli , agarose gel electrophoresis, and PCR, the instructions of [ 17 ] were followed. Genomic PCR fragments of the positive clones were sequenced by Sanger sequencing at Eurofins BIOMI Ltd. (Gödöllő, Hungary). DNA sequences of selected clones are presented in the Supplementary material 2. EMBOSS Transeq ( https://www.ebi.ac.uk/jdispatcher/st/emboss_transeq ) was used to translate DNA sequences to peptide sequences. Sequence comparisons were performed with Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ). Anthocyanin content of tuber skins A simplified method of [ 18 ] was applied for the peeled skins of freshly harvested tubers as described in [ 10 ]. Statistical analysis The significance of differences ( p < 0.05) between two groups of data was detected by one-way ANOVA with a post hoc Tukey’s HSD test. Results Sequence comparison of GI.04 and GI.12 of Désirée The coding sequences of GI.04 and GI.12 were sequenced and first compared to the corresponding S. tuberosum group Phureja sequences (https://spuddb.uga.edu/). This comparison revealed that GI.04 of Désirée possesses a 21-bp deletion compared with the corresponding Phureja CDS, resulting in the lack of amino acids (a.a.) from 31 to 38 of Phureja. All the other nucleotides, with the exception of a G/T base pair change, were identical to the Phureja CDS. GI.12 of Désirée was even more similar to the corresponding Phureja sequence than GI.04 , with changes of only six bps that resulted in the alteration of three a.a. in the protein sequence. The existence of the interaction site with the LOV domain proteins and the presence of the NLS in the GI proteins of Désirée were tested via comparison with the Arabidopsis GI protein. Both Désirée GI proteins were slightly shorter than the Arabidopsis GI protein as GI.04 and GI.12 were composed of 1166 and 1171 a.a., respectively, versus the 1173 a.a. of the Arabidopsis GI protein. The LOV domain binding sites were highly conserved in both Désirée GI proteins. In contrast, the a.a. sequences of the NLS site varied. The four NLSs of Arabidopsis have a consensus sequence of K (K/R) X (K/R) [3], whereas in GI.04, only two sites correspond to it. GI.12 was even more diverse, with no sequences similar to the Arabidopsis NLS consensus sequence (Fig. S1). Generation and characterisation of GI.04 Désirée mutants To evaluate the function of GI.04 in the potato cv. Désirée, gene-specific mutants were generated using the CRISPR/Cas9 genome editing system. Since GI.04 and GI.12 are 84% identical at the transcript level, in the first experiment, the gRNAs were designed near the 3' end of the GI.04 gene, where the difference between GI.04 and GI.12 was the greatest. A single Cas9 construct containing two gRNAs (Table S2; gRNA1 and gRNA2) to increase the possibility of obtaining large deletions and disrupting gene structure and function was constructed and used for Désirée transformation via A. tumefaciens . Transgenic plants were obtained by antibiotic selection and the genomic DNA of 82 plants was tested for mutations with PCR using a primer pair surrounding the gRNA1–gRNA2 region. In this way, 29 mutants carrying large deletions visible on agarose gels were identified. Considering the size and number of fragments with large deletions, three lines were selected for further studies. On the basis of the lack of PCR products and the results of the qPCR assay using a primer pair corresponding to the two gRNA sequences (Fig. S2A and B), all three mutants, designated eGI.04/1, eGI.04/2 and eGI.04/3, were expected to carry mutations in all four copies of GI.04 in the tetraploid Désirée. Localisation of mutations in the three selected eGI.04 mutants was carried out by cloning the mutated PCR fragments generated with different primer combinations into a cloning vector from which they were reamplified, Sanger sequenced and compared to the sequence of the corresponding Désirée fragment. Large deletions extending even to 499 bp were detected in each mutant. Furthermore, there was a duplicated segment in eGI.04/2, and only two types of mutations were detected, whereas eGI.04/3 was a line with eight different mutated fragments (Fig. S2C). The effects of mutations on GI.04 at the protein level were investigated by translating the nucleotide sequences to a.a. sequences and comparing them to the Désirée sequence. With one exception, the deletions caused frame shifts and the generation of an early stop codon, resulting in C-terminal-truncated proteins. The only exception was a copy of GI.04 in the eGI.04/1 mutant, in which after a 92-a.a. deletion, the original sequence was retained. Nevertheless, none of the deletions extended to the LOV binding domain or the putative NLS region (Fig. 1A and B). The morphology and tuberisation of the eGI.04 mutants were tested after in vitro propagation under greenhouse conditions in pots. The mutants, especially eGI.04/1, were visibly shorter than Désirée, but no significant difference in the time of tuberisation or tuber yield was detected (Fig. 2). Since the lack of changes in tuberisation might have been explained by the integrity of the LOV binding domain and NLS, a new set of mutants with gRNAs targeting this functionally important part of GI.04 were generated (Table S2; gRNA3 and gRNA4). On the basis of PCR and qPCR tests using the gRNA oligos as primers, three null mutants, mGI.04/1, mGI.04/2 and mGI.04/3, were obtained from 148 tested transgenic plants (Fig. S3A and B). The localisation of the mutations revealed that all three mutants carried deletions only at the position of gRNA4 (Fig. S3C). However, the integrity of the LOV and NLS domains was destroyed by these mutations (Fig. 1A and C). Out of the three mutants, only mGI.04/1 differed from Désirée in phenotype, as it was shorter and remained green, whereas Désirée and the other two mutants already presented signs of senescence at the end of the vegetation period. Nevertheless, in terms of tuberisation, there was no significant difference between the mutants and the Désirée plants (Fig. 3). Mutations at the protein level in combination with the phenotype of the GI.04 mutant plants are shown in Fig. S4. Generation and characterisation of GI.12 Désirée mutants In the first experiment, as in the case of GI.04 , gRNAs (Table S2; gRNA5 and gRNA6) were designed for that part of GI.12 , which showed the least similarity to GI.04 . The genomic DNA of 82 transgenic plants was tested for the presence of large deletions using a primer pair surrounding the gRNA5–gRNA6 region. Eighteen plants with visibly shorter PCR fragments detected via agarose gel electrophoresis were obtained, three of which were selected and propagated in vitro for further studies. Using the oligos corresponding to the two gRNAs as primers in the PCR and qPCR assays, two mutants, eGI.12/1 and eGI.12/2, appeared to be null mutants, whereas in eGI.12/3, a PCR product was detected either on agarose gel or by qPCR, albeit with lower intensity than in Désirée (Fig. S5A and B). The localisation of the mutations was carried out in the same way as that for the GI.04 mutants. Large deletions extending to both gRNA sequences were found in eGI.12/1, whereas only gRNA5 and the region surrounding it were missing in eGI.12/2. Sequencing the PCR fragments obtained from eGI.12/3 resulted in the identification of two large deletions and a lack of 2‒3 bps in the middle of the sequence corresponding to gRNA5 in two copies of GI.12 . This finding explains the production of a low amount of PCR fragment with the gRNA primers (Fig. S5C). Translation of the nucleotide acid sequences to a.a. sequences revealed that eGI.12/1 and eGI.12/2 were similar to each other, as both carried two truncated proteins and two copies of GI.12 with deletions of 48 and 54 a.a., respectively, whereas three truncated proteins and one GI.12 copy with a deletion of 53 a.a. were present in eGI.12/3. Like in the case of GI.04, which was targeted by gRNAs at a similar position, none of the mutations extended to the LOV domain or the putative NLS region (Fig. 4A and B). Greenhouse tests of eGI.12 mutants revealed similar morphological changes in eGI.12/1 and eGI.12/2 as in eGI.04/1. These two mutants were shorter and presented signs of senescence later than Désirée. eGI.12/3 was similar to Désirée, which was unexpected, as this mutant had three copies of truncated proteins, whereas the others had only two. The initiation of tuberisation was not altered in any of the mutants, but the yield of eGI.12/2 was significantly lower than that of Désirée in three consecutive plant tests each with 8 plants/line (Fig. 5). The LOV binding domain and NLS region in GI.12 were targeted with the gRNA pair gRNA7 and gRNA8. These gRNAs were very effective. In the PCR and qPCR tests, when the corresponding oligos were used as primers, 14 null mutants were found among the 22 mutants examined. Mutations were mapped in three mutants designated mGI.12/1, mGI12/2 and mGI12/3 (Fig. S6). DNA sequence analysis detected only very short deletions extending only to 1–6 nucleotides in the sequences corresponding to the gRNAs in mGI.12/1 and in three GI.12 copies of the other two mutants. However, these short deletions were at the 3’ end of the genomic DNA sequence corresponding to gRNA7, which could explain the lack of PCR products obtained via the use of gRNA7 and gRNA8 as primers. The fourth copy of GI.12 in mGI12/2 had a large deletion starting in gRNA8 and going 3’ toward the end of the GI.12 coding sequence, whereas there was a deletion including gRNA7 in one copy of GI.12 in mGI.12/3. Deletions detected at the nucleic acid level resulted in four copies of the truncated GI.12 protein in mGI.12/1 and three copies of the truncated protein in the mGI.12/2 and mGI.12/3 mutants. In these two mutants, however, one GI.12 copy was almost unaffected, with only one a.a. change and one a.a. deletion (Fig. 4C). The plants of all three mGI.12 mutant lines were shorter than those of Désirée and presented early senescence, which was most pronounced in mGI.12/1. This mutant had a greater number of tubers than did Désirée at the early stage of tuberisation and, despite its short lifetime, had a yield similar to that of Désirée (Fig. 6). Mutations at the protein level in combination with the phenotype of the GI.12 mutant plants are shown in Fig. S7. Generation and characterisation of GI.04-GI.12 double Désirée mutants To obtain Désirée mutants affected by both GI genes, cotransformation with A. tumefaciens strains carrying the Cas9 construct with gRNA3 and gRNA4 targeting the LOV–NLS domain of GI.04 and the Cas9 construct with gRNA7 and gRNA8 targeting the LOV–NLS domain of GI.12 was performed. The two strains were mixed in equal amounts before being used for Désirée infection. Transgenic lines obtained by antibiotic selection were assessed for deletions in both GI.04 and GI.12 using the gRNA oligos as primers. On the basis of this test, no null mutant for both GI genes was present among the 216 transgenic plants tested. Considering the intensity and size of the PCR fragments generated via the gRNA primers and other primer pair combinations, three mutants, mGI.412/1, mGI.412/2 and mGI.412/3, were selected for mutation mapping. Although with the gRNA3–gRNA4 primer pair, a PCR fragment was detected in mGI.412/1, the DNA sequence analysis revealed large deletions in three copies of GI.04 and loss of one bp in the gRNA4 segment in the fourth copy. In line with the lack of the PCR fragment with the gRNA7–gRNA8 primer pair, few bp deletions corresponding to gRNA sequences were identified in GI.12. Thus, mGI.412/1 carried mutations in each copy of GI.04 and GI.12 . The second mutant, mGI.412/2, was a null mutant for GI.04 with deletions in the gRNA4 segment and a few bp change in one copy of the gRNA8 segment in GI.12. In mGI.412/3, three copies of GI.04 carried deletions in the gRNA4 region, but the fourth copy was wild-type, and only point mutations were present in two copies of GI.12 , while two copies were non-mutated (Figs. S8 and S9). Two GI.04 protein copies were truncated, and two had deletions, whereas two GI.12 copies were truncated, and two copies had only one a.a. substitution and one a.a. deletion in mGI412/1. The mGI.412/2 mutant had deletions in three copies and truncation in one copy of GI.04, but GI.12 was almost unaffected, as it had only a.a. changes in two copies. In the mGI.412/3 mutant, three different truncated GI.04 proteins were encoded by the mutant alleles, whereas wild-type GI.04 was synthesised from the fourth copy, one copy of GI.12 was truncated, one copy had a one a.a. change and two copies were wild-type (Fig. 7). The double mutant plants were shorter than Désirée and had a shorter life cycle. In correlation with the genotype of the plants, the effect of mutations was the most pronounced in mGI.412/1 (Fig. S9). This mutant tuberised earlier than did Désirée; however, owing to its smaller size and shorter life time, its tuber yield was also significantly lower than that of Désirée (Fig. 8). Anthocyanin content of tuber skins Earlier, it was demonstrated that a reduction in the GI.04 transcript level by approximately 50% led to a reduction in the anthocyanin content in tuber skins [10]. On the basis of this finding, the anthocyanin content of tubers harvested from the GI mutants was investigated. However, no significant change in skin colour was detected (Fig. S11). Discussion Efficiency of Cas9-mediated mutagenesis in the potato cv. Désirée The CRISPR/Cas technology has already been widely adopted to increase the efficiency of breeding programs through rapid and precise modification of the plant genome. This technology has also been successfully used in potato for enhancing quality traits, changing tuber skin colour and starch composition, reducing glycoalkaloid content and preventing enzymatic browning of tuber cuts, as well as for resistance breeding [ 19 ]. Although the presence of four copies of genes in the tetraploid (2n = 4x = 48) genome of commercial potato varieties makes it difficult to edit the genome, we were successful in generating several null mutants in one of the two GI genes of the potato cv. Désirée. Although Cas9 is present in these mutants, they are stable since the sequences corresponding to the gRNAs are completely or partially missing, or at least differ in sequence. The type of mutation generated depended on the gRNAs used to target the selected gene. For example, large deletions were obtained with gRNA1, gRNA2 and gRNA5, whereas small deletions extending to a few base pairs dominated in the case of gRNA7 and gRNA8. Duplications were detected in eGI.04/2, and 92 bp were replaced by an unknown sequence in mGI.04/1. The size and type of Cas9-induced mutations are determined by cellular DNA repair mechanisms. The local sequence context, such as the chromatin state and gene expression levels, strongly influences editing outcomes [ 20 , 21 ]. Since we found different types of mutations within the same gene, it could not be the chromatin structure or the level of expression that caused the difference. For CRISPR/Cas nucleases, recognition of target sequences requires a short protospacer adjacent motif (PAM) located outside the targeted sequence. The relative affinities of Cas9–gRNA for different PAM sequences positively correlate with the efficiency of gene editing [ 22 ]. Thus, the difference in PAM sequences may influence the rate and type of mutations. This can be especially true if we consider that no mutations could be introduced into the gRNA3 target site, whereas the gRNA7–gRNA8 pair was very active, producing null mutants with a 14 to 22 ratio. Eight different mutations were identified in eGI.04/3. Although it cannot be excluded that this is due to chromosomal duplication, it is very probable that the eGI.04/3 plants are mosaics, or in other words, chimeras. Transgenic plant chimeras generated by Agrobacterium -mediated transformation have been described for many species. For example, to test the efficiency of Cas9 -mediated mutagenesis, the PHYTOENE DESATURASE ( PDS ) gene, which is involved in carotenoid biosynthesis, was targeted as a model gene, and plants with green‒white sections on their leaves were obtained [23 and references in it]. All these plants are mosaics. The formation of chimeric plants from a group of cells rather than from a single cell has already been elegantly demonstrated [ 24 ]. Our transgenic plants were obtained via callus regeneration. Thus, the eGI.04/3 mutant likely originated from at least two cells. In contrast to eGI.04/3, only two types of mutations were detected in eGI.04/2. Although the PCR fragments of more than 20 clones were sequenced, the possibility of the existence of wild-type or differently mutated GI.04 alleles in eGI.04/2 cannot be ruled out. As an alternative explanation, we can suppose that the mutations in two chromosomes were formed before the S phase of cell division, and then, the mitosis separated the duplicated chromosomes in such a way that two copies of the mutated chromosomes were introduced into one of the two newly formed nuclei, leading to a cell with two chromosomes carrying identical mutations. Effects of GI mutations on the phenotype and tuberisation of Désirée plants In Arabidopsis , GI was initially discovered as a “supervital” mutant with a late-flowering phenotype [ 25 ]. Subsequent studies identified several gi mutant alleles, each of which influences distinct biological processes on the basis of their location in the GI coding sequence [ 26 ]. In our study, mutations were introduced into two distinct regions of GI.04 and GI.12 in the potato cv. Désirée. Corresponding to the location of sequences targeted by RNAs, the mutations truncated or introduced deletions into GI.04 around the 567–632 and 816–869 a.a. positions, whereas in GI.12, these were around the 618–694 and 834–863 a.a. positions. Plants of one GI.04 mutant line and two GI.12 lines with truncated proteins and deletions in the 816–869 and 834–863 a.a. regions, respectively, were shorter and remained green for a longer time than Désirée. Previously, it was demonstrated that ELF3 of Arabidopsis interacts with the GI at the N-terminal (1–507 a.a.) and C-terminal (801–1173 a.a.) regions and that a mutation in ELF3 disturbs the pattern of GI cyclic accumulation [ 6 ]. Thus, the lack or weakness of the GI–ELF3 interaction may have changed the life cycle and morphology of eGI.04/1, eGI.12/1 and eGI.12/2 mutant plants. Interestingly, the complete loss of the GI.04 C-terminal region in eGI.04/2 and eGI.04/3 resulted only in plant shortening but not in a change in the duration of the vegetation period. The phenotype of eGI.12/3 did not differ from the phenotype of Désirée, which might be explained by the presence of a GI.12 allele with only one a.a. deletion in eGI.12/3. It was previously shown that the LOV domain protein LKP2 binds to the 563–789 a.a. region of Arabidopsis GI [ 5 ]. This part is well conserved both in GI.04 and GI.12 and has the same motives detected in Arabidopsis . In contrast, the NLS sequences identified in Arabidopsis GI, with two exceptions in GI.04, are not present in the GI proteins of Désirée. This fact, however, does not exclude the possibility that the GI proteins of Désirée also reside in the nucleus, as several non-classical NLSs are known from different species [ 27 ]. Moreover, the GI genes of tomato ( S. lycopersicum ), SlGI.04 and SlGI.12 , encode almost identical a.a. sequences with the GI genes of potato, and it has been shown very recently that SlGI.04 and SlGI.12 reside in the nucleus [ 28 ]. GI.04 and GI.12 mutants with truncation or deletion in the 567–632 a.a. and the 618–694 a.a. region, respectively, differ in phenotype. The mGI.04/1 mutant, which has two truncated proteins and the largest deletion in the binding sites of LOV domain proteins, has the short plant and delayed senescence phenotype, whereas the other two mGI.04 mutants do not differ from Désirée. In contrast, all three mGI.12 mutants presented signs of early senescence. Similar to the mGI.04/1 plants, all mGI.12 plants were shorter than the control plants. The mutant phenotype is the most pronounced in mGI.12/1, which has four copies of truncated GI.12 lacking the majority of LOV domain binding sites, whereas mGI.12/2 and mGI.12/3 carry only three truncated copies and an almost wild-type GI.12 allele. Owing to this truncation, it is highly probable that the blue-light receptors cannot bind to the truncated mGI proteins in Désirée, which leads to perturbation of oscillation of the plant circadian clock and changes in the growth and life cycle length of the plants. Isolation of double GI mutants was attempted via cotransformation. One mutant, mGI.412/1, carried mutations in all four GI.04 and GI.12 alleles; however, mutations in two GI.12 copies led to only minor changes at the protein level. Since this was the only mutant that carried mutations in each GI allele out of the 216 tested ones, we assume that the complete loss of GI function would be lethal for the plants. In correlation with the extent of mutations, mGI.412/1 plants presented the greatest phenotypical changes compared to Désirée plants. The mGI.412/1 plants were very short and died very early. The phenotypes of the mGI.412/2 and mGI.412/3 mutants were similar to that of the mGI.412/1 mutant, with a lower intensity. Thus, we concluded that, as in Arabidopsis , the GI proteins in Désirée influence the developmental processes and life cycles of the plants. Despite the 84% identity at the transcript level, mutations in the LOV domain binding site resulted in different changes in the phenotypes of the plants. Early senescence was characteristic of only the mGI.12 and mGI.412 mutants, while the mGI.04 mutants were similar to Désirée or even remained green for a longer time. In silico promoter analysis revealed binding sites for EVENING ELEMENT and ABSCISIC ACID RESPONSE ELEMENT-LIKE elements related to circadian regulation in both GI promoters. However, the two genes differ in several other cis- acting regulatory elements as well as in organ-specific expression and responses to abiotic stresses [ 9 ], which may explain the different effects of mutations at the phenotype level. CDF1 was found to regulate tuberisation and plant life cycle length. The truncated variants of CDF1 exhibit enhanced protein stability caused by the inability to bind to GI and FKF1. The overexpression of truncated CDF1 leads to early tuberisation and a short life cycle in potato [ 29 , 30 ]. In our study, the putative binding site of FKF1, the LOV domain binding site, was mutated in mGI mutants, which might have led to the accumulation of CDF1 and the formation of a similar phenotype to that of potatoes overexpressing the truncated CDF1. Nevertheless, only mGI.12/1 and mGI.412/1, the mutants with the shortest lifetimes, tuberised significantly earlier than Désirée. Compared with that of Désirée, the vegetative development of mGI.412.1 was so much shorter that it reduced the tuber yield. Antisense repression of approximately 50% of Désirée GI.04 did not influence tuber formation or yield but did cause a reduction in tuber colour and anthocyanin content [ 10 ]. In contrast, none of the GI mutants presented significantly lower amounts of anthocyanins in their tuber skin than did Désirée tubers. This result suggests that those domains of GI proteins that might be important in terms of the regulation of anthocyanin synthesis remained intact in the GI mutants. Conclusions The CRISPR/Cas9 system can be successfully used even in tetraploid potato to generate mutations in all four copies of a gene. Cotransformation can be used to obtain mutations in two genes simultaneously. Large as well as small deletions, inversions or nucleotide substitutions can be obtained. The efficiency of targeted mutagenesis, however, may depend on the PAM sequence adjacent to the site targeted by the gRNA. Analysing the effects of mutations revealed that both GI genes, GI.04 and GI.12 , influence plant development. However, the influence of GI.12 is more pronounced than the influence of GI.04 . Certain mutations at 3’ to the LOV–NLS domain reduced the size and increased the lifetime of Désirée plants, whereas mutations in LOV-NLS domain of GI.12 shortened the life cycle length of plants and, in certain cases, led to an increase in the earliness of tuberisation. In the absence of the GI.04 protein, mutations in GI.12 not only shorten the size of the plants and duration of the vegetative period but also result in a reduction in tuber yield. Nevertheless, one of the GI.12 mutants, mGI.12/1, tuberised earlier than the control without a yield penalty under greenhouse conditions. After further detailed investigation, this mutant might be a good candidate for breeding purposes. Declarations Aknowledgements The authors are grateful to M. Kiss for the excellent assistance in propagation, transformation and greenhouse growth of potato plants. Ethics approval and consent to participate Not applicable. This study does not involve any human or animal testing. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was financially supported by the National Research, Development and Innovation Office – NKFIH (grant number: K_146328). Author Contribution F.K.-R., K.O., Z.G.T. and V.V. performed the experiments, analysed the data, reviewed and edited the manuscript. Z.B. conceptualised the research topic, analysed the data, drew the figures and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors are grateful to M. Kiss for the excellent assistance in propagation, transformation and greenhouse growth of potato plants. Data Availability DNA sequences of the *GIGANTEA* genes *GI.04* and *GI.12* analysed during the current study are available in the DDBJ repository under the accession numbers LC914694 and LC914695, respectively. DNA sequences of the PCR fragments obtained from the genomic DNA of mutant lines are deposited in the ZENODO database and can be cited using the DOI 10.5281/zenodo.18593396. All research data and materials supporting the results and analysis of the article could be shared upon request. References Dutta M, Mali S, Raturi V, Zinta G. Transcriptional and post–transcriptional regulation of tuberization in potato ( Solanum tuberosum L). J Plant Growth Reg. 2024;43:1–24. https://doi.org/10.1007/s00344-023-11053-5 . Liu L, Xie Y, Yahaya BS, Wu F. GIGANTEA unveiled: Exploring its diverse roles and mechanisms. 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Curr Opin Biotechnol. 2019;55:68–73. https://doi.org/10.1016/j.copbio.2018.07.005 . Mekler V, Kuznedelov K, Severinov K. Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences. J Biol Chem. 2020;295:6509–17. https://doi.org/10.1074/jbc.RA119.012239 . Bánfalvi Z, Csákvári E, Villányi V, Kondrák M. Generation of transgene-free PDS mutants in potato by Agrobacterium -mediated transformation. BMC Biotechnol. 2020;20:1–10. https://doi.org/10.1186/S12896-020-00621-2/TABLES/3 . Zhu XY, Zhao M, Ma S, Ge YM, Zhang MF, Chen LP. Induction and origin of adventitious shoots from chimeras of Brassica juncea and Brassica oleracea . Plant Cell Rep. 2007;26:1727–32. https://doi.org/10.1007/s00299-007-0398-4 . Rédei GP. Supervital mutants of Arabidopsis. Genetics. 1962;47:443–60. Mishra P, Panigrahi KC. GIGANTEA—An emerging story. Front Plant Sci. 2015;6:8. https://doi.org/10.3389/fpls.2015.00008 . 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Supplementary Files Supplementaryfile1Tables.pdf Supplementaryfile2GIsequences.pdf Supplementaryfile3Figures.pptx Supplementaryfile4Originalphotos.pptx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers invited by journal 25 Feb, 2026 Editor invited by journal 24 Feb, 2026 Editor assigned by journal 11 Feb, 2026 Submission checks completed at journal 10 Feb, 2026 First submitted to journal 10 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8542741","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598140367,"identity":"e77c67e0-51d2-41c4-aff3-684345db31ed","order_by":0,"name":"Flóra Karsai-Rektenwald","email":"","orcid":"","institution":"Magyar Agrár- és Élettudományi Egyetem","correspondingAuthor":false,"prefix":"","firstName":"Flóra","middleName":"","lastName":"Karsai-Rektenwald","suffix":""},{"id":598140372,"identity":"36b26807-7754-4029-a3c9-76e4bf37be40","order_by":1,"name":"Khongorzul Odgerel","email":"","orcid":"","institution":"Magyar Agrár- és Élettudományi Egyetem","correspondingAuthor":false,"prefix":"","firstName":"Khongorzul","middleName":"","lastName":"Odgerel","suffix":""},{"id":598140374,"identity":"422ce31e-b41c-45fb-83fa-64f88f443bc8","order_by":2,"name":"Zoltán Gábor Tóth","email":"","orcid":"","institution":"Magyar Agrár- és Élettudományi Egyetem","correspondingAuthor":false,"prefix":"","firstName":"Zoltán","middleName":"Gábor","lastName":"Tóth","suffix":""},{"id":598140376,"identity":"63841f36-d879-45cb-a968-a11a4d9e1f10","order_by":3,"name":"Vanda Villányi","email":"","orcid":"","institution":"Magyar Agrár- és Élettudományi Egyetem","correspondingAuthor":false,"prefix":"","firstName":"Vanda","middleName":"","lastName":"Villányi","suffix":""},{"id":598140377,"identity":"484ad663-4790-4b1a-bde6-815646bf3641","order_by":4,"name":"Zsófia Bánfalvi","email":"data:image/png;base64,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","orcid":"","institution":"Magyar Agrár- és Élettudományi Egyetem","correspondingAuthor":true,"prefix":"","firstName":"Zsófia","middleName":"","lastName":"Bánfalvi","suffix":""}],"badges":[],"createdAt":"2026-01-07 14:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8542741/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8542741/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103724126,"identity":"36ace20f-0ac0-4419-87fa-e4c839124a07","added_by":"auto","created_at":"2026-03-02 07:56:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1628228,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the GI.04 protein in the \u003cem\u003eS. tuberosum \u003c/em\u003ecv. Désirée (\u003cstrong\u003eA\u003c/strong\u003e) and its mutants generated by targeted mutagenesis using the gRNAs corresponding to the amino acids labelled in yellow (\u003cstrong\u003eB\u003c/strong\u003e) and green (\u003cstrong\u003eC\u003c/strong\u003e), respectively. The LOV domain indicates the binding site of the proteins carrying the LOV (\u003cu\u003eL\u003c/u\u003eight-\u003cu\u003eO\u003c/u\u003exygen-\u003cu\u003eV\u003c/u\u003eoltage) domain. The nuclear localisation signal is abbreviated as NLS. The letters from “a” to “h” indicate the different GI.04 alleles detected in the mutant plants. Black lines represent the wild-type amino acid sequences, whereas red lines represent mutated sequences. Deletions are labelled by black boxes.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/f4f1d035f6b8f0804446a009.png"},{"id":103724242,"identity":"1795f662-d321-4c48-8b95-7ac8763f0395","added_by":"auto","created_at":"2026-03-02 07:56:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":339072,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and tuberisation of the eGI.04 mutants compared to Désirée (DES). Photos of the plants were taken at the end of the vegetation period. The tubers harvested from the photographed plants are shown below the plants. At the early stage of tuberisation, at 7 weeks after transferring the \u003cem\u003ein vitro \u003c/em\u003eplantlets into the pots, the number of tubers was counted. The bars indicate the number of tubers/plant, whereas the dots indicate the % of tuberising plants. The tuber yield was tested at the end of the vegetation period. Three consecutive plant tests were performed each with 8 plants/line. The average tuber yield of DES was 33.0±3.4, 24.3±6.4 and 23.1±1.7 g fresh weight/plant in the 1\u003csup\u003est\u003c/sup\u003e, 2\u003csup\u003end \u003c/sup\u003eand 3\u003csup\u003erd\u003c/sup\u003e experiment, respectively, and regarded as 100% for comparison. The standard deviations are indicated by the error bars. Means presented with the same letter are not significantly different at \u003cem\u003ep ≤ \u003c/em\u003e0.05.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/1e063ef9d9891a745234d405.png"},{"id":103724344,"identity":"edc0c824-1bd5-45cf-8c74-de8846c4c652","added_by":"auto","created_at":"2026-03-02 07:56:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":356705,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and tuberisation of the mGI.04 mutants compared to Désirée (DES). At the early stage of tuberisation, data are from one experiment with 8 plants/line, whereas the tuber yield was tested in two consecutive plant tests, with 8 and 16 plants/line, respectively. The photos are from the 1\u003csup\u003est \u003c/sup\u003eexperiment. The average tuber yield of DES was 21.2±2.5 and 26.8±2.0 g fresh weight/plant and regarded as 100% for comparison. See the Fig. 2 legend for more details.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/1bbbc21fafe2afb70e2e8402.png"},{"id":103724178,"identity":"d7376b61-9687-4cbb-9e04-d8884586ba3b","added_by":"auto","created_at":"2026-03-02 07:56:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":126238,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the GI.12 protein in the \u003cem\u003eS. tuberosum \u003c/em\u003ecv. Désirée (\u003cstrong\u003eA\u003c/strong\u003e) and its mutants generated by targeted mutagenesis using the gRNAs corresponding to the amino acids labelled in yellow (\u003cstrong\u003eB\u003c/strong\u003e) and green (\u003cstrong\u003eC\u003c/strong\u003e), respectively. Abbreviations and labels are as in Fig. 1.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/d98e5fa06850d506c7d88b27.png"},{"id":103724204,"identity":"699f1360-6ce8-4135-9edd-0d351996466a","added_by":"auto","created_at":"2026-03-02 07:56:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":343947,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and tuberisation of the eGI.12 mutants compared to ‘Désirée’ (DES). At the early stage of tuberisation, data are from one experiment with 8 plants/line, whereas the tuber yield was tested in three consecutive plant tests each with 8 plants/line. The average tuber yield of DES was 32.9±4.5, 24.3±6.4 and 22.7±1.9 g fresh weight/plant in the 1\u003csup\u003est\u003c/sup\u003e, 2\u003csup\u003end \u003c/sup\u003eand 3\u003csup\u003erd\u003c/sup\u003e experiment, respectively, and regarded as 100% for comparison. See the Fig. 2 legend for more details.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/2a85b4c07c4ad1cb59f1dee3.png"},{"id":103724233,"identity":"15f3a303-982c-4719-a9b9-738e0a119c32","added_by":"auto","created_at":"2026-03-02 07:56:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":340400,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and tuberisation of the mGI.12 mutants compared to Désirée (DES). At the early stage of tuberisation, data are from one experiment with 16 plants/line. The tuber yield was tested in two consecutive plant tests, with 8 and 16 plants/line, respectively. The photos are from the 1\u003csup\u003est \u003c/sup\u003eexperiment. The average tuber yield of DES was 19.4±2.0 and 24.2±3.6 g fresh weight/plant in the 1\u003csup\u003est \u003c/sup\u003eand 2\u003csup\u003end \u003c/sup\u003eexperiment, respectively, and regarded as 100% for comparison. See the Fig. 2 legend for more details. \u0026nbsp;\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/521cd37d380fe392d535ed76.png"},{"id":103724123,"identity":"225ffa53-4169-42e8-9302-5460f176e341","added_by":"auto","created_at":"2026-03-02 07:56:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1888063,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the GI.04 and GI.12 proteins in the \u003cem\u003eS. tuberosum \u003c/em\u003ecv. Désirée (\u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e) and its double mutants (\u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e) generated by targeted mutagenesis using the gRNAs corresponding to the amino acids labelled in green. Abbreviations and labels are as in Fig. 1.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/23b8cf6b73b1d9962cc56e35.png"},{"id":103724243,"identity":"fa37c282-c44a-47c5-9771-e0cdd1b0354e","added_by":"auto","created_at":"2026-03-02 07:56:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":253059,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype and tuberisation of the mGI.412 mutants compared to ‘Désirée’ (DES). All data are from two experiments with 16 plants/line in each. The photos of five representative plants and their tubers are from the 1\u003csup\u003est \u003c/sup\u003eexperiment. The average tuber yield of DES was 18.0±5.0 and 16.23±4.6 g fresh weight/plant in the 1\u003csup\u003est \u003c/sup\u003eand 2\u003csup\u003end \u003c/sup\u003eexperiment, respectively, and regarded as 100% for comparison. See the Fig. 2 legend for more details.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/f7832796d2568634e29e893c.png"},{"id":104399743,"identity":"2c60d4b2-31ac-4569-8b53-54055f296c9a","added_by":"auto","created_at":"2026-03-11 12:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6156437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/681cb0ae-f5bc-45bb-9be9-8eeed2418eed.pdf"},{"id":103724264,"identity":"901f52ff-0989-4a77-b552-5a74e8829702","added_by":"auto","created_at":"2026-03-02 07:56:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":114398,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1Tables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/ed72a864ca853fe4caf162a7.pdf"},{"id":103724365,"identity":"233ca661-1e31-4e9a-bd9a-c6aa20264761","added_by":"auto","created_at":"2026-03-02 07:57:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":242504,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2GIsequences.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/6923b5c091c7b60095e83f85.pdf"},{"id":103724181,"identity":"d90b8480-d911-47b0-bf54-75b6c99fdf0c","added_by":"auto","created_at":"2026-03-02 07:56:16","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4698046,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile3Figures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/e3a74b6821888d3ced01dce3.pptx"},{"id":103724244,"identity":"958268ae-8b7c-46a7-b8ae-5c6978754349","added_by":"auto","created_at":"2026-03-02 07:56:27","extension":"pptx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17448707,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile4Originalphotos.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8542741/v1/1773e3550d0c60cb3a7cdb16.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the role of the two GIGANTEA genes in the life cycle length and tuberisation of the potato cultivar Désirée","fulltext":[{"header":"Background","content":"\u003cp\u003eEarliness of tuberisation is an important agronomic trait. Early potato varieties withstand various biotic and abiotic stresses and complete their life cycle before stress becomes a serious constraint and, therefore, are more profitable for growers.\u003c/p\u003e \u003cp\u003eTuberisation is a complex developmental process influenced by environmental factors. The molecular mechanisms that control tuber formation have been investigated in detail over the past few decades in \u003cem\u003eS. tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e and reported that the stolon-to-tuber transition is governed by mobile RNAs and proteins, phytohormones, a plethora of small RNAs and their targets. In the wild \u003cem\u003eandigena\u003c/em\u003e subspecies, tuberisation is strictly dependent on short days (SDs). This control is mediated by the potato SELF-PRUNING 6A (SP6A) protein, which acts as the main phloem-transported tuberigen signal. \u003cem\u003eSP6A\u003c/em\u003e is negatively controlled by CONSTANS (CO), whereas \u003cem\u003eCO\u003c/em\u003e transcription is increased through proteolytic degradation of CYCLING DOF FACTOR 1 (CDF1), which is mediated by the FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1/GIGANTEA (FKF1/GI) complex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Thus, GI is indirectly involved in light-dependent tuberisation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGI\u003c/em\u003e encodes a plant-specific protein with pleiotropic functions, including flowering, photoperiodic response, phytochrome B signalling, circadian clock regulation, and carbohydrate metabolism. GI interacts with hormonal signalling pathways, balances stress responses, promotes growth and optimises plant productivity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, GI is comprised of 1173 amino acids. It has a nuclear localisation signal (NLS) and predominantly resides in the nucleus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, it is also localised in the cytosol, where it stabilises ZEITLUPE (ZTL), an F-box protein that binds to GI through its light-oxygen voltage-sensing (LOV) blue-light-absorbing domain [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition to ZTL, two other LOV domain containing blue light receptors, LKP2 and FKF1, also bind to the middle region of GI (residues 563\u0026ndash;789) to prevent the degradation of TOC1 and PRR5, thereby contributing to the correct oscillation of the plant circadian clock, whereas GI binding to FKF1 facilitates the degradation of CDF flowering repressors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The E3 ubiquitin-ligase COP1 also interacts with GI through the clock-associated protein ELF3, which acts as a substrate adaptor protein to accelerate GI destabilisation and degradation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the function of GI in tuberisation has been demonstrated in \u003cem\u003eS. tuberosum\u003c/em\u003e ssp. \u003cem\u003eandigena\u003c/em\u003e, the current commercial potato varieties from Europe and America have genetic closeness to ssp. \u003cem\u003etuberosum\u003c/em\u003e, compared with the tubers of ssp. \u003cem\u003eandigena\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The introduction of potato into temperate latitudes was accompanied by alterations in its photoperiodic requirements, i.e., tuberisation became day-length independent [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, even though the role of the GI in tuberisation is well demonstrated in the SD-tuberising ssp. \u003cem\u003eandigena\u003c/em\u003e, its role in tuberisation in tetraploid commercial cultivars, such as cv. D\u0026eacute;sir\u0026eacute;e, is still elusive. Previously, we demonstrated that there are two copies of \u003cem\u003eGI\u003c/em\u003e genes in potato homologous to \u003cem\u003eArabidopsis GI\u003c/em\u003e located on chromosomes 4 and 12 (\u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e) with different regulatory elements in their promoter region; however, with 84% identity at the transcriptional level[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The antisense repression of \u003cem\u003eGI.04\u003c/em\u003e by approximately 50% affected the transcription of the genes involved in the circadian clock, flowering, starch synthesis, and stress responses in the leaves of D\u0026eacute;sir\u0026eacute;e plants; however, it did not influence tuber formation or yield but did cause a reduction in tuber colour [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the function of the \u003cem\u003eGI\u003c/em\u003e genes in the potato cv. D\u0026eacute;sir\u0026eacute;e, targeted mutations were introduced into \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e and simultaneously into both genes via the CRIPR/Cas9 gene editing system. Here, we report that both \u003cem\u003eGI\u003c/em\u003e genes influence the development and life cycle length of D\u0026eacute;sir\u0026eacute;e plants and, in certain cases, mutations in \u003cem\u003eGI.12\u003c/em\u003e can lead not only to plant size reduction and shortening of the vegetation period but also to earlier onset of tuberisation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material, growing conditions and phenotyping\u003c/h2\u003e \u003cp\u003eThe potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.) cultivar D\u0026eacute;sir\u0026eacute;e from Fritz Lange KG (Bad Schwartau, Germany), cultivated in tissue culture at the Max Planck Institute of Molecular Plant Physiology (Golm, Germany), was used as the starting material and propagated \u003cem\u003ein vitro\u003c/em\u003e from stem segments in the rooting medium RM (MS without vitamins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]) containing 2% (w/v) sucrose and 0.8% agar in 40-ml tubes closed with paper plugs at 24\u0026deg;C under a 16 h/8 h day/night cycle and a light intensity of 75 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor plant phenotyping, 4-week-old \u003cem\u003ein vitro\u003c/em\u003e plantlets were transferred into 18x14 cm pots filled with sterile Tabaksubstrat soil A200 (Stender GmbH, Schermbeck, Germany) and grown further under greenhouse conditions. From the middle of November until the middle of March, the ambient light conditions were supplemented with artificial lighting by sodium lamps to provide a minimum of 12 h light conditions. The temperature regime varied from 18 to 24\u0026deg;C. The soil humidity reached approximately 80%, which was provided by regular watering. Pesticides and fungicides were regularly applied for pest and fungal pathogen control.\u003c/p\u003e \u003cp\u003eThe morphological characteristics of the plants were observed visually and documented via photos. The earliness of tuberisation was assessed by carefully tipping the plants out of the pots and counting the number of tubers seven weeks after planting. After counting, the plants were replanted into the pots and grown further in the greenhouse. At the end of the vegetation period, the tubers were harvested, weighed, and peeled for anthocyanin measurement.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning and sequencing the coding sequences of D\u0026eacute;sir\u0026eacute;e\u003c/b\u003e \u003cb\u003eGI.04\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eGI.12\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRNA was isolated from the leaves of \u003cem\u003ein vitro-\u003c/em\u003egrown D\u0026eacute;sir\u0026eacute;e plants at 4 h after the beginning of the light period using the method of [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and cDNA was synthesised with the Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Fisher Scientific, Waltham, MA, USA). The \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e coding sequences (CDSs) were obtained with the help of the GI04 attB1\u0026ndash;GI04 attB2 and GI12 attB1\u0026ndash;GI12 attB2 primer pairs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively, in PCR reactions using the CloneAmp HiFi PCR Premix (Takara Bio Inc., Shiga, Japan) and following the manufacturer\u0026rsquo;s instructions. The PCR products were introduced into the pDONR\u0026trade;221 vector with Gateway\u0026trade; cloning (Thermo Fisher Scientific, Waltham, MA, USA) and Sanger sequenced at Eurofins BIOMI Ltd. (G\u0026ouml;d\u0026ouml;llő, Hungary) in fragments generated with the M13, GI04 640\u0026ndash;2070, GI04 2000\u0026ndash;3400, GI12 630\u0026ndash;2100 and GI12 2030\u0026ndash;3440 forward‒reverse primer pairs (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTargeted mutagenesis of\u003c/b\u003e \u003cb\u003eGI.04\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eGI.12\u003c/b\u003e \u003cb\u003ein D\u0026eacute;sir\u0026eacute;e\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTargeted mutagenesis was carried out using pCBC-DT1T2 as gRNA template and the \u003cem\u003eCas9\u003c/em\u003e delivery vector pKSE401 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Gene-specific gRNAs were designed using the CRISPOR tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the oligos were synthesised by Integrated DNA Technologies (Coralville, Iowa, USA). For the gRNA sequences, see Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e LBA4404-mediated leaf transformation was performed according to the protocol published in [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. After transformation, the \u003cem\u003eAgrobacterium\u003c/em\u003e was eliminated by adding 500 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cefotaxime to the medium. Kanamycin at a concentration of 50 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used for the regeneration, rooting and propagation of the transgenic plants \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection and mapping of the mutations in transgenic Désirée plants\u003c/h3\u003e\n\u003cp\u003eGenomic DNA from \u003cem\u003ein vitro\u003c/em\u003e-grown plants was isolated using the method of Shure et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Selection of mutated plants was performed by PCR using primer pairs corresponding to gRNAs, and the amplified fragments were detected on agarose gels. The lack of or lower amounts of PCR products than those obtained from D\u0026eacute;sir\u0026eacute;e were supported by qPCR using PowerTrack\u0026trade; SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) in a LightCycler-96 (Roche, Basel, Switzerland) following the manufacturer\u0026rsquo;s instructions. \u003cem\u003eACTIN\u003c/em\u003e was used as a reference gene [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For the localisation of mutations, gene-specific PCR primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) surrounding the region restricted by gRNA sequences were designed using the Primer-BLAST tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The PCR products amplified from the genomic DNA were subsequently cloned using the pGEM-T-Easy (Promega, Madison, WI, USA), CloneJet or TOPO Cloning Kit for Sequencing (Thermo Fisher Scientific, Waltham, MA, USA) and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α competent cells. Transformed cells were selected on antibiotic-containing LB plates, and plasmid DNA was isolated. For basic molecular techniques, e.g., plasmid DNA isolation, transformation of \u003cem\u003eE. coli\u003c/em\u003e, agarose gel electrophoresis, and PCR, the instructions of [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were followed. Genomic PCR fragments of the positive clones were sequenced by Sanger sequencing at Eurofins BIOMI Ltd. (G\u0026ouml;d\u0026ouml;llő, Hungary). DNA sequences of selected clones are presented in the Supplementary material 2. EMBOSS Transeq (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/st/emboss_transeq\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/st/emboss_transeq\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to translate DNA sequences to peptide sequences. Sequence comparisons were performed with Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/msa/clustalo\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/msa/clustalo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAnthocyanin content of tuber skins\u003c/h3\u003e\n\u003cp\u003eA simplified method of [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] was applied for the peeled skins of freshly harvested tubers as described in [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe significance of differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between two groups of data was detected by one-way ANOVA with a post hoc Tukey\u0026rsquo;s HSD test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eSequence comparison of GI.04 and GI.12 of D\u0026eacute;sir\u0026eacute;e\u003c/h2\u003e\n \u003cp\u003eThe coding sequences of \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e were sequenced and first compared to the corresponding \u003cem\u003eS. tuberosum\u003c/em\u003e group Phureja sequences (https://spuddb.uga.edu/). This comparison revealed that \u003cem\u003eGI.04\u003c/em\u003e of D\u0026eacute;sir\u0026eacute;e possesses a 21-bp deletion compared with the corresponding Phureja CDS, resulting in the lack of amino acids (a.a.) from 31 to 38 of Phureja. All the other nucleotides, with the exception of a G/T base pair change, were identical to the Phureja CDS. \u003cem\u003eGI.12\u003c/em\u003e of D\u0026eacute;sir\u0026eacute;e was even more similar to the corresponding Phureja sequence than \u003cem\u003eGI.04\u003c/em\u003e, with changes of only six bps that resulted in the alteration of three a.a. in the protein sequence.\u003c/p\u003e\n \u003cp\u003eThe existence of the interaction site with the LOV domain proteins and the presence of the NLS in the GI proteins of D\u0026eacute;sir\u0026eacute;e were tested via comparison with the \u003cem\u003eArabidopsis\u003c/em\u003e GI protein. Both D\u0026eacute;sir\u0026eacute;e GI proteins were slightly shorter than the \u003cem\u003eArabidopsis\u003c/em\u003e GI protein as GI.04 and GI.12 were composed of 1166 and 1171 a.a., respectively, versus the 1173 a.a. of the \u003cem\u003eArabidopsis\u003c/em\u003e GI protein. The LOV domain binding sites were highly conserved in both D\u0026eacute;sir\u0026eacute;e GI proteins. In contrast, the a.a. sequences of the NLS site varied. The four NLSs of \u003cem\u003eArabidopsis\u003c/em\u003e have a consensus sequence of K (K/R) X (K/R) [3], whereas in GI.04, only two sites correspond to it. GI.12 was even more diverse, with no sequences similar to the \u003cem\u003eArabidopsis\u003c/em\u003e NLS consensus sequence (Fig. S1).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration and characterisation of\u003c/strong\u003e \u003cstrong\u003eGI.04\u003c/strong\u003e \u003cstrong\u003eD\u0026eacute;sir\u0026eacute;e mutants\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo evaluate the function of \u003cem\u003eGI.04\u003c/em\u003e in the potato cv. D\u0026eacute;sir\u0026eacute;e, gene-specific mutants were generated using the CRISPR/Cas9 genome editing system. Since \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e are 84% identical at the transcript level, in the first experiment, the gRNAs were designed near the 3\u0026apos; end of the \u003cem\u003eGI.04\u003c/em\u003e gene, where the difference between \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e was the greatest. A single Cas9 construct containing two gRNAs (Table S2; gRNA1 and gRNA2) to increase the possibility of obtaining large deletions and disrupting gene structure and function was constructed and used for D\u0026eacute;sir\u0026eacute;e transformation via \u003cem\u003eA. tumefaciens\u003c/em\u003e. Transgenic plants were obtained by antibiotic selection and the genomic DNA of 82 plants was tested for mutations with PCR using a primer pair surrounding the gRNA1\u0026ndash;gRNA2 region. In this way, 29 mutants carrying large deletions visible on agarose gels were identified. Considering the size and number of fragments with large deletions, three lines were selected for further studies. On the basis of the lack of PCR products and the results of the qPCR assay using a primer pair corresponding to the two gRNA sequences (Fig. S2A and B), all three mutants, designated eGI.04/1, eGI.04/2 and eGI.04/3, were expected to carry mutations in all four copies of \u003cem\u003eGI.04\u003c/em\u003e in the tetraploid D\u0026eacute;sir\u0026eacute;e.\u003c/p\u003e\n \u003cp\u003eLocalisation of mutations in the three selected eGI.04 mutants was carried out by cloning the mutated PCR fragments generated with different primer combinations into a cloning vector from which they were reamplified, Sanger sequenced and compared to the sequence of the corresponding D\u0026eacute;sir\u0026eacute;e fragment. Large deletions extending even to 499 bp were detected in each mutant. Furthermore, there was a duplicated segment in eGI.04/2, and only two types of mutations were detected, whereas eGI.04/3 was a line with eight different mutated fragments (Fig. S2C).\u003c/p\u003e\n \u003cp\u003eThe effects of mutations on GI.04 at the protein level were investigated by translating the nucleotide sequences to a.a. sequences and comparing them to the D\u0026eacute;sir\u0026eacute;e sequence. With one exception, the deletions caused frame shifts and the generation of an early stop codon, resulting in C-terminal-truncated proteins. The only exception was a copy of GI.04 in the eGI.04/1 mutant, in which after a 92-a.a. deletion, the original sequence was retained. Nevertheless, none of the deletions extended to the LOV binding domain or the putative NLS region (Fig. 1A and B).\u003c/p\u003e\n \u003cp\u003eThe morphology and tuberisation of the eGI.04 mutants were tested after \u003cem\u003ein vitro\u003c/em\u003e propagation under greenhouse conditions in pots. The mutants, especially eGI.04/1, were visibly shorter than D\u0026eacute;sir\u0026eacute;e, but no significant difference in the time of tuberisation or tuber yield was detected (Fig. 2).\u003c/p\u003e\n \u003cp\u003eSince the lack of changes in tuberisation might have been explained by the integrity of the LOV binding domain and NLS, a new set of mutants with gRNAs targeting this functionally important part of GI.04 were generated (Table S2; gRNA3 and gRNA4). On the basis of PCR and qPCR tests using the gRNA oligos as primers, three null mutants, mGI.04/1, mGI.04/2 and mGI.04/3, were obtained from 148 tested transgenic plants (Fig. S3A and B). The localisation of the mutations revealed that all three mutants carried deletions only at the position of gRNA4 (Fig. S3C). However, the integrity of the LOV and NLS domains was destroyed by these mutations (Fig. 1A and C). Out of the three mutants, only mGI.04/1 differed from D\u0026eacute;sir\u0026eacute;e in phenotype, as it was shorter and remained green, whereas D\u0026eacute;sir\u0026eacute;e and the other two mutants already presented signs of senescence at the end of the vegetation period. Nevertheless, in terms of tuberisation, there was no significant difference between the mutants and the D\u0026eacute;sir\u0026eacute;e plants (Fig. 3).\u003c/p\u003e\n \u003cp\u003eMutations at the protein level in combination with the phenotype of the GI.04 mutant plants are shown in Fig. S4.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration and characterisation of\u003c/strong\u003e \u003cstrong\u003eGI.12\u003c/strong\u003e \u003cstrong\u003eD\u0026eacute;sir\u0026eacute;e mutants\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn the first experiment, as in the case of \u003cem\u003eGI.04\u003c/em\u003e, gRNAs (Table S2; gRNA5 and gRNA6) were designed for that part of \u003cem\u003eGI.12\u003c/em\u003e, which showed the least similarity to \u003cem\u003eGI.04\u003c/em\u003e. The genomic DNA of 82 transgenic plants was tested for the presence of large deletions using a primer pair surrounding the gRNA5\u0026ndash;gRNA6 region. Eighteen plants with visibly shorter PCR fragments detected via agarose gel electrophoresis were obtained, three of which were selected and propagated \u003cem\u003ein vitro\u003c/em\u003e for further studies. Using the oligos corresponding to the two gRNAs as primers in the PCR and qPCR assays, two mutants, eGI.12/1 and eGI.12/2, appeared to be null mutants, whereas in eGI.12/3, a PCR product was detected either on agarose gel or by qPCR, albeit with lower intensity than in D\u0026eacute;sir\u0026eacute;e (Fig. S5A and B).\u003c/p\u003e\n \u003cp\u003eThe localisation of the mutations was carried out in the same way as that for the GI.04 mutants. Large deletions extending to both gRNA sequences were found in eGI.12/1, whereas only gRNA5 and the region surrounding it were missing in eGI.12/2. Sequencing the PCR fragments obtained from eGI.12/3 resulted in the identification of two large deletions and a lack of 2‒3 bps in the middle of the sequence corresponding to gRNA5 in two copies of \u003cem\u003eGI.12\u003c/em\u003e. This finding explains the production of a low amount of PCR fragment with the gRNA primers (Fig. S5C).\u003c/p\u003e\n \u003cp\u003eTranslation of the nucleotide acid sequences to a.a. sequences revealed that eGI.12/1 and eGI.12/2 were similar to each other, as both carried two truncated proteins and two copies of GI.12 with deletions of 48 and 54 a.a., respectively, whereas three truncated proteins and one GI.12 copy with a deletion of 53 a.a. were present in eGI.12/3. Like in the case of GI.04, which was targeted by gRNAs at a similar position, none of the mutations extended to the LOV domain or the putative NLS region (Fig. 4A and B).\u003c/p\u003e\n \u003cp\u003eGreenhouse tests of eGI.12 mutants revealed similar morphological changes in eGI.12/1 and eGI.12/2 as in eGI.04/1. These two mutants were shorter and presented signs of senescence later than D\u0026eacute;sir\u0026eacute;e. eGI.12/3 was similar to D\u0026eacute;sir\u0026eacute;e, which was unexpected, as this mutant had three copies of truncated proteins, whereas the others had only two. The initiation of tuberisation was not altered in any of the mutants, but the yield of eGI.12/2 was significantly lower than that of D\u0026eacute;sir\u0026eacute;e in three consecutive plant tests each with 8 plants/line (Fig. 5).\u003c/p\u003e\n \u003cp\u003eThe LOV binding domain and NLS region in \u003cem\u003eGI.12\u003c/em\u003e were targeted with the gRNA pair gRNA7 and gRNA8. These gRNAs were very effective. In the PCR and qPCR tests, when the corresponding oligos were used as primers, 14 null mutants were found among the 22 mutants examined.\u003c/p\u003e\n \u003cp\u003eMutations were mapped in three mutants designated mGI.12/1, mGI12/2 and mGI12/3 (Fig. S6). DNA sequence analysis detected only very short deletions extending only to 1\u0026ndash;6 nucleotides in the sequences corresponding to the gRNAs in mGI.12/1 and in three \u003cem\u003eGI.12\u003c/em\u003e copies of the other two mutants. However, these short deletions were at the 3\u0026rsquo; end of the genomic DNA sequence corresponding to gRNA7, which could explain the lack of PCR products obtained via the use of gRNA7 and gRNA8 as primers. The fourth copy of \u003cem\u003eGI.12\u003c/em\u003e in mGI12/2 had a large deletion starting in gRNA8 and going 3\u0026rsquo; toward the end of the \u003cem\u003eGI.12\u003c/em\u003e coding sequence, whereas there was a deletion including gRNA7 in one copy of \u003cem\u003eGI.12\u003c/em\u003e in mGI.12/3.\u003c/p\u003e\n \u003cp\u003eDeletions detected at the nucleic acid level resulted in four copies of the truncated GI.12 protein in mGI.12/1 and three copies of the truncated protein in the mGI.12/2 and mGI.12/3 mutants. In these two mutants, however, one GI.12 copy was almost unaffected, with only one a.a. change and one a.a. deletion (Fig. 4C).\u003c/p\u003e\n \u003cp\u003eThe plants of all three mGI.12 mutant lines were shorter than those of D\u0026eacute;sir\u0026eacute;e and presented early senescence, which was most pronounced in mGI.12/1. This mutant had a greater number of tubers than did D\u0026eacute;sir\u0026eacute;e at the early stage of tuberisation and, despite its short lifetime, had a yield similar to that of D\u0026eacute;sir\u0026eacute;e (Fig. 6).\u003c/p\u003e\n \u003cp\u003eMutations at the protein level in combination with the phenotype of the GI.12 mutant plants are shown in Fig. S7.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGeneration and characterisation of\u003c/strong\u003e \u003cstrong\u003eGI.04-GI.12\u003c/strong\u003e \u003cstrong\u003edouble D\u0026eacute;sir\u0026eacute;e mutants\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo obtain D\u0026eacute;sir\u0026eacute;e mutants affected by both \u003cem\u003eGI\u003c/em\u003e genes, cotransformation with \u003cem\u003eA. tumefaciens\u003c/em\u003e strains carrying the Cas9 construct with gRNA3 and gRNA4 targeting the LOV\u0026ndash;NLS domain of \u003cem\u003eGI.04\u003c/em\u003e and the Cas9 construct with gRNA7 and gRNA8 targeting the LOV\u0026ndash;NLS domain of \u003cem\u003eGI.12\u003c/em\u003e was performed. The two strains were mixed in equal amounts before being used for D\u0026eacute;sir\u0026eacute;e infection. Transgenic lines obtained by antibiotic selection were assessed for deletions in both \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e using the gRNA oligos as primers. On the basis of this test, no null mutant for both \u003cem\u003eGI\u003c/em\u003e genes was present among the 216 transgenic plants tested. Considering the intensity and size of the PCR fragments generated via the gRNA primers and other primer pair combinations, three mutants, mGI.412/1, mGI.412/2 and mGI.412/3, were selected for mutation mapping.\u003c/p\u003e\n \u003cp\u003eAlthough with the gRNA3\u0026ndash;gRNA4 primer pair, a PCR fragment was detected in mGI.412/1, the DNA sequence analysis revealed large deletions in three copies of \u003cem\u003eGI.04\u003c/em\u003e and loss of one bp in the gRNA4 segment in the fourth copy. In line with the lack of the PCR fragment with the gRNA7\u0026ndash;gRNA8 primer pair, few bp deletions corresponding to gRNA sequences were identified in \u003cem\u003eGI.12.\u003c/em\u003e Thus, mGI.412/1 carried mutations in each copy of \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e. The second mutant, mGI.412/2, was a null mutant for \u003cem\u003eGI.04\u003c/em\u003e with deletions in the gRNA4 segment and a few bp change in one copy of the gRNA8 segment in \u003cem\u003eGI.12.\u003c/em\u003e In mGI.412/3, three copies of \u003cem\u003eGI.04\u003c/em\u003e carried deletions in the gRNA4 region, but the fourth copy was wild-type, and only point mutations were present in two copies of \u003cem\u003eGI.12\u003c/em\u003e, while two copies were non-mutated (Figs. S8 and S9).\u003c/p\u003e\n \u003cp\u003eTwo GI.04 protein copies were truncated, and two had deletions, whereas two GI.12 copies were truncated, and two copies had only one a.a. substitution and one a.a. deletion in mGI412/1. The mGI.412/2 mutant had deletions in three copies and truncation in one copy of GI.04, but GI.12 was almost unaffected, as it had only a.a. changes in two copies. In the mGI.412/3 mutant, three different truncated GI.04 proteins were encoded by the mutant alleles, whereas wild-type GI.04 was synthesised from the fourth copy, one copy of GI.12 was truncated, one copy had a one a.a. change and two copies were wild-type (Fig. 7).\u003c/p\u003e\n \u003cp\u003eThe double mutant plants were shorter than D\u0026eacute;sir\u0026eacute;e and had a shorter life cycle. In correlation with the genotype of the plants, the effect of mutations was the most pronounced in mGI.412/1 (Fig. S9). This mutant tuberised earlier than did D\u0026eacute;sir\u0026eacute;e; however, owing to its smaller size and shorter life time, its tuber yield was also significantly lower than that of D\u0026eacute;sir\u0026eacute;e (Fig. 8).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnthocyanin content of tuber skins\u003c/h3\u003e\n\u003cp\u003eEarlier, it was demonstrated that a reduction in the \u003cem\u003eGI.04\u003c/em\u003e transcript level by approximately 50% led to a reduction in the anthocyanin content in tuber skins [10]. On the basis of this finding, the anthocyanin content of tubers harvested from the GI mutants was investigated. However, no significant change in skin colour was detected (Fig. S11).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEfficiency of Cas9-mediated mutagenesis in the potato cv. D\u0026eacute;sir\u0026eacute;e\u003c/h2\u003e \u003cp\u003eThe CRISPR/Cas technology has already been widely adopted to increase the efficiency of breeding programs through rapid and precise modification of the plant genome. This technology has also been successfully used in potato for enhancing quality traits, changing tuber skin colour and starch composition, reducing glycoalkaloid content and preventing enzymatic browning of tuber cuts, as well as for resistance breeding [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although the presence of four copies of genes in the tetraploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;48) genome of commercial potato varieties makes it difficult to edit the genome, we were successful in generating several null mutants in one of the two \u003cem\u003eGI\u003c/em\u003e genes of the potato cv. D\u0026eacute;sir\u0026eacute;e. Although \u003cem\u003eCas9\u003c/em\u003e is present in these mutants, they are stable since the sequences corresponding to the gRNAs are completely or partially missing, or at least differ in sequence.\u003c/p\u003e \u003cp\u003eThe type of mutation generated depended on the gRNAs used to target the selected gene. For example, large deletions were obtained with gRNA1, gRNA2 and gRNA5, whereas small deletions extending to a few base pairs dominated in the case of gRNA7 and gRNA8. Duplications were detected in eGI.04/2, and 92 bp were replaced by an unknown sequence in mGI.04/1. The size and type of Cas9-induced mutations are determined by cellular DNA repair mechanisms. The local sequence context, such as the chromatin state and gene expression levels, strongly influences editing outcomes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since we found different types of mutations within the same gene, it could not be the chromatin structure or the level of expression that caused the difference. For CRISPR/Cas nucleases, recognition of target sequences requires a short protospacer adjacent motif (PAM) located outside the targeted sequence. The relative affinities of Cas9\u0026ndash;gRNA for different PAM sequences positively correlate with the efficiency of gene editing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, the difference in PAM sequences may influence the rate and type of mutations. This can be especially true if we consider that no mutations could be introduced into the gRNA3 target site, whereas the gRNA7\u0026ndash;gRNA8 pair was very active, producing null mutants with a 14 to 22 ratio.\u003c/p\u003e \u003cp\u003eEight different mutations were identified in eGI.04/3. Although it cannot be excluded that this is due to chromosomal duplication, it is very probable that the eGI.04/3 plants are mosaics, or in other words, chimeras. Transgenic plant chimeras generated by \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation have been described for many species. For example, to test the efficiency of \u003cem\u003eCas9\u003c/em\u003e-mediated mutagenesis, the \u003cem\u003ePHYTOENE DESATURASE\u003c/em\u003e (\u003cem\u003ePDS\u003c/em\u003e) gene, which is involved in carotenoid biosynthesis, was targeted as a model gene, and plants with green‒white sections on their leaves were obtained [23 and references in it]. All these plants are mosaics. The formation of chimeric plants from a group of cells rather than from a single cell has already been elegantly demonstrated [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our transgenic plants were obtained via callus regeneration. Thus, the eGI.04/3 mutant likely originated from at least two cells.\u003c/p\u003e \u003cp\u003eIn contrast to eGI.04/3, only two types of mutations were detected in eGI.04/2. Although the PCR fragments of more than 20 clones were sequenced, the possibility of the existence of wild-type or differently mutated \u003cem\u003eGI.04\u003c/em\u003e alleles in eGI.04/2 cannot be ruled out. As an alternative explanation, we can suppose that the mutations in two chromosomes were formed before the S phase of cell division, and then, the mitosis separated the duplicated chromosomes in such a way that two copies of the mutated chromosomes were introduced into one of the two newly formed nuclei, leading to a cell with two chromosomes carrying identical mutations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of\u003c/b\u003e \u003cb\u003eGI\u003c/b\u003e \u003cb\u003emutations on the phenotype and tuberisation of D\u0026eacute;sir\u0026eacute;e plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eGI\u003c/em\u003e was initially discovered as a \u0026ldquo;supervital\u0026rdquo; mutant with a late-flowering phenotype [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Subsequent studies identified several \u003cem\u003egi\u003c/em\u003e mutant alleles, each of which influences distinct biological processes on the basis of their location in the \u003cem\u003eGI\u003c/em\u003e coding sequence [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, mutations were introduced into two distinct regions of \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e in the potato cv. D\u0026eacute;sir\u0026eacute;e. Corresponding to the location of sequences targeted by RNAs, the mutations truncated or introduced deletions into GI.04 around the 567\u0026ndash;632 and 816\u0026ndash;869 a.a. positions, whereas in GI.12, these were around the 618\u0026ndash;694 and 834\u0026ndash;863 a.a. positions.\u003c/p\u003e \u003cp\u003ePlants of one GI.04 mutant line and two GI.12 lines with truncated proteins and deletions in the 816\u0026ndash;869 and 834\u0026ndash;863 a.a. regions, respectively, were shorter and remained green for a longer time than D\u0026eacute;sir\u0026eacute;e. Previously, it was demonstrated that ELF3 of \u003cem\u003eArabidopsis\u003c/em\u003e interacts with the GI at the N-terminal (1\u0026ndash;507 a.a.) and C-terminal (801\u0026ndash;1173 a.a.) regions and that a mutation in \u003cem\u003eELF3\u003c/em\u003e disturbs the pattern of GI cyclic accumulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, the lack or weakness of the GI\u0026ndash;ELF3 interaction may have changed the life cycle and morphology of eGI.04/1, eGI.12/1 and eGI.12/2 mutant plants. Interestingly, the complete loss of the GI.04 C-terminal region in eGI.04/2 and eGI.04/3 resulted only in plant shortening but not in a change in the duration of the vegetation period. The phenotype of eGI.12/3 did not differ from the phenotype of D\u0026eacute;sir\u0026eacute;e, which might be explained by the presence of a GI.12 allele with only one a.a. deletion in eGI.12/3.\u003c/p\u003e \u003cp\u003eIt was previously shown that the LOV domain protein LKP2 binds to the 563\u0026ndash;789 a.a. region of \u003cem\u003eArabidopsis\u003c/em\u003e GI [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This part is well conserved both in GI.04 and GI.12 and has the same motives detected in \u003cem\u003eArabidopsis\u003c/em\u003e. In contrast, the NLS sequences identified in \u003cem\u003eArabidopsis\u003c/em\u003e GI, with two exceptions in GI.04, are not present in the GI proteins of D\u0026eacute;sir\u0026eacute;e. This fact, however, does not exclude the possibility that the GI proteins of D\u0026eacute;sir\u0026eacute;e also reside in the nucleus, as several non-classical NLSs are known from different species [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, the \u003cem\u003eGI\u003c/em\u003e genes of tomato (\u003cem\u003eS. lycopersicum\u003c/em\u003e), \u003cem\u003eSlGI.04\u003c/em\u003e and \u003cem\u003eSlGI.12\u003c/em\u003e, encode almost identical a.a. sequences with the \u003cem\u003eGI\u003c/em\u003e genes of potato, and it has been shown very recently that SlGI.04 and SlGI.12 reside in the nucleus [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGI.04 and GI.12 mutants with truncation or deletion in the 567\u0026ndash;632 a.a. and the 618\u0026ndash;694 a.a. region, respectively, differ in phenotype. The mGI.04/1 mutant, which has two truncated proteins and the largest deletion in the binding sites of LOV domain proteins, has the short plant and delayed senescence phenotype, whereas the other two mGI.04 mutants do not differ from D\u0026eacute;sir\u0026eacute;e. In contrast, all three mGI.12 mutants presented signs of early senescence. Similar to the mGI.04/1 plants, all mGI.12 plants were shorter than the control plants. The mutant phenotype is the most pronounced in mGI.12/1, which has four copies of truncated GI.12 lacking the majority of LOV domain binding sites, whereas mGI.12/2 and mGI.12/3 carry only three truncated copies and an almost wild-type GI.12 allele. Owing to this truncation, it is highly probable that the blue-light receptors cannot bind to the truncated mGI proteins in D\u0026eacute;sir\u0026eacute;e, which leads to perturbation of oscillation of the plant circadian clock and changes in the growth and life cycle length of the plants.