NOVEL KINASE FOR TREATING AND PREVENTING FUNGAL INFECTIONS, AND USE THEREOF

TECHNICAL FIELD

The preset invention relates to novel kinases for preventing and treating pathogenic fungal infection and the use thereof. Moreover, the present invention relates to a method for screening an antifungal agent, which comprises measuring the amount or activity of a Cryptococcus neoformans pathogenicity-regulating kinase protein or the expression level of a gene encoding the protein and to an antifungal pharmaceutical composition comprising an inhibitor against a Cryptococcus neoformans pathogenicity-regulating kinase protein or a gene encoding the protein.

BACKGROUND ART

Cryptococcus neoformans is a pathogenic fungus which is ubiquitously distributed in diverse natural environments, including soil, tree and bird guano, and uses various hosts ranging from lower eukaryotes to aquatic and terrestrial animals (Lin, X. & Heitman, J. The biology of the Cryptococcus neoformans species complex. Annu. Rev. Microbiol. 60, 69-105, 2006). Cryptococcus neoformans is the leading cause of fungal meningoencephalitis deaths and is known to cause approximately one million new infections and approximately 600,000 deaths worldwide each year (Park, B. J. et al. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23, 525-530, doi:10.1097/QAD.0b013e328322ffac, 2009). However, limited therapeutic options are available for treatment of systemic cryptococcosis (Perfect, J. R. et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of America. Clin Infect Dis 50, 291-322, doi:10.1086/649858, 2010). Meanwhile, C. neoformans is regarded as an ideal fungal model system for basidiomycetes, owing to the availability of completely sequenced and well-annotated genome databases, a classical genetic dissection method through sexual differentiation, efficient methods of reverse and forward genetics, and a variety of heterologous host model systems (Idnurm, A. et al. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat. Rev. Microbiol. 3, 753-764, 2005).

Extensive studies have been conducted over several decades to understand the mechanisms underlying the pathogenicity of C. neoformans. Besides efforts to analyze the functions of individual genes and proteins, recent large-scale functional genetic analyses have provided comprehensive insights into the overall biological circuitry of C. neoformans. However, the signaling and metabolic pathways responsible for the general biological characteristics and pathogenicity of C. neoformans have not yet been fully elucidated. This is mainly because the functions of kinases, which have a central role in signaling pathways and are responsible for the activation or expression of transcription factors (TFs), have not been fully characterized on a genome-wide scale. In general, kinases play pivotal roles in growth, cell cycle control, differentiation, development, the stress response and many other cellular functions, affecting about 30% of cellular proteins by phosphorylation (Cohen, P. The regulation of protein function by multisite phosphorylation-a 25 year update. Trends Biochem Sci 25, 596-601, 2000). Furthermore, kinases are considered to be a protein class representing a major target in drug development, as their activity is easily inhibited by small molecules such as compounds, or antibodies (Rask-Andersen, M., Masuram, S. & Schioth, H. B. The druggable genome: Evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu Rev Pharmacol Toxicol 54, 9-26, doi:10.1146/annurev-pharmtox-011613-135943, 2014). Therefore, the systematic functional profiling of fungal kinases in human fungal pathogens is in high demand to identify virulence-related kinases that could be further developed as antifungal drug targets.

Accordingly, the present inventors performed systematic functional profiling of the kinome networks in C. neoformans and Basidiomycetes by constructing a high-quality library of 226 signature-tagged gene-deletion strains through homologous recombination methods for 114 putative kinases, and examining their phenotypic traits under 30 distinct in vitro growth conditions, including growth, differentiation, stress responses, antifungal resistance and virulence-factor production (capsule, melanin and urease). Furthermore, the present inventors investigated their pathogenicity and infectivity potential in insect and murine host models.

DISCLOSURE
Technical Problem

It is an object of the present invention to provide novel kinases for prevention and treatment of pathogenic fungal infection and the use thereof. Furthermore, the present invention is intended to provide a method of screening an antifungal agent by measuring the amount or activity of a Cryptococcus neoformans pathogenicity-regulating kinase protein or the expression level of a gene encoding the protein. The present invention is also intended to provide an antifungal pharmaceutical composition comprising an inhibitor and/or activator of a Cryptococcus neoformans pathogenicity-regulating kinase protein or a gene encoding the protein. The present invention is also intended to provide a method for screening a drug candidate for treating and preventing cryptococcosis or meningoencephalitis. The present invention is also intended to provide a pharmaceutical composition for treatment and prevention of cryptococcosis or meningoencephalitis. The present invention is also intended to provide a method for diagnosing fungal infection.

Technical Solution

To achieve the above objects, the present invention provides novel pathogenicity-regulating kinase proteins. Specifically, the novel pathogenicity-regulating kinase proteins according to the present invention include, but are not limited to, Fpk1, Bck1, Ga183, Kic1, Vps15, Ipk1, Mec1, Urk1, Yak1, Pos5, Irk1, Hs1101, Irk2, Mps1, Sat4, Irk3, Cdc7, Irk4, Swe102, Vrk1, Fbp26, Psk201, Ypk101, Pan3, Ssk2, Utr1, Pho85, Bud32, Tco6, Arg5, 6, Ssn3, Irk6, Dak2, Rim15, Dak202a, Snf101, Mpk2, Cmk1, Irk7, Cbk1, Kic102, Mkk2, Cka1, and Bub1.

The present invention also provides a method for screening an antifungal agent, comprising the steps of: (a) bringing a sample to be analyzed into contact with a cell containing a pathogenicity-regulating kinase protein; (b) measuring the amount or activity of the protein; and (c) determining that the sample is an antifungal agent, when the amount or activity of the protein is measured to be down-regulated or up-regulated.

The present invention also provides a method for screening an antifungal agent, comprising the steps of: (a) bringing a sample to be analyzed into contact with a cell containing a gene encoding a pathogenicity-regulating kinase protein; (b) measuring the expression level of the gene; and (c) determining that the sample is an antifungal agent, when the expression level of the gene is measured to be down-regulated or up-regulated.

In the present invention, the cell that is used in screening of the antifungal agent may be a fungal cell, for example, a Cryptococcus neoformans cell.

In the present invention, the antifungal agent may be an agent for treating and preventing meningoencephalitis or cryptococcosis, but is not limited thereto.

In the present invention, a BLAST matrix for 60 pathogenicity-related kinases was constructed using the CFGF (Comparative Fungal Genomics Platform) (http://cfgp.riceblast.snu.ac.kr) database, and the pathogenicity-related 60 kinase protein sequence was queried. As a result, orthologue proteins were retrieved and matched from the genome database from the 35 eukaryotic species. To determine the orthologue proteins, each protein sequence was analyzed by BLAST and reverse-BLAST using genome databases (CGD; Candida genome database for C. albicans, Broad institute database for Fusarium graminearum and C. neoformans). 21 kinases were related to pathogenicity in both F. graminearum and C. neoformans. 13 kinases were related to pathogenicity of C. neoformans and C. albicans. Among them, five kinases, including Sch9, Snf1, Pka1, Hog1 and Swe1, were related to virulence of all the three fungal pathogenic strains. Genes in the pathogenicity network according to the present invention were classified by the predicted biological functions listed in the information of their Gene Ontology (GO) term. Six kinases (Arg5/6, Ipk1, Irk2, Irk4, Irk6 and vrk1) did not have any functionally related genes in CryptoNet (http://www.inetbio.org/cryptonet).

As used herein, the team “sample” means an unknown candidate that is used in screening to examine whether it influences the expression level of a gene or the amount or activity of a protein. Examples of the sample include, but are not limited to, chemical substances, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

The team “antifungal agent” as used herein is meant to include inorganic antifungal agents, organic natural extract-based antifungal agents, organic aliphatic compound-based antifungal agents, and organic aromatic compound-based antifungal agents, which serve to inhibit the propagation of bacteria and/or fungi. Examples of the inorganic antifungal agents include, but are not limited to, chlorine compounds (especially sodium hypochlorite), peroxides (especially hydrogen peroxide), boric acid compounds (especially boric acid and sodium borate), copper compounds (especially copper sulfate), zinc compounds (especially zinc sulfate and zinc chloride), sulfur-based compounds (especially sulfur, calcium sulfate, and hydrated sulfur), calcium compounds (especially calcