Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors

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Results We performed whole-genome sequencing of CRFK and PG-4 cells. We analyzed the resulting data to infer the phenotype of the individual from which the cells were derived, specifically for the coat color, coat length, coat pattern, and iris color. Our data suggested that CRFK cells originated from a cat with long chocolate-brown fur lacking stripes and with non-blue irises; PG-4 cells originated from a cat with long bicolored white and dark brown fur without stripes, and with non-blue irises. Analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs. Conclusions Variants of the genes affecting coat phenotype and iris color may influence physiological functions throughout the body. These results shed light on the previously unknown genetic background of commonly used feline cultured cells and the phenotype of the donor individuals. These insights may facilitate a more accurate interpretation of data derived from feline cultured cells and inform guidelines to establish cell lines with various genotypes (for example, disease-associated variants) through genome editing, which will help elucidate genetic factors affecting resistance to infectious diseases and cancer, the risk of internal organ afflictions, and genetic diseases such as deafness in cats with white fur and blue irises. genomics coat pattern iris color RNA sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Domestic cats ( Felis catus ) are widely kept as companion animals around the world, and the phenotypic diversity of their coat color, coat pattern, coat length, and iris color has been a topic of interest to breeders and scientists alike. Recent advances in genomics have markedly improved our understanding of the genetic basis controlling these diverse phenotypes, and the respective genotype-phenotype relationships are being increasingly elucidated. 1 Examples include the TYR gene, which is the color locus associated with albinism and a color point phenotype 2 – 4 ; the TYRP1 gene, conveying brown coat color 2 , 5 ; the MC1R genes, associated with in red coat color 6 ; the KIT gene, determining white coat and white spotting 7 – 11 ; the ARHGAP36 gene, involved in orange coat color 12 , 13 ; the MLPH gene, causing dilute pigmentation 14 ; the ASIP gene, controlling the expression of the agouti pattern 15 ; the LVRN gene, determining the type of tabby pattern 16 ; the Dkk4 gene, associated with the ticked tabby pattern 17 ; the FGF5 gene, involved in long-fur variants 18 , 19 ; the KRT71 and LPAR6 genes, involved in hairless and rexing (curly hair) phenotypes 20 – 22 ; and the PAX3 gene, which is associated with blue irises 23 , 24 . Furthermore, technological advancements such as long-read sequencing facilitated the construction of a more continuous and accurate draft genome of the domestic cat 25 – 27 , and a plethora of genetic polymorphisms associated with disease have been identified 17 , 25 , 28 – 32 . Immortalized cell lines are an indispensable tool in current biomedical and veterinary science. Crandell-Rees Feline Kidney (CRFK) cells, derived from the kidneys of a domestic cat 33 – 34 , have been used for numerous virological studies, including feline calicivirus, 35–37 feline immunodeficiency virus 38 – 41 , feline coronavirus 42 – 45 , and various feline endogenous retroviruses such as RD114 46–48 , as well as for vaccine development 49 – 51 . PG-4 cells, derived from feline fetal astrocytes, have also played an important role in retrovirus research 52 – 53 . These cell lines have been passaged for decades in laboratories worldwide and have facilitated numerous scientific breakthroughs. However, despite their widespread use, no detailed information on the genetic background of the donor individuals, particularly with regard to their phenotype, such as coat and iris color, is available. Genomic information on such cell lines is crucial for a deeper understanding of their biological properties, and elucidating the phenotypes of their donors may provide new insights into the influence of specific genotypes on cell properties. In this study, we performed whole-genome sequencing of CRFK and PG-4 cell lines. The sequencing data were used for in-depth analysis of the nucleotide sequences of genes known to be associated with coat and iris phenotypes (i.e., TYR , TYRP1 , MC1R , KIT , ARHGAP36 , MLPH , ASIP , LVRN , DKK4 , FGF5 , KRT71 , LPAR6 , and PAX3 ; Table 1 ). Based on the genotyping results for each gene, we inferred the previously unknown phenotypes of the donor individuals. Furthermore, analysis of publicly available RNA-seq data confirmed that these genes were expressed in skin and eyes as well as in various other tissues throughout the body. Our results provide fundamental insights into the genetic background of widely used feline cell lines, offering valuable information that will be beneficial for interpreting the results of future research using these cell lines, such as informing guidelines to establish cell lines with various genotypes, including disease-associated genotypes, through genome editing. Table 1 Phenotypic traits of coat and iris conferred by DNA variants, and genotypes of CRFK and PG-4 cells. List of genes and phenotypes focused on in this study, mutation position on CDS coordinate and on genomic coordinate provided by Fca126_mat1.0 and AnAms1.0, and a summary of genotypes of CRFK and PG-4 cells obtained by the present study. Locus Gene Phenotype (Alleles) Mutation in CDS (or related noncoding region): F.catus_Fca126_mat1.0 Genome: F.catus_Fca126_mat1.0 Genome: AnAms1.0 Genotype in CRFK Genotype in PG-4 Color TYR 2 – 4 Burmese (c b ) c.679G > T D1: g.44022819C > A D1: g.46081417C > A + / + + / + Siamese (c s ) c.904G > A D1: g.44013031C > T D1: g.46071661C > T + / + + / c s Albino (c a ) c.939del D1: g.44012996del D1: g.46071626del + / + + / + Mocha (c m ) c.820_936delinsAATCTC D1: g.44012836_44012998delinsGAGATT D1: g.46071466_46071628delinsGAGATT + / + + / + Brown TYRP1 2,5 Chocolate (b ch ) c.1261 + 5G > A D4: g.38142265G > A D4: g.39391308G > A + / + + / + Chocolate (b c ) c.8C > G D4: g.38129873C > G D4: g.39378919C > G b c / b c + / + Cinnamon (b i ) c.298C > T D4: g.38130163C > T D4: g.39379209C > T + / + + / + Amber MC1R 6 Red (e) c.250G > A E2: g.61570294G > A E2: g.62623146G > A + / + + / + White KIT 7 – 11 White (w w ) (Intron 1: FERV1-LTR insertion) B1: g.161388687_161388688insFERV1-LTR B1: g.163130991_163130992insFERV1-LTR + / + + / + White spotted (w s ) (Intron 1: FERV1 insertion) B1: g.161388687_161388688insFERV1 B1: g.163130991_163130992insFERV1 + / + + / w s White glove (w g ) c.1035_1036delinsCA B1: g.161337385_161337386delinsTG B1: g.163079561_163079562delinsTG + / + + / + Salmiak (w a ) (KIT-KDR Intergenic region: 95 kb deletion) B1: g.161142880_161237957del B1: g.162885414_162980052del + / + + / + Orange Arhgap36 12,13 Orange (o) (Intron 1: 5 kb deletion) (Lacking) X: g.109186183_109191258del + / + + / + Dilution MLPH 14 Dilute (d) c.83del C1: g.218197448del C1: g.219535339del + / d + / + Agouti ASIP 15 Nonagouti (a) c.123_124del A3: g.24831083-24831084del A3: g.25283750_25283751del a / a a / a Tabby LVRN 16 Blotched tabby (t b ) c.176C > A A1: g.94477275C > A A1: g.97133509C > A + / + + / + Blotched tabby (t b ) c.416C > A A1: g.94477515C > A A1: g.97133749C > A + / + + / t b Blotched tabby (t b ) c.682G > A A1: g.94477781G > A A1: g.97134015G > A + / + + / + Blotched tabby (t b ) c.2522G > A A1: g.94536796G > A A1: g.97192983G > A t b / t b + / t b Ticked Dkk4 17 Ticked (ti) c.188G > A B1: g.40429492G > A B1: g.41805975G > A + / + + / + Ticked (ti) c.53C > T B1: g.40428846C > T B1: g.41805329C > T + / + + / + Longhair FGF5 18,19 Long fur (l) c.356_357insT B1: g.139634675_139634676insA B1: g.141368532_141368533insA + / + + / + Long fur (l) c.406C > T B1: g.139645946G > A B1: g.141379803G > A + / + + / + Long fur (l) c.474del B1: g.139634261del B1: g.141368118del + / + + / l Long fur (l) c.475A > C B1: g.139634260T > G B1: g.141368117T > G l / l l / + Long fur (l) c.577G > A B1: g.139634158C > T B1: g.141368015C > T + / + + / + Hairless Rexing KRT71 20,21 Hairless (hr) c.816 + 1G > A B4: g.78941699C > T B4: g.80471148C > T + / + + / + Rexing (re) c.1108-4_1184del, c.1184_1185insAGTTGGAG, and c.1196_1197insT B4: g.78939390_78939470del, B4: g.78939389_78939390insCTCCAACT, and B4: g.78939377_78939378insA B4: g.80468919_80468919del, B4: g.80468918_80468919insCTCCAACT, and B4: g.80468826_80468827insA + / + + / + Rexing (re) c.445-1G > C B4: g.78943283C > G B4: g.80472733C > G + / + + / + Rexing LPAR6 22 Rexing (re) c.250_253del A1: g.22865220_22865223del A1: g.23372953_23372956del + / + + / + Blue iris PAX3 23,24 Blue iris (bi) (Inrton4: FERV1-LTR homolog insertion) C1: g.205833101_205833102insN[395] C1: g.207180017_207180018insN[395] + / + + / + Blue iris (bi) (Intron4: RD114-LTR homolog insertion) C1: g.205834854_205834855insN[433] C1: g.207181773_207181774insN[433] + / + + / + Methods Cell lines and culture conditions CRFK cells (JCRB9035, RRID: CVCL_2426) and a feline sarcoma-positive leukemia-negative (S + L−) astrocyte cells termed PG-4 (S + L-) (JCRB9125, RRID: CVCL_3322) were obtained from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan). CRFK cells were cultured in Dulbecco's Modified Eagle Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 IU/mL), streptomycin (100 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA), and 1% non-essential amino acids (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37°C and 5% CO₂. PG-4 cells were cultured in McCoy’s 5A Modified Medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), penicillin (100 IU/mL), and streptomycin (100 ng/mL) (Sigma-Aldrich) at 37°C and 5% CO₂. For cell passaging, cells were washed using BASIC DPBS (no calcium or magnesium; Gibco) and then treated with TrypLE™ Express Enzyme (1X; Gibco) for 5 min at 37°C and 5% CO₂ conditions. Paired-end short-read whole-genome sequencing Genomic DNA was extracted from logarithmically growing cells (approximately 1×10⁶ cells) using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity (A260/A280 ratio) of the extracted DNA were measured using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Genomic DNA extracted from CRFK cells and PG-4 cells was used for library preparation and paired-end sequencing (150 bp read length) at Novogene (Beijing, China) using a Rapid Plus DNA Lib Prep Kit for Illumina V2 (Illumina) and an Illumina NovaSeq X Plus platform (Illumina). Library quality control was performed by Novogene using qPCR and fragment size analysis according to Novogene’s standard protocols. The raw sequence data were obtained as .fastq files. Mapping of paired-end reads and quantification Adapter sequences were trimmed, and low-quality reads were removed from the original .fastq files using fastp software (ver. 0.21.0) with default parameters. Reads in processed .fastq files were mapped to the reference genome sequence of Felis catus , F.catus_Fca126_mat1.0 (RefSeq assembly GCF_018350175.1) or AnAms1.0 (Genbank assembly GCA_013340865.2) 27 using bwa mem software (ver. 0.7.19) with the setting “-R "@RG\tID:sample1\tSM:sample1\tLB:lib1\tPL:ILLUMINA"” and default parameters otherwise. Mapping results were obtained as .sam files. Text data in .sam files were transformed to binary data in sorted .bam files using Samtools (ver. 1.7) with default parameters. Index files were created for the obtained .bam files in the form of .bam.bai files. Small variations in coat morphology-related genes The variations of coat morphology-related phenotypes of the donor cats from which CRFK and PG-4 cell lines originated were inferred based on the comparisons between the wild-type sequences and mapped sequences on variant positions of each coat morphology-related gene (Table 1 ). Coat phenotype-related polymorphisms in TYR , TYRP1 , MC1R , KIT , MLPH , ASIP , LVRN , Dkk4 , FGF5 , KRT71 , and LPAR6 genes, which relate to phenotypes such as the color, brown, red, white glove, dilution, agouti, tabby, ticked, long-fur, hairless, and rexing (Table 1 ), were examined using alignments of paired-end sequence reads with wild-type sequences. Large deletions in intron 1 of ARHGAP36 and the intergenic region between KIT and KDR genes The deletion of a part of intron 1 of the ARHGAP36 gene (X: 109,186,183_109,191,258 in AnAms1.0; Table 1 ) was reported to produce orange coat color 12 , 13 , and that of the intergenic region between the KIT and KDR genes (B1: 161142880_161237957 in F.catus_Fca126_mat1.0, Table 1 ) produces salmiak coat color 11 . The occurrence of these deletions in CRFK and PG-4 was estimated by comparing the mapped read number distributions between these regions and their neighboring regions with the same region length. It should be noted that F.catus_Fca126_mat1.0 has a deletion of the genomic region corresponding to X: 109186183_109191258 in AnAms1.0 (Table 1 ). Thus, AnAms1.0 (or felCat9 25 ) was used as the reference genome for analysis of the orange locus. Insertions of FERV1-LTR and RD114-LTR into PAX3 The insertions of homologous sequences of the long terminal repeat (LTR) of the feline endogenous retrovirus 1 (FERV1), termed FERV1-LTR, into a specific region in intron 4 of PAX3 (C1: g.205833101_205833102 in F.catus_Fca126_mat1.0, Table 1 ) or the LTR of RD114 retrovirus, termed RD114-LTR, into another specific region in the same intron (C1: g.205834854_205834855 in F.catus_Fca126_mat1.0, Table 1 ) are associated with blue iris color in cats. 23 , 24 The occurrence of such insertions was estimated by extracting the genome reads that were mapped in both FERV1-LTR and intron 4 of PAX3 or both RD114-LTR sequences and intron 4 of PAX3 . The genome sequence of RD114-LTR was obtained from the NCBI database 54 . Long-read whole-genome sequencing Genomic DNA of CRFK cells and PG-4 cells was extracted for long-read genome sequencing using the NucleoBond HMW DNA kit (TaKaRa, Tokyo, Japan), and libraries for the Oxford Nanopore Technologies (ONT, UK) sequencer were constructed using a ligation library preparation kit (SQK-LSK114, ONT). All procedures were performed according to the manufacturer’s instructions, and the quality and molecular weight of the genomic DNA were measured using the Qubit fluorometric quantification system (Thermo Fisher Scientific). Sequencing was conducted using a PromethION sequencer (PromethION 2 Solo, ONT) and R10.4.1 flow cells. POD5 files were basecalled to .fastq files using MinKNOW software v25.05.14. Insertions of FERV1 homolog into KIT examined by long-read sequencing The insertion of FERV1 into a specific region of intron 1 of the KIT gene (B1: 161388687_161388688 in F.catus_Fca126_mat1.0, Table 1 ) and that of FERV1-LTR make the coat color of cats white-spotted and white throughout, respectively 8 . The occurrence of such insertions in CRFK and PG-4 cells was estimated by mapping the reads of long-read sequencing to the genome sequence named KIT-FERV1 sequence using minimap2 (ver. 2.30), where the KIT-FERV1 sequence is a part of the KIT intron 1 sequence with the insertion of the FERV1 sequence, as shown in Supplementary Fig. 1 of the study by David et al 8 . Transcriptome data analysis of coat phenotype- and iris color-related genes in various tissues of domestic cats Mapped RNA-seq data on the F.catus_Fca126_mat1.0 were obtained in .bam and .bam.bai formats for various tissues from the Ensembl public database 55 , specifically, the file transfer protocol (ftp) site 56 . The annotation file of the F.catus_Fca126_mat1.0 in gene transfer format (GTF) was obtained from the NCBI database (Bethesda, MD, USA). In the file, the chromosome numbers were converted from NC_058368.1, NC_058369.1, etc. to A1, A2, etc. Using FeatureCounts with the options—M-O—fraction, 57 read counts data for each gene described in the GTF file were obtained from the .bam and .bam.bai formatted files. This count data could also be obtained from a file “F.catus_Fca126_mat1.0.ENA_gene_exp_mat_by_FeatureCounts.csv” deposited in F.catlas 58 . From these counts, the transcript level of each gene was calculated by dividing the read count by the total exon length. The total transcript level was defined as the sum of all the transcript levels of the genes. Based on this result, the transcripts per million (TPM) of each gene were calculated as 1,000,000*(transcript level)/(total transcript level). Results Short- and long-read whole-genome sequencing For CRFK and PG-4 cells, the total output of raw sequence data of 150 bp paired-end sequencing was 60.6 Gb and 60.1 Gb, and the [average] \(\:\:\pm\:\:\) [standard deviation] of mapping depth of the reads for F.catus_Fca126_mat1.0 were \(\:23.59\pm\:9.79\) and \(\:23.45\pm\:8.17\) , respectively; the total output of raw sequence data of long-read sequencing was 9.6 Gb and 11.8 Gb, N50 values were 23.5 kb and 38.2 kb, and the [average] \(\:\:\pm\:\:\) [standard deviation] of mapping depth of reads for F.catus_Fca126_mat1.0 were \(\:3.91\pm\:3.11\) and \(\:4.79\pm\:3.34\) , respectively. The average and standard deviations specified above were estimated through the entire genome region without the regions with singular sequences where the read coverage exhibited > 100. Predicted phenotype of the CRFK cell donor The mapping of paired-end reads from the whole-genome sequences of the CRFK cell revealed that most coat morphology-related genes were conserved (Table 1 ). However, the following variations were identified in mapped genome reads from CRFK cells: a homozygous mutation of c.123_124del of the ASIP gene (the nonagouti allele; Fig. 1 ), a homozygous mutation of c.475A > C of the FGF5 gene (the long coat allele; Fig. 2 ), a homozygous mutation of c.2522G > A of the LVRN gene (the blotched-tabby allele; Fig. 3 ), a homozygous mutation of c.8C > G of the TYRP1 gene (the chocolate-blown allele; Fig. 4 ), and a heterozygous mutation of c.83delT of the MLPH gene (the dilute allele; Fig. 5 ). We found no 5 kbp deletion at intron 1 of the ARHGAP36 gene and no 95 kbp deletion at the intergenic region between the KIT and KDR genes. We also found no genome reads that could be mapped in both the PAX3 intron 4 and FERV1-LTR or both the PAX3 intron 4 gene and RD114-LTR sequences. Additionally, four reads from the long-read sequencing mapped to the KIT-FERV1 sequence, but these reads contained no insertions of FERV1 and FERV1-LTR (Fig. 6 ). The abovementioned homozygous mutation of the TYRP1 gene is known to exhibit chocolate-brown coat color 2 . Additionally, the homozygous mutation of the FGF5 gene was known to exhibit long fur 18 . However, the mutation of the LVRN did not influence coat morphology in this case because of the mutation of the ASIP gene that suppresses the stripe pattern on the cat’s skin coat, and the mutant genotype of the MLPH gene was recessive. 1 Therefore, the cat from which the CRFK cells originated is expected to have had long, chocolate brown, unstriped fur and non-blue eyes (Fig. 7 ). Predicted phenotype of the PG-4 cell donor Mapped paired-end reads from the whole-genome sequences of the PG-4 cell confirmed that most coat morphology-related genes were conserved (Table 1 ) but identified following variations: a homozygous mutation of c.123_124del of the ASIP gene (Fig. 1 ), a compound heterozygous mutation of c.474delT and c.475A > C of the FGF5 gene (Fig. 2 ) where one allele exhibits c.474delT and the other allele exhibits c.475A > C, the heterozygous mutation of c.2522G > A of LVRN gene (Fig. 3 ), a heterozygous mutation of c.904G > A of the TYR gene (the Siamese-color allele; Fig. 8 ), and a homozygous mutation of c.416C > A of the LVRN gene (Fig. 9 ). We found no large deletions at intron 1 of the ARHGAP36 gene and the intergenic regions between the KIT and KDR genes, as was also the case for CRFK. We also found no genome reads that could be mapped in both the PAX3 intron 4 and FERV1-LTR or both the PAX3 intron 4 gene and RD114-LTR sequences. We found that six long-read sequences mapped to the KIT-FERV1 sequence, and four of them contained the insertions of the more than 7 kbp FERV1 homolog sequence (Fig. 6 ). This suggested the PG-4 genome contains the heterozygous insertion of the entire FERV1 homolog sequence. Note that the mutant genotype of the TYR gene was recessive, and the two heterozygous mutations of the LVRN do not influence coat morphology in this case because of the mutation of the ASIP gene, the same as in the case of CRFK 1 . However, the effect of FERV1 is known to be dominant 8 . Additionally, the compound heterozygous mutation of the FGF5 gene produces long fur 18 . Therefore, the cat from which the PG-4 cells originated is expected to have had white and dark brown, unstriped, long fur and non-blue irises (Fig. 7 ). Expression features of coat phenotype- and iris color-related genes in various tissues The analysis of publicly available RNA-seq data from various organs of domestic cats in the Ensembl database ( https://ftp.ensembl.org/pub/data_files/felis_catus/F.catus_Fca126_mat1.0/rnaseq/ ) revealed that coat phenotype-related genes are expressed in various organs at levels (TPM) comparable to or greater than those in the skin (Fig. 4 , Table S2 ). For example, TYR , TYRP1 , and MLPH were highly expressed in the ear tip, retina, and optic nerve, and MC1R , KIT , DKK4 , and FGF5 were highly expressed in the brain. KIT , DKK4 , and LPAR6 tend to be expressed throughout the body, with prominent expression in the reproductive organs. Additionally, ARHGAP36 was highly expressed in the spinal cord, and ASIP , along with MLPH , was highly expressed in the lungs. The iris color-related gene PAX3 was also highly expressed in the cerebellum, in addition to visual organs, and, along with DKK4 , in the kidney and embryonal tissue. Discussion We performed whole-genome sequencing of CRFK, a cultured feline kidney cell line, and PG-4, a feline fetal astrocyte-derived cell line, and analyzed the genome data to determine the coat color, hair length, coat pattern, and iris color of the donor individuals. The results suggested that the CRFK cell line originated from a cat with long dark brown hair without stripes and non-blue irises, and the PG-4 cell line came from a cat with long fur, dark brown and white bicolor coat without stripes, and non-blue irises. Coat morphology and iris color are phenotypes that can be readily identified and used to predict genotypes. However, the expressions of these genes are not limited to the skin and eyes; rather, they are expressed in various tissues throughout the body (Fig. 10 ). Therefore, differences in these genotypes may contribute to disease susceptibility in specific organs. The effects of these genetic variations are likely weaker than those of the rearing environment and may not be significant, especially in young, healthy individuals. However, as domestic cats have become longer-lived in recent years, individual-specific risks may become more apparent, and the underlying genotypes may exert increasing effects as the cats age. Therefore, elucidating the relationships between genotypes underlying readily recognizable phenotypes and disease risk is crucial from the perspectives of both medical care and welfare, such as prevention and symptom suppression/alleviation. Results from research on cultured cells to elucidate disease mechanisms and develop therapeutic drugs must be interpreted with caution, i.e., not only on the experimental results obtained using established, immortalized cells but also with regard to their genotype and expression profiles of the genes harboring polymorphisms in various tissues and organs. However, so far, such information has not been available, even for certain frequently used cell lines such as CRFK. The present study provides such information and facilitates a more comprehensive interpretation of research on feline cultured cell lines. Furthermore, with the availability of detailed genomic information, genome editing can be applied to these cell lines to establish various derivative lines that differ only in specific aspects, e.g., regarding coat morphology or other traits, and compare their properties. This is expected to advance research on the regulatory mechanisms of coat color, for example, the validation of the role of the novel KIT isoform caused by FERV1-LTR insertion in the formation of white coat 59 , as well as the relationships between the visually identifiable phenotypes examined in the present study and disease risk and treatment methods. Simultaneously, such detailed genomic information can inform guidelines for establishing cell lines with various disease-associated genotypes through genome editing. Establishing such derivative cell lines can help elucidate genetic factors associated with resistance to infectious diseases and cancer, the risk of internal organ afflictions, as well as genetic diseases such as deafness in cats with white fur and blue irises. It should be noted that the karyotype of CRFK differs from that of the normal domestic cat genome. 33 It is thus possible that the karyotype of PG-4 cells, which has not been reported, differs from the normal domestic cat genome. However, using only whole-genome analysis of the present sequence read data, approximately 60 Gb paired-end sequencing data and 10 Gb long-read data, it was difficult to detect such large structural variations. Similarly, it was difficult to identify the accurate sequences and locations of various long repeat sequences, endogenous retroviral sequences, and their homologs because of the deficiency of the read coverage, particularly in the long-read coverage. However, these sequences may also be associated with various diseases, as exemplified by the involvement of FERV1-LTR in deafness in cats 8 , 23 , 24 and the positive and negative roles of enFeLV-LTR and enFeLV-env in resistance to exogenous feline leukemia virus 60 – 62 . The larger volume of ongoing long-read sequencing is expected to resolve these issues in the future. Conclusions We performed whole-genome sequencing of CRFK and PG-4 cells and inferred the phenotype of the donors of these cells. We suggested that CRFK cells originated from a cat with long, chocolate-brown fur lacking stripes and non-blue irises; PG-4 cells originated from a cat with long, bicolored white and dark brown fur without stripes, and with non-blue irises. Additionally, analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs, indicating that variants of these genes, which affect coat phenotype and iris color, may influence physiological functions throughout the body. These insights may facilitate a more accurate interpretation of data derived from feline cultured cells and inform guidelines to establish cell lines with various disease-associated variants through genome editing, which will help elucidate genetic factors affecting resistance to infectious diseases and cancer, the risk of internal organ afflictions, and genetic diseases. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Conflicting interests The authors have no conflicting interests to declare. Funding This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grants (award numbers 24K01783 (T. I.) and 24K09248 (A. A.)). Author Contribution Conceptualization: A.A., Data curation: T.K., A.K., R.H., and T.I., Formal analysis: G.T. and R.G., Funding acquisition: A.A., Investigation: G.T., R.G., T.K., R.H., and A.A., Methodology: G.T., R.G., T.K., T.I., and A.A., Project administration: N.S. and A.A., Supervision: N.S. and A.A., Validation: R.G. and A.A., Visualization: T.G., N.S., and A.A., Writing - original draft: A.A., Writing - review & editing: T.G., T.K., T.I., and N.S. Acknowledgements Not applicable. Data Availability These raw read data of the short and long read sequencings were deposited in the DDBJ Sequence Read Archive (DRA) with BioProject accession PRJDB37530 (DRR748391-DRR748394, DRR786948, and DRR786949). All other data generated or analyzed during this study were included in this published article (and Supplementary Information files). References Lyons LA. DNA mutations of the cat: The good, the bad and the ugly: The good, the bad and the ugly. 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Supplementary Files TableS1.xlsx TableS2.xlsx CRFKPG4genomeCAHGsup.docx Cite Share Download PDF Status: Published Journal Publication published 06 Mar, 2026 Read the published version in Companion Animal Health and Genetics → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviews received at journal 20 Nov, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers invited by journal 17 Nov, 2025 Editor assigned by journal 17 Nov, 2025 Submission checks completed at journal 14 Nov, 2025 First submitted to journal 09 Nov, 2025 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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14:08:29","extension":"pdf","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142916,"visible":true,"origin":"","legend":"","description":"","filename":"fig7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/a7d37bd676776a25c0d67bcb.pdf"},{"id":96803562,"identity":"7da82103-ab02-4d5a-b414-b5d515798f49","added_by":"auto","created_at":"2025-11-26 08:59:08","extension":"pdf","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39236,"visible":true,"origin":"","legend":"","description":"","filename":"fig8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/dbc4487e3b9194e5784510e1.pdf"},{"id":96916727,"identity":"d09a0d0c-0a4b-4425-a091-018b27109556","added_by":"auto","created_at":"2025-11-27 14:08:51","extension":"pdf","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38695,"visible":true,"origin":"","legend":"","description":"","filename":"fig9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/a235825b1cfe7880a0a069d6.pdf"},{"id":96803565,"identity":"4c74c4d5-a8e2-4c5a-85d1-4e498151e6cb","added_by":"auto","created_at":"2025-11-26 08:59:08","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158737,"visible":true,"origin":"","legend":"","description":"","filename":"3af7502aa4c84153aa684f043612c6b81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/e72172a6f4731c98a3060871.xml"},{"id":96803568,"identity":"2803f4f6-e5e1-480f-8bad-caf8944e3687","added_by":"auto","created_at":"2025-11-26 08:59:08","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172350,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/7a8431e61600b367ed7f437b.html"},{"id":96803539,"identity":"fc51b9a4-dbec-4dda-899b-6d897cfb5b1d","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with wild-type (WT) and mutant sequences of ASIP genes. \u003c/strong\u003eAlignment results of genome sequencing reads of around ASIP: c.123_124; CRFK (a) and PG-4 (b) cells. Left string indicates the name of sequenced read, and red colored symbols indicate coat phenotype-related mutations\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/38be30ca31079f6195eb50c1.png"},{"id":96803542,"identity":"aa8e8f45-80af-4d3e-9f02-f26b4535b19d","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of FGF5 genes. \u003c/strong\u003eAlignment results of genome sequencing reads around FGF5: c.474 and c.475; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/116a165aca74025d84579422.png"},{"id":96803540,"identity":"857af9c3-561d-4f11-acfd-52018acd704f","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72600,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of LVRN genes. \u003c/strong\u003eAlignment results of genome sequencing reads around LVRN: c.2522; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/ebd4b5c2def0970c7f557945.png"},{"id":96803546,"identity":"396d8eae-86c4-4484-b212-e360102b2790","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":171471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of TYRP1 genes. \u003c/strong\u003eAlignment results of genome sequencing reads around TYRP1: c.8; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/b0e29a2f217b7c26326d986d.png"},{"id":96916830,"identity":"477e1225-d32d-46ed-b561-1d5d60fee462","added_by":"auto","created_at":"2025-11-27 14:08:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":198442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of MLPH genes. \u003c/strong\u003eAlignment results of genome sequencing reads around MLPH: c.83; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/390bce079ffbc668e8f6333e.png"},{"id":96803544,"identity":"90b64aff-c5ab-4ed4-9fc8-5ca9b1402087","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":137419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParts of upper and lower streams of alignment results of long-read sequencing reads of CRFK and PG-4 cells with WT and KIT-FERV1 sequences of KIT intron 1. \u003c/strong\u003eAlignment results of long-read genome sequencing reads from CRFK and PG-4 cells to the KIT-FERV1 that contains sequence around the region B1: g.161388687_161388688 (F.catus_Fca126_mat1.0). Left string indicates [the name of cell (CRFK or PG-4): name of sequenced read], and red colored sequences indicate FERV1 and aligned sequences with it. Four long reads from CRFK could align, but no reads contained the FERV1 homologue, while 6 reads from PG-4 could align, and four of the six reads contained the FERV1 homologue. This figure shows only a part of the results at the upper and lower streams of the KIT-FERV1 sequence. See Table S1 for the alignment result with the full-length KIT-FERV1 sequence\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/32d7894104f5eefab8e863a2.png"},{"id":96803550,"identity":"2b7342b4-7840-49ca-8e13-8cda82025f3a","added_by":"auto","created_at":"2025-11-26 08:59:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":144565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExample illustrations of inferred original cats of CRFK and PG-4 cells. \u003c/strong\u003eAn example illustration of the inferred phenotypes of coat morphologies and iris colors of the original cats of the CRFK cell (a) and the PG-4 cell (b). CRFK cells are expected to originate from a cat with long, chocolate-brown fur, lacking stripes, and with non-blue irises. PG-4 cells were expected to be derived from a cat with long, white and dark brown bicolored fur without stripes and non-blue irises\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/50fe9585b2d772a6d3b8319c.png"},{"id":96918382,"identity":"42e16fdd-a005-4df7-9b36-144557847238","added_by":"auto","created_at":"2025-11-27 14:11:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":209404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of TYR genes. \u003c/strong\u003eAlignment results of genome sequencing reads around TYR: c.904; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/8cad4325e5887cc5b6902d77.png"},{"id":96915762,"identity":"c77b4494-aad6-4f88-9408-4447c01d141d","added_by":"auto","created_at":"2025-11-27 14:07:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":192056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlignment results of whole genome sequencing reads of CRFK and PG-4 cells with WT and mutant sequences of LVRN genes. \u003c/strong\u003eAlignment results of genome sequencing reads around LVRN: c. 416; CRFK (a) and PG-4 (b) cells. Left strings and symbol colors are the same as in Figure 1\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/91f8400d69f49b4c48f5ec97.png"},{"id":96803545,"identity":"a12f7e14-5756-4429-b767-c88af91c1f97","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":119676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of coat phenotype-related and iris color-related genes in various tissues. \u003c/strong\u003eThe expression level of each gene in each tissue is quantified as log\u003csub\u003e2\u003c/sub\u003e(transcripts per million+1). The length of the red bar for gene A for each tissue is proportional to the [expression levels of gene A in each tissue] / [the maximum expression level of gene A among all focused tissues]. Most coat phenotype-related genes were found to exhibit similar or higher expressions in various tissues other than skin, and the iris color-related gene (PAX3) was also found to exhibit similar or higher expressions in a variety of tissues other than the opt-functional organs\u003c/p\u003e","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/4c5c3062f4e91fa85023727e.png"},{"id":104250725,"identity":"2260b96e-70d3-41b4-8e7a-0e4840147fc6","added_by":"auto","created_at":"2026-03-09 16:06:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3443014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/364539e6-a05d-40b9-a993-be8515827ac0.pdf"},{"id":96803547,"identity":"e3019a00-b79c-44b9-ae89-cd100b41a589","added_by":"auto","created_at":"2025-11-26 08:59:07","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":35496,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/4765da6469a5ee12d9661ce9.xlsx"},{"id":96803570,"identity":"fa68ea41-7e60-425a-a6a4-2a61f88ec5a8","added_by":"auto","created_at":"2025-11-26 08:59:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20910198,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/4150a8d11384886d2676e267.xlsx"},{"id":96916881,"identity":"1fa9054c-70ec-4b9d-a3f1-500ba2fd547a","added_by":"auto","created_at":"2025-11-27 14:09:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13981,"visible":true,"origin":"","legend":"","description":"","filename":"CRFKPG4genomeCAHGsup.docx","url":"https://assets-eu.researchsquare.com/files/rs-8072344/v1/3edc7adf029a53fa92cb236f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors","fulltext":[{"header":"Background","content":"\u003cp\u003eDomestic cats (\u003cem\u003eFelis catus\u003c/em\u003e) are widely kept as companion animals around the world, and the phenotypic diversity of their coat color, coat pattern, coat length, and iris color has been a topic of interest to breeders and scientists alike. Recent advances in genomics have markedly improved our understanding of the genetic basis controlling these diverse phenotypes, and the respective genotype-phenotype relationships are being increasingly elucidated.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Examples include the \u003cem\u003eTYR\u003c/em\u003e gene, which is the color locus associated with albinism and a color point phenotype\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eTYRP1\u003c/em\u003e gene, conveying brown coat color\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eMC1R\u003c/em\u003e genes, associated with in red coat color\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eKIT\u003c/em\u003e gene, determining white coat and white spotting\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eARHGAP36\u003c/em\u003e gene, involved in orange coat color\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eMLPH\u003c/em\u003e gene, causing dilute pigmentation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eASIP\u003c/em\u003e gene, controlling the expression of the agouti pattern\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eLVRN\u003c/em\u003e gene, determining the type of tabby pattern\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eDkk4\u003c/em\u003e gene, associated with the ticked tabby pattern\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eFGF5\u003c/em\u003e gene, involved in long-fur variants\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; the \u003cem\u003eKRT71\u003c/em\u003e and \u003cem\u003eLPAR6\u003c/em\u003e genes, involved in hairless and rexing (curly hair) phenotypes\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e; and the \u003cem\u003ePAX3\u003c/em\u003e gene, which is associated with blue irises\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Furthermore, technological advancements such as long-read sequencing facilitated the construction of a more continuous and accurate draft genome of the domestic cat \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and a plethora of genetic polymorphisms associated with disease have been identified \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImmortalized cell lines are an indispensable tool in current biomedical and veterinary science. Crandell-Rees Feline Kidney (CRFK) cells, derived from the kidneys of a domestic cat\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, have been used for numerous virological studies, including feline calicivirus,\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e feline immunodeficiency virus\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, feline coronavirus\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, and various feline endogenous retroviruses such as RD114\u003csup\u003e46\u0026ndash;48\u003c/sup\u003e, as well as for vaccine development\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. PG-4 cells, derived from feline fetal astrocytes, have also played an important role in retrovirus research\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. These cell lines have been passaged for decades in laboratories worldwide and have facilitated numerous scientific breakthroughs. However, despite their widespread use, no detailed information on the genetic background of the donor individuals, particularly with regard to their phenotype, such as coat and iris color, is available. Genomic information on such cell lines is crucial for a deeper understanding of their biological properties, and elucidating the phenotypes of their donors may provide new insights into the influence of specific genotypes on cell properties.\u003c/p\u003e\u003cp\u003eIn this study, we performed whole-genome sequencing of CRFK and PG-4 cell lines. The sequencing data were used for in-depth analysis of the nucleotide sequences of genes known to be associated with coat and iris phenotypes (i.e., \u003cem\u003eTYR\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, \u003cem\u003eMC1R\u003c/em\u003e, \u003cem\u003eKIT\u003c/em\u003e, \u003cem\u003eARHGAP36\u003c/em\u003e, \u003cem\u003eMLPH\u003c/em\u003e, \u003cem\u003eASIP\u003c/em\u003e, \u003cem\u003eLVRN\u003c/em\u003e, \u003cem\u003eDKK4\u003c/em\u003e, \u003cem\u003eFGF5\u003c/em\u003e, \u003cem\u003eKRT71\u003c/em\u003e, \u003cem\u003eLPAR6\u003c/em\u003e, and \u003cem\u003ePAX3\u003c/em\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on the genotyping results for each gene, we inferred the previously unknown phenotypes of the donor individuals. Furthermore, analysis of publicly available RNA-seq data confirmed that these genes were expressed in skin and eyes as well as in various other tissues throughout the body. Our results provide fundamental insights into the genetic background of widely used feline cell lines, offering valuable information that will be beneficial for interpreting the results of future research using these cell lines, such as informing guidelines to establish cell lines with various genotypes, including disease-associated genotypes, through genome editing.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ePhenotypic traits of coat and iris conferred by DNA variants, and genotypes of CRFK and PG-4 cells.\u003c/b\u003e List of genes and phenotypes focused on in this study, mutation position on CDS coordinate and on genomic coordinate provided by Fca126_mat1.0 and AnAms1.0, and a summary of genotypes of CRFK and PG-4 cells obtained by the present study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLocus\u003c/em\u003e\u003c/p\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhenotype\u003c/p\u003e\u003cp\u003e(Alleles)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMutation in\u003c/p\u003e\u003cp\u003eCDS (or related noncoding region):\u003c/p\u003e\u003cp\u003eF.catus_Fca126_mat1.0\u003c/p\u003e\u003cp\u003eGenome: F.catus_Fca126_mat1.0\u003c/p\u003e\u003cp\u003eGenome: AnAms1.0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003cp\u003ein\u003c/p\u003e\u003cp\u003eCRFK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003cp\u003ein\u003c/p\u003e\u003cp\u003ePG-4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eColor\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTYR\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBurmese\u003c/p\u003e\u003cp\u003e(c\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.679G\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eD1: g.44022819C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eD1: g.46081417C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSiamese\u003c/p\u003e\u003cp\u003e(c\u003csup\u003es\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.904G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eD1: g.44013031C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eD1: g.46071661C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / c\u003csup\u003es\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlbino\u003c/p\u003e\u003cp\u003e(c\u003csup\u003ea\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.939del\u003c/p\u003e\u003cp\u003eD1: g.44012996del \u003c/p\u003e\u003cp\u003eD1: g.46071626del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMocha\u003c/p\u003e\u003cp\u003e(c\u003csup\u003em\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.820_936delinsAATCTC\u003c/p\u003e\u003cp\u003eD1: g.44012836_44012998delinsGAGATT\u003c/p\u003e\u003cp\u003eD1: g.46071466_46071628delinsGAGATT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBrown\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTYRP1\u003csup\u003e2,5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChocolate\u003c/p\u003e\u003cp\u003e(b\u003csup\u003ech\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1261\u0026thinsp;+\u0026thinsp;5G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eD4: g.38142265G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eD4: g.39391308G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChocolate\u003c/p\u003e\u003cp\u003e(b\u003csup\u003ec\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.8C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003cp\u003eD4: g.38129873C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003cp\u003eD4: g.39378919C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003csup\u003ec\u003c/sup\u003e / b\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCinnamon\u003c/p\u003e\u003cp\u003e(b\u003csup\u003ei\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.298C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eD4: g.38130163C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eD4: g.39379209C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAmber\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMC1R\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRed\u003c/p\u003e\u003cp\u003e(e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.250G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eE2: g.61570294G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eE2: g.62623146G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eWhite\u003c/em\u003e\u003c/p\u003e\u003cp\u003eKIT\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003cp\u003e(w\u003csup\u003ew\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Intron 1: FERV1-LTR insertion)\u003c/p\u003e\u003cp\u003eB1: g.161388687_161388688insFERV1-LTR\u003c/p\u003e\u003cp\u003eB1: g.163130991_163130992insFERV1-LTR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite spotted\u003c/p\u003e\u003cp\u003e(w\u003csup\u003es\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Intron 1: FERV1 insertion)\u003c/p\u003e\u003cp\u003eB1: g.161388687_161388688insFERV1\u003c/p\u003e\u003cp\u003eB1: g.163130991_163130992insFERV1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / w\u003csup\u003es\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003cp\u003eglove\u003c/p\u003e\u003cp\u003e(w\u003csup\u003eg\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1035_1036delinsCA\u003c/p\u003e\u003cp\u003eB1: g.161337385_161337386delinsTG\u003c/p\u003e\u003cp\u003eB1: g.163079561_163079562delinsTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSalmiak\u003c/p\u003e\u003cp\u003e(w\u003csup\u003ea\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(KIT-KDR Intergenic region: 95 kb deletion) \u003c/p\u003e\u003cp\u003eB1: g.161142880_161237957del\u003c/p\u003e\u003cp\u003eB1: g.162885414_162980052del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOrange\u003c/em\u003e\u003c/p\u003e\u003cp\u003eArhgap36\u003c/p\u003e\u003cp\u003e\u003csup\u003e12,13\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOrange\u003c/p\u003e\u003cp\u003e(o)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Intron 1: 5 kb deletion)\u003c/p\u003e\u003cp\u003e(Lacking)\u003c/p\u003e\u003cp\u003eX: g.109186183_109191258del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eDilution\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMLPH\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDilute\u003c/p\u003e\u003cp\u003e(d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.83del\u003c/p\u003e\u003cp\u003eC1: g.218197448del\u003c/p\u003e\u003cp\u003eC1: g.219535339del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / d\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAgouti\u003c/em\u003e\u003c/p\u003e\u003cp\u003eASIP\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNonagouti\u003c/p\u003e\u003cp\u003e(a)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.123_124del\u003c/p\u003e\u003cp\u003eA3: g.24831083-24831084del\u003c/p\u003e\u003cp\u003eA3: g.25283750_25283751del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea / a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea / a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTabby\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLVRN\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlotched tabby\u003c/p\u003e\u003cp\u003e(t\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.176C\u0026thinsp;\u0026gt;\u0026thinsp;A \u003c/p\u003e\u003cp\u003eA1: g.94477275C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.97133509C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlotched tabby\u003c/p\u003e\u003cp\u003e(t\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.416C\u0026thinsp;\u0026gt;\u0026thinsp;A \u003c/p\u003e\u003cp\u003eA1: g.94477515C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.97133749C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / t\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlotched tabby\u003c/p\u003e\u003cp\u003e(t\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.682G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.94477781G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.97134015G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlotched tabby\u003c/p\u003e\u003cp\u003e(t\u003csup\u003eb\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.2522G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.94536796G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eA1: g.97192983G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003et\u003csup\u003eb\u003c/sup\u003e / t\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / t\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTicked\u003c/em\u003e\u003c/p\u003e\u003cp\u003eDkk4\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTicked\u003c/p\u003e\u003cp\u003e(ti)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.188G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eB1: g.40429492G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eB1: g.41805975G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTicked\u003c/p\u003e\u003cp\u003e(ti)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.53C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eB1: g.40428846C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eB1: g.41805329C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eLonghair\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFGF5\u003csup\u003e18,19\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLong fur\u003c/p\u003e\u003cp\u003e(l)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.356_357insT\u003c/p\u003e\u003cp\u003eB1: g.139634675_139634676insA\u003c/p\u003e\u003cp\u003eB1: g.141368532_141368533insA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLong fur\u003c/p\u003e\u003cp\u003e(l)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.406C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eB1: g.139645946G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eB1: g.141379803G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLong fur\u003c/p\u003e\u003cp\u003e(l)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.474del\u003c/p\u003e\u003cp\u003eB1: g.139634261del\u003c/p\u003e\u003cp\u003eB1: g.141368118del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / l\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLong fur\u003c/p\u003e\u003cp\u003e(l)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.475A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003cp\u003eB1: g.139634260T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003cp\u003eB1: g.141368117T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003el / l\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003el / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLong fur\u003c/p\u003e\u003cp\u003e(l)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.577G\u0026thinsp;\u0026gt;\u0026thinsp;A \u003c/p\u003e\u003cp\u003eB1: g.139634158C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eB1: g.141368015C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHairless\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eRexing\u003c/em\u003e\u003c/p\u003e\u003cp\u003eKRT71\u003c/p\u003e\u003cp\u003e\u003csup\u003e20,21\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHairless\u003c/p\u003e\u003cp\u003e(hr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.816\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e\u003cp\u003eB4: g.78941699C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003cp\u003eB4: g.80471148C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRexing\u003c/p\u003e\u003cp\u003e(re)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.1108-4_1184del, \u003c/p\u003e\u003cp\u003ec.1184_1185insAGTTGGAG, \u003c/p\u003e\u003cp\u003eand c.1196_1197insT\u003c/p\u003e\u003cp\u003eB4: g.78939390_78939470del, \u003c/p\u003e\u003cp\u003eB4: g.78939389_78939390insCTCCAACT, \u003c/p\u003e\u003cp\u003eand B4: g.78939377_78939378insA\u003c/p\u003e\u003cp\u003eB4: g.80468919_80468919del,\u003c/p\u003e\u003cp\u003eB4: g.80468918_80468919insCTCCAACT,\u003c/p\u003e\u003cp\u003eand B4: g.80468826_80468827insA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRexing\u003c/p\u003e\u003cp\u003e(re)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.445-1G\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e\u003cp\u003eB4: g.78943283C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003cp\u003eB4: g.80472733C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eRexing\u003c/em\u003e\u003c/p\u003e\u003cp\u003eLPAR6\u003csup\u003e22\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRexing\u003c/p\u003e\u003cp\u003e(re)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec.250_253del\u003c/p\u003e\u003cp\u003eA1: g.22865220_22865223del \u003c/p\u003e\u003cp\u003eA1: g.23372953_23372956del\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eBlue iris\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePAX3\u003csup\u003e23,24\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlue iris\u003c/p\u003e\u003cp\u003e(bi)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Inrton4: FERV1-LTR homolog insertion)\u003c/p\u003e\u003cp\u003eC1: g.205833101_205833102insN[395]\u003c/p\u003e\u003cp\u003eC1: g.207180017_207180018insN[395]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlue iris\u003c/p\u003e\u003cp\u003e(bi)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(Intron4: RD114-LTR homolog insertion)\u003c/p\u003e\u003cp\u003eC1: g.205834854_205834855insN[433]\u003c/p\u003e\u003cp\u003eC1: g.207181773_207181774insN[433]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+ / +\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and culture conditions\u003c/h2\u003e\u003cp\u003eCRFK cells (JCRB9035, RRID: CVCL_2426) and a feline sarcoma-positive leukemia-negative (S\u0026thinsp;+\u0026thinsp;L\u0026minus;) astrocyte cells termed PG-4 (S\u0026thinsp;+\u0026thinsp;L-) (JCRB9125, RRID: CVCL_3322) were obtained from the JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan). CRFK cells were cultured in Dulbecco's Modified Eagle Medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 IU/mL), streptomycin (100 ng/mL) (Sigma-Aldrich, St. Louis, MO, USA), and 1% non-essential amino acids (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37\u0026deg;C and 5% CO₂. PG-4 cells were cultured in McCoy\u0026rsquo;s 5A Modified Medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), penicillin (100 IU/mL), and streptomycin (100 ng/mL) (Sigma-Aldrich) at 37\u0026deg;C and 5% CO₂. For cell passaging, cells were washed using BASIC DPBS (no calcium or magnesium; Gibco) and then treated with TrypLE\u0026trade; Express Enzyme (1X; Gibco) for 5 min at 37\u0026deg;C and 5% CO₂ conditions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePaired-end short-read whole-genome sequencing\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from logarithmically growing cells (approximately 1\u0026times;10⁶ cells) using the DNeasy Blood \u0026amp; Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity (A260/A280 ratio) of the extracted DNA were measured using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Genomic DNA extracted from CRFK cells and PG-4 cells was used for library preparation and paired-end sequencing (150 bp read length) at Novogene (Beijing, China) using a Rapid Plus DNA Lib Prep Kit for Illumina V2 (Illumina) and an Illumina NovaSeq X Plus platform (Illumina). Library quality control was performed by Novogene using qPCR and fragment size analysis according to Novogene\u0026rsquo;s standard protocols. The raw sequence data were obtained as .fastq files.\u003c/p\u003e\n\u003ch3\u003eMapping of paired-end reads and quantification\u003c/h3\u003e\n\u003cp\u003eAdapter sequences were trimmed, and low-quality reads were removed from the original .fastq files using fastp software (ver. 0.21.0) with default parameters. Reads in processed .fastq files were mapped to the reference genome sequence of \u003cem\u003eFelis catus\u003c/em\u003e, F.catus_Fca126_mat1.0 (RefSeq assembly GCF_018350175.1) or AnAms1.0 (Genbank assembly GCA_013340865.2)\u003csup\u003e27\u003c/sup\u003e using bwa mem software (ver. 0.7.19) with the setting \u0026ldquo;-R \"@RG\\tID:sample1\\tSM:sample1\\tLB:lib1\\tPL:ILLUMINA\"\u0026rdquo; and default parameters otherwise. Mapping results were obtained as .sam files. Text data in .sam files were transformed to binary data in sorted .bam files using Samtools (ver. 1.7) with default parameters. Index files were created for the obtained .bam files in the form of .bam.bai files.\u003c/p\u003e\n\u003ch3\u003eSmall variations in coat morphology-related genes\u003c/h3\u003e\n\u003cp\u003eThe variations of coat morphology-related phenotypes of the donor cats from which CRFK and PG-4 cell lines originated were inferred based on the comparisons between the wild-type sequences and mapped sequences on variant positions of each coat morphology-related gene (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Coat phenotype-related polymorphisms in \u003cem\u003eTYR\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, \u003cem\u003eMC1R\u003c/em\u003e, \u003cem\u003eKIT\u003c/em\u003e, \u003cem\u003eMLPH\u003c/em\u003e, \u003cem\u003eASIP\u003c/em\u003e, \u003cem\u003eLVRN\u003c/em\u003e, \u003cem\u003eDkk4\u003c/em\u003e, \u003cem\u003eFGF5\u003c/em\u003e, \u003cem\u003eKRT71\u003c/em\u003e, and \u003cem\u003eLPAR6\u003c/em\u003e genes, which relate to phenotypes such as the color, brown, red, white glove, dilution, agouti, tabby, ticked, long-fur, hairless, and rexing (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), were examined using alignments of paired-end sequence reads with wild-type sequences.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLarge deletions in intron 1 of ARHGAP36 and the intergenic region between\u003c/b\u003e \u003cb\u003eKIT\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eKDR\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe deletion of a part of intron 1 of the \u003cem\u003eARHGAP36\u003c/em\u003e gene (X: 109,186,183_109,191,258 in AnAms1.0; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was reported to produce orange coat color\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and that of the intergenic region between the \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eKDR\u003c/em\u003e genes (B1: 161142880_161237957 in F.catus_Fca126_mat1.0, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) produces salmiak coat color\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The occurrence of these deletions in CRFK and PG-4 was estimated by comparing the mapped read number distributions between these regions and their neighboring regions with the same region length. It should be noted that F.catus_Fca126_mat1.0 has a deletion of the genomic region corresponding to X: 109186183_109191258 in AnAms1.0 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, AnAms1.0 (or felCat9\u003csup\u003e25\u003c/sup\u003e) was used as the reference genome for analysis of the orange locus.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInsertions of FERV1-LTR and RD114-LTR into\u003c/b\u003e \u003cb\u003ePAX3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe insertions of homologous sequences of the long terminal repeat (LTR) of the feline endogenous retrovirus 1 (FERV1), termed FERV1-LTR, into a specific region in intron 4 of \u003cem\u003ePAX3\u003c/em\u003e (C1: g.205833101_205833102 in F.catus_Fca126_mat1.0, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) or the LTR of RD114 retrovirus, termed RD114-LTR, into another specific region in the same intron (C1: g.205834854_205834855 in F.catus_Fca126_mat1.0, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are associated with blue iris color in cats.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The occurrence of such insertions was estimated by extracting the genome reads that were mapped in both FERV1-LTR and intron 4 of \u003cem\u003ePAX3\u003c/em\u003e or both RD114-LTR sequences and intron 4 of \u003cem\u003ePAX3\u003c/em\u003e. The genome sequence of RD114-LTR was obtained from the NCBI database\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eLong-read whole-genome sequencing\u003c/h3\u003e\n\u003cp\u003eGenomic DNA of CRFK cells and PG-4 cells was extracted for long-read genome sequencing using the NucleoBond HMW DNA kit (TaKaRa, Tokyo, Japan), and libraries for the Oxford Nanopore Technologies (ONT, UK) sequencer were constructed using a ligation library preparation kit (SQK-LSK114, ONT). All procedures were performed according to the manufacturer\u0026rsquo;s instructions, and the quality and molecular weight of the genomic DNA were measured using the Qubit fluorometric quantification system (Thermo Fisher Scientific). Sequencing was conducted using a PromethION sequencer (PromethION 2 Solo, ONT) and R10.4.1 flow cells. POD5 files were basecalled to .fastq files using MinKNOW software v25.05.14.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInsertions of FERV1 homolog into\u003c/b\u003e \u003cb\u003eKIT\u003c/b\u003e \u003cb\u003eexamined by long-read sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe insertion of FERV1 into a specific region of intron 1 of the \u003cem\u003eKIT\u003c/em\u003e gene (B1: 161388687_161388688 in F.catus_Fca126_mat1.0, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and that of FERV1-LTR make the coat color of cats white-spotted and white throughout, respectively \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The occurrence of such insertions in CRFK and PG-4 cells was estimated by mapping the reads of long-read sequencing to the genome sequence named KIT-FERV1 sequence using minimap2 (ver. 2.30), where the KIT-FERV1 sequence is a part of the \u003cem\u003eKIT\u003c/em\u003e intron 1 sequence with the insertion of the FERV1 sequence, as shown in Supplementary Fig.\u0026nbsp;1 of the study by David et al\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome data analysis of coat phenotype- and iris color-related genes in various tissues of domestic cats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMapped RNA-seq data on the F.catus_Fca126_mat1.0 were obtained in .bam and .bam.bai formats for various tissues from the Ensembl public database\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, specifically, the file transfer protocol (ftp) site\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The annotation file of the F.catus_Fca126_mat1.0 in gene transfer format (GTF) was obtained from the NCBI database (Bethesda, MD, USA). In the file, the chromosome numbers were converted from NC_058368.1, NC_058369.1, etc. to A1, A2, etc.\u003c/p\u003e\u003cp\u003eUsing FeatureCounts with the options\u0026mdash;M-O\u0026mdash;fraction,\u003csup\u003e57\u003c/sup\u003e read counts data for each gene described in the GTF file were obtained from the .bam and .bam.bai formatted files. This count data could also be obtained from a file \u0026ldquo;F.catus_Fca126_mat1.0.ENA_gene_exp_mat_by_FeatureCounts.csv\u0026rdquo; deposited in F.catlas\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. From these counts, the transcript level of each gene was calculated by dividing the read count by the total exon length. The total transcript level was defined as the sum of all the transcript levels of the genes. Based on this result, the transcripts per million (TPM) of each gene were calculated as 1,000,000*(transcript level)/(total transcript level).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eShort- and long-read whole-genome sequencing\u003c/h2\u003e\u003cp\u003eFor CRFK and PG-4 cells, the total output of raw sequence data of 150 bp paired-end sequencing was 60.6 Gb and 60.1 Gb, and the [average]\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e[standard deviation] of mapping depth of the reads for F.catus_Fca126_mat1.0 were \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:23.59\\pm\\:9.79\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:23.45\\pm\\:8.17\\)\u003c/span\u003e\u003c/span\u003e, respectively; the total output of raw sequence data of long-read sequencing was 9.6 Gb and 11.8 Gb, N50 values were 23.5 kb and 38.2 kb, and the [average]\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e[standard deviation] of mapping depth of reads for F.catus_Fca126_mat1.0 were \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:3.91\\pm\\:3.11\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:4.79\\pm\\:3.34\\)\u003c/span\u003e\u003c/span\u003e, respectively. The average and standard deviations specified above were estimated through the entire genome region without the regions with singular sequences where the read coverage exhibited \u0026gt;\u0026thinsp;100.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePredicted phenotype of the CRFK cell donor\u003c/h3\u003e\n\u003cp\u003eThe mapping of paired-end reads from the whole-genome sequences of the CRFK cell revealed that most coat morphology-related genes were conserved (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the following variations were identified in mapped genome reads from CRFK cells: a homozygous mutation of c.123_124del of the \u003cem\u003eASIP\u003c/em\u003e gene (the \u003cem\u003enonagouti\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a homozygous mutation of c.475A\u0026thinsp;\u0026gt;\u0026thinsp;C of the \u003cem\u003eFGF5\u003c/em\u003e gene (the \u003cem\u003elong coat\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a homozygous mutation of c.2522G\u0026thinsp;\u0026gt;\u0026thinsp;A of the \u003cem\u003eLVRN\u003c/em\u003e gene (the \u003cem\u003eblotched-tabby\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), a homozygous mutation of c.8C\u0026thinsp;\u0026gt;\u0026thinsp;G of the \u003cem\u003eTYRP1\u003c/em\u003e gene (the \u003cem\u003echocolate-blown\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and a heterozygous mutation of c.83delT of the \u003cem\u003eMLPH\u003c/em\u003e gene (the \u003cem\u003edilute\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We found no 5 kbp deletion at intron 1 of the \u003cem\u003eARHGAP36\u003c/em\u003e gene and no 95 kbp deletion at the intergenic region between the \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eKDR\u003c/em\u003e genes. We also found no genome reads that could be mapped in both the \u003cem\u003ePAX3\u003c/em\u003e intron 4 and FERV1-LTR or both the \u003cem\u003ePAX3\u003c/em\u003e intron 4 gene and RD114-LTR sequences. Additionally, four reads from the long-read sequencing mapped to the KIT-FERV1 sequence, but these reads contained no insertions of FERV1 and FERV1-LTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe abovementioned homozygous mutation of the \u003cem\u003eTYRP1\u003c/em\u003e gene is known to exhibit chocolate-brown coat color\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Additionally, the homozygous mutation of the \u003cem\u003eFGF5\u003c/em\u003e gene was known to exhibit long fur\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, the mutation of the \u003cem\u003eLVRN\u003c/em\u003e did not influence coat morphology in this case because of the mutation of the \u003cem\u003eASIP\u003c/em\u003e gene that suppresses the stripe pattern on the cat\u0026rsquo;s skin coat, and the mutant genotype of the \u003cem\u003eMLPH\u003c/em\u003e gene was recessive.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Therefore, the cat from which the CRFK cells originated is expected to have had long, chocolate brown, unstriped fur and non-blue eyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePredicted phenotype of the PG-4 cell donor\u003c/h2\u003e\u003cp\u003eMapped paired-end reads from the whole-genome sequences of the PG-4 cell confirmed that most coat morphology-related genes were conserved (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but identified following variations: a homozygous mutation of c.123_124del of the \u003cem\u003eASIP\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a compound heterozygous mutation of c.474delT and c.475A\u0026thinsp;\u0026gt;\u0026thinsp;C of the \u003cem\u003eFGF5\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) where one allele exhibits c.474delT and the other allele exhibits c.475A\u0026thinsp;\u0026gt;\u0026thinsp;C, the heterozygous mutation of c.2522G\u0026thinsp;\u0026gt;\u0026thinsp;A of \u003cem\u003eLVRN\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), a heterozygous mutation of c.904G\u0026thinsp;\u0026gt;\u0026thinsp;A of the \u003cem\u003eTYR\u003c/em\u003e gene (the \u003cem\u003eSiamese-color\u003c/em\u003e allele; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), and a homozygous mutation of c.416C\u0026thinsp;\u0026gt;\u0026thinsp;A of the \u003cem\u003eLVRN\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). We found no large deletions at intron 1 of the \u003cem\u003eARHGAP36\u003c/em\u003e gene and the intergenic regions between the \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eKDR\u003c/em\u003e genes, as was also the case for CRFK. We also found no genome reads that could be mapped in both the \u003cem\u003ePAX3\u003c/em\u003e intron 4 and FERV1-LTR or both the \u003cem\u003ePAX3\u003c/em\u003e intron 4 gene and RD114-LTR sequences.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe found that six long-read sequences mapped to the KIT-FERV1 sequence, and four of them contained the insertions of the more than 7 kbp FERV1 homolog sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This suggested the PG-4 genome contains the heterozygous insertion of the entire FERV1 homolog sequence.\u003c/p\u003e\u003cp\u003eNote that the mutant genotype of the \u003cem\u003eTYR\u003c/em\u003e gene was recessive, and the two heterozygous mutations of the \u003cem\u003eLVRN\u003c/em\u003e do not influence coat morphology in this case because of the mutation of the \u003cem\u003eASIP\u003c/em\u003e gene, the same as in the case of CRFK\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the effect of \u003cem\u003eFERV1\u003c/em\u003e is known to be dominant\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Additionally, the compound heterozygous mutation of the \u003cem\u003eFGF5\u003c/em\u003e gene produces long fur\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, the cat from which the PG-4 cells originated is expected to have had white and dark brown, unstriped, long fur and non-blue irises (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eExpression features of coat phenotype- and iris color-related genes in various tissues\u003c/h2\u003e\u003cp\u003eThe analysis of publicly available RNA-seq data from various organs of domestic cats in the Ensembl database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftp.ensembl.org/pub/data_files/felis_catus/F.catus_Fca126_mat1.0/rnaseq/\u003c/span\u003e\u003cspan address=\"https://ftp.ensembl.org/pub/data_files/felis_catus/F.catus_Fca126_mat1.0/rnaseq/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed that coat phenotype-related genes are expressed in various organs at levels (TPM) comparable to or greater than those in the skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). For example, \u003cem\u003eTYR\u003c/em\u003e, \u003cem\u003eTYRP1\u003c/em\u003e, and \u003cem\u003eMLPH\u003c/em\u003e were highly expressed in the ear tip, retina, and optic nerve, and \u003cem\u003eMC1R\u003c/em\u003e, \u003cem\u003eKIT\u003c/em\u003e, \u003cem\u003eDKK4\u003c/em\u003e, and \u003cem\u003eFGF5\u003c/em\u003e were highly expressed in the brain. \u003cem\u003eKIT\u003c/em\u003e, \u003cem\u003eDKK4\u003c/em\u003e, and \u003cem\u003eLPAR6\u003c/em\u003e tend to be expressed throughout the body, with prominent expression in the reproductive organs. Additionally, \u003cem\u003eARHGAP36\u003c/em\u003e was highly expressed in the spinal cord, and \u003cem\u003eASIP\u003c/em\u003e, along with \u003cem\u003eMLPH\u003c/em\u003e, was highly expressed in the lungs. The iris color-related gene \u003cem\u003ePAX3\u003c/em\u003e was also highly expressed in the cerebellum, in addition to visual organs, and, along with \u003cem\u003eDKK4\u003c/em\u003e, in the kidney and embryonal tissue.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe performed whole-genome sequencing of CRFK, a cultured feline kidney cell line, and PG-4, a feline fetal astrocyte-derived cell line, and analyzed the genome data to determine the coat color, hair length, coat pattern, and iris color of the donor individuals. The results suggested that the CRFK cell line originated from a cat with long dark brown hair without stripes and non-blue irises, and the PG-4 cell line came from a cat with long fur, dark brown and white bicolor coat without stripes, and non-blue irises.\u003c/p\u003e\u003cp\u003eCoat morphology and iris color are phenotypes that can be readily identified and used to predict genotypes. However, the expressions of these genes are not limited to the skin and eyes; rather, they are expressed in various tissues throughout the body (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Therefore, differences in these genotypes may contribute to disease susceptibility in specific organs. The effects of these genetic variations are likely weaker than those of the rearing environment and may not be significant, especially in young, healthy individuals. However, as domestic cats have become longer-lived in recent years, individual-specific risks may become more apparent, and the underlying genotypes may exert increasing effects as the cats age. Therefore, elucidating the relationships between genotypes underlying readily recognizable phenotypes and disease risk is crucial from the perspectives of both medical care and welfare, such as prevention and symptom suppression/alleviation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eResults from research on cultured cells to elucidate disease mechanisms and develop therapeutic drugs must be interpreted with caution, i.e., not only on the experimental results obtained using established, immortalized cells but also with regard to their genotype and expression profiles of the genes harboring polymorphisms in various tissues and organs. However, so far, such information has not been available, even for certain frequently used cell lines such as CRFK. The present study provides such information and facilitates a more comprehensive interpretation of research on feline cultured cell lines.\u003c/p\u003e\u003cp\u003eFurthermore, with the availability of detailed genomic information, genome editing can be applied to these cell lines to establish various derivative lines that differ only in specific aspects, e.g., regarding coat morphology or other traits, and compare their properties. This is expected to advance research on the regulatory mechanisms of coat color, for example, the validation of the role of the novel \u003cem\u003eKIT\u003c/em\u003e isoform caused by FERV1-LTR insertion in the formation of white coat\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, as well as the relationships between the visually identifiable phenotypes examined in the present study and disease risk and treatment methods. Simultaneously, such detailed genomic information can inform guidelines for establishing cell lines with various disease-associated genotypes through genome editing. Establishing such derivative cell lines can help elucidate genetic factors associated with resistance to infectious diseases and cancer, the risk of internal organ afflictions, as well as genetic diseases such as deafness in cats with white fur and blue irises.\u003c/p\u003e\u003cp\u003eIt should be noted that the karyotype of CRFK differs from that of the normal domestic cat genome.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e It is thus possible that the karyotype of PG-4 cells, which has not been reported, differs from the normal domestic cat genome. However, using only whole-genome analysis of the present sequence read data, approximately 60 Gb paired-end sequencing data and 10 Gb long-read data, it was difficult to detect such large structural variations. Similarly, it was difficult to identify the accurate sequences and locations of various long repeat sequences, endogenous retroviral sequences, and their homologs because of the deficiency of the read coverage, particularly in the long-read coverage. However, these sequences may also be associated with various diseases, as exemplified by the involvement of FERV1-LTR in deafness in cats\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and the positive and negative roles of enFeLV-LTR and enFeLV-env in resistance to exogenous feline leukemia virus\u003csup\u003e\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The larger volume of ongoing long-read sequencing is expected to resolve these issues in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe performed whole-genome sequencing of CRFK and PG-4 cells and inferred the phenotype of the donors of these cells. We suggested that CRFK cells originated from a cat with long, chocolate-brown fur lacking stripes and non-blue irises; PG-4 cells originated from a cat with long, bicolored white and dark brown fur without stripes, and with non-blue irises. Additionally, analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs, indicating that variants of these genes, which affect coat phenotype and iris color, may influence physiological functions throughout the body. These insights may facilitate a more accurate interpretation of data derived from feline cultured cells and inform guidelines to establish cell lines with various disease-associated variants through genome editing, which will help elucidate genetic factors affecting resistance to infectious diseases and cancer, the risk of internal organ afflictions, and genetic diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflicting interests\u003c/h2\u003e\u003cp\u003eThe authors have no conflicting interests to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grants (award numbers 24K01783 (T. I.) and 24K09248 (A. A.)).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: A.A., Data curation: T.K., A.K., R.H., and T.I., Formal analysis: G.T. and R.G., Funding acquisition: A.A., Investigation: G.T., R.G., T.K., R.H., and A.A., Methodology: G.T., R.G., T.K., T.I., and A.A., Project administration: N.S. and A.A., Supervision: N.S. and A.A., Validation: R.G. and A.A., Visualization: T.G., N.S., and A.A., Writing - original draft: A.A., Writing - review \u0026amp; editing: T.G., T.K., T.I., and N.S.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThese raw read data of the short and long read sequencings were deposited in the DDBJ Sequence Read Archive (DRA) with BioProject accession PRJDB37530 (DRR748391-DRR748394, DRR786948, and DRR786949). All other data generated or analyzed during this study were included in this published article (and Supplementary Information files).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLyons LA. DNA mutations of the cat: The good, the bad and the ugly: The good, the bad and the ugly. J Feline Med Surg. 2015;17:203\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/1098612X15571878\u003c/span\u003e\u003cspan address=\"10.1177/1098612X15571878\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchmidt-Kuntzel A, Eizirik E, O\u0026rsquo;Brien SJ, et al. Tyrosinase and tyrosinase related protein 1 alleles specify domestic cat coat color phenotypes of the Albino and Brown loci. J Hered. 2005;96:289\u0026ndash;301.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLyons LA, Imes DL, Rah HC, et al. 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J Virol. 2024;98:e01771\u0026ndash;23.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"companion-animal-health-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cgae","sideBox":"Learn more about [Canine Medicine and Genetics](https://cgejournal.biomedcentral.com/)","snPcode":"40575","submissionUrl":"https://submission.nature.com/new-submission/40575/3","title":"Companion Animal Health and Genetics","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"genomics, coat pattern, iris color, RNA sequencing","lastPublishedDoi":"10.21203/rs.3.rs-8072344/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8072344/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCrandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived) have been in use and have been passaged for decades in laboratories worldwide; however, no detailed information on the genetic background of the donor individuals is available, particularly regarding phenotype characteristics such as coat and iris color.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe performed whole-genome sequencing of CRFK and PG-4 cells. We analyzed the resulting data to infer the phenotype of the individual from which the cells were derived, specifically for the coat color, coat length, coat pattern, and iris color. Our data suggested that CRFK cells originated from a cat with long chocolate-brown fur lacking stripes and with non-blue irises; PG-4 cells originated from a cat with long bicolored white and dark brown fur without stripes, and with non-blue irises. Analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eVariants of the genes affecting coat phenotype and iris color may influence physiological functions throughout the body. These results shed light on the previously unknown genetic background of commonly used feline cultured cells and the phenotype of the donor individuals. These insights may facilitate a more accurate interpretation of data derived from feline cultured cells and inform guidelines to establish cell lines with various genotypes (for example, disease-associated variants) through genome editing, which will help elucidate genetic factors affecting resistance to infectious diseases and cancer, the risk of internal organ afflictions, and genetic diseases such as deafness in cats with white fur and blue irises.\u003c/p\u003e","manuscriptTitle":"Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 08:59:03","doi":"10.21203/rs.3.rs-8072344/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T21:23:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T18:23:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279460674566976839764904834295246633318","date":"2025-12-02T17:17:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T13:11:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90316870738404342297486265145582050987","date":"2025-11-20T11:09:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-17T23:56:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-17T23:55:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-14T06:44:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Companion Animal Health and Genetics","date":"2025-11-10T03:26:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"companion-animal-health-and-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cgae","sideBox":"Learn more about [Canine Medicine and Genetics](https://cgejournal.biomedcentral.com/)","snPcode":"40575","submissionUrl":"https://submission.nature.com/new-submission/40575/3","title":"Companion Animal Health and Genetics","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dacca7de-141c-4e76-9a88-ee977ca729de","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:03:30+00:00","versionOfRecord":{"articleIdentity":"rs-8072344","link":"https://doi.org/10.1186/s40575-026-00150-9","journal":{"identity":"companion-animal-health-and-genetics","isVorOnly":false,"title":"Companion Animal Health and Genetics"},"publishedOn":"2026-03-06 15:58:23","publishedOnDateReadable":"March 6th, 2026"},"versionCreatedAt":"2025-11-26 08:59:03","video":"","vorDoi":"10.1186/s40575-026-00150-9","vorDoiUrl":"https://doi.org/10.1186/s40575-026-00150-9","workflowStages":[]},"version":"v1","identity":"rs-8072344","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8072344","identity":"rs-8072344","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}