\u003c/p\u003e \u003cp\u003eIsolation of double GI mutants was attempted via cotransformation. One mutant, mGI.412/1, carried mutations in all four \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e alleles; however, mutations in two \u003cem\u003eGI.12\u003c/em\u003e copies led to only minor changes at the protein level. Since this was the only mutant that carried mutations in each \u003cem\u003eGI\u003c/em\u003e allele out of the 216 tested ones, we assume that the complete loss of GI function would be lethal for the plants.\u003c/p\u003e \u003cp\u003eIn correlation with the extent of mutations, mGI.412/1 plants presented the greatest phenotypical changes compared to D\u0026eacute;sir\u0026eacute;e plants. The mGI.412/1 plants were very short and died very early. The phenotypes of the mGI.412/2 and mGI.412/3 mutants were similar to that of the mGI.412/1 mutant, with a lower intensity. Thus, we concluded that, as in \u003cem\u003eArabidopsis\u003c/em\u003e, the GI proteins in D\u0026eacute;sir\u0026eacute;e influence the developmental processes and life cycles of the plants.\u003c/p\u003e \u003cp\u003eDespite the 84% identity at the transcript level, mutations in the LOV domain binding site resulted in different changes in the phenotypes of the plants. Early senescence was characteristic of only the mGI.12 and mGI.412 mutants, while the mGI.04 mutants were similar to D\u0026eacute;sir\u0026eacute;e or even remained green for a longer time. In silico promoter analysis revealed binding sites for EVENING ELEMENT and ABSCISIC ACID RESPONSE ELEMENT-LIKE elements related to circadian regulation in both \u003cem\u003eGI\u003c/em\u003e promoters. However, the two genes differ in several other \u003cem\u003ecis-\u003c/em\u003eacting regulatory elements as well as in organ-specific expression and responses to abiotic stresses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which may explain the different effects of mutations at the phenotype level.\u003c/p\u003e \u003cp\u003eCDF1 was found to regulate tuberisation and plant life cycle length. The truncated variants of CDF1 exhibit enhanced protein stability caused by the inability to bind to GI and FKF1. The overexpression of truncated CDF1 leads to early tuberisation and a short life cycle in potato [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, the putative binding site of FKF1, the LOV domain binding site, was mutated in mGI mutants, which might have led to the accumulation of CDF1 and the formation of a similar phenotype to that of potatoes overexpressing the truncated CDF1. Nevertheless, only mGI.12/1 and mGI.412/1, the mutants with the shortest lifetimes, tuberised significantly earlier than D\u0026eacute;sir\u0026eacute;e. Compared with that of D\u0026eacute;sir\u0026eacute;e, the vegetative development of mGI.412.1 was so much shorter that it reduced the tuber yield.\u003c/p\u003e \u003cp\u003eAntisense repression of approximately 50% of D\u0026eacute;sir\u0026eacute;e \u003cem\u003eGI.04\u003c/em\u003e did not influence tuber formation or yield but did cause a reduction in tuber colour and anthocyanin content [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, none of the GI mutants presented significantly lower amounts of anthocyanins in their tuber skin than did D\u0026eacute;sir\u0026eacute;e tubers. This result suggests that those domains of GI proteins that might be important in terms of the regulation of anthocyanin synthesis remained intact in the GI mutants.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe CRISPR/Cas9 system can be successfully used even in tetraploid potato to generate mutations in all four copies of a gene. Cotransformation can be used to obtain mutations in two genes simultaneously. Large as well as small deletions, inversions or nucleotide substitutions can be obtained. The efficiency of targeted mutagenesis, however, may depend on the PAM sequence adjacent to the site targeted by the gRNA. Analysing the effects of mutations revealed that both \u003cem\u003eGI\u003c/em\u003e genes, \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e, influence plant development. However, the influence of \u003cem\u003eGI.12\u003c/em\u003e is more pronounced than the influence of \u003cem\u003eGI.04\u003c/em\u003e. Certain mutations at 3\u0026rsquo; to the LOV\u0026ndash;NLS domain reduced the size and increased the lifetime of D\u0026eacute;sir\u0026eacute;e plants, whereas mutations in LOV-NLS domain of \u003cem\u003eGI.12\u003c/em\u003e shortened the life cycle length of plants and, in certain cases, led to an increase in the earliness of tuberisation. In the absence of the GI.04 protein, mutations in \u003cem\u003eGI.12\u003c/em\u003e not only shorten the size of the plants and duration of the vegetative period but also result in a reduction in tuber yield. Nevertheless, one of the GI.12 mutants, mGI.12/1, tuberised earlier than the control without a yield penalty under greenhouse conditions. After further detailed investigation, this mutant might be a good candidate for breeding purposes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eAknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to M. Kiss for the excellent assistance in propagation, transformation and greenhouse growth of potato plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This study does not involve any human or animal testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by the National Research, Development and Innovation Office \u0026ndash; NKFIH (grant number: K_146328).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eF.K.-R., K.O., Z.G.T. and V.V. performed the experiments, analysed the data, reviewed and edited the manuscript. Z.B. conceptualised the research topic, analysed the data, drew the figures and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors are grateful to M. Kiss for the excellent assistance in propagation, transformation and greenhouse growth of potato plants.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eDNA sequences of the *GIGANTEA* genes *GI.04* and *GI.12* analysed during the current study are available in the DDBJ repository under the accession numbers LC914694\u0026nbsp;and LC914695, respectively. DNA sequences of the PCR fragments obtained from the genomic DNA of mutant lines are deposited in the ZENODO database and can be cited using the DOI 10.5281/zenodo.18593396. All research data and materials supporting the results and analysis of the article could be shared upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDutta M, Mali S, Raturi V, Zinta G. 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New Phytol. 2024;242:2541\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.19525\u003c/span\u003e\u003cspan address=\"10.1111/nph.19525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CRISPR/Cas9, Mutation mapping, Plant development, Solanum tuberosum, Tuber skin colour","lastPublishedDoi":"10.21203/rs.3.rs-8542741/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8542741/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e Earliness of tuberisation is an important agronomic trait. It was demonstrated earlier that GIGANTEA (GI), a plant-specific nuclear protein that regulates multiple processes, is indirectly involved in tuberisation in a diploid potato. Commercial potatoes, including the cultivar D\u0026eacute;sir\u0026eacute;e, are tetraploids and carry two copies of \u003cem\u003eGI\u003c/em\u003e genes, designated \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e. The aim of our study was to explore the role of the two \u003cem\u003eGI\u003c/em\u003e genes in D\u0026eacute;sir\u0026eacute;e in relation to tuberisation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e To obtain information on \u003cem\u003eGI.04\u003c/em\u003e and \u003cem\u003eGI.12\u003c/em\u003e functions in D\u0026eacute;sir\u0026eacute;e, mutations were introduced into the two genes individually and simultaneously using the CRISPR/Cas9 system. Two different segments of the genes were targeted by gRNAs. PCR was used for mutant identification. Three mutants from each mutagenesis were selected, and the mutations were localised at the DNA sequence level. The phenotype and tuberisation of the plants were tested by growing the plants in pots in a greenhouse. The individual mutations affecting all four copies of the genes, in general, reduced plant size. Plants of one GI.04 mutant line and two GI.12 mutant lines with truncated proteins and deletions in the 816\u0026ndash;869 and 834\u0026ndash;863 amino acid (a.a.) regions, respectively, were shorter and remained green for a longer time than D\u0026eacute;sir\u0026eacute;e. GI.04 and GI.12 mutants with truncation or deletion in the 567\u0026ndash;632 a.a. and 618\u0026ndash;694 a.a. regions, respectively, differ in phenotype; one GI.04 mutant had longer, whereas all three GI.12 mutants and the double mutants had shorter life cycles. However, only one of the GI.12 mutants and one of the double mutants tuberised earlier than D\u0026eacute;sir\u0026eacute;e. The tuber yield of the double mutant with the shortest life time was lower than that of D\u0026eacute;sir\u0026eacute;e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e Both \u003cem\u003eGI\u003c/em\u003e genes of D\u0026eacute;sir\u0026eacute;e influence the development and life cycle length of plants. The influence of GI.12 is more pronounced than the influence of GI.04. In conjunction with the shortened lifetime, the onset of tuberisation occurs earlier.\u003c/p\u003e","manuscriptTitle":"Exploring the role of the two GIGANTEA genes in the life cycle length and tuberisation of the potato cultivar Désirée","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 07:53:49","doi":"10.21203/rs.3.rs-8542741/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-23T02:07:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26754225008354334519272949623749736163","date":"2026-04-10T09:35:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T03:33:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198531731776579460378845091561188588342","date":"2026-03-23T04:48:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T09:54:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-24T06:20:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T08:48:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-10T14:38:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-02-10T13:37:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09ea9ffd-caf5-4d88-8467-ec020083c8ab","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T07:53:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 07:53:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8542741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8542741","identity":"rs-8542741","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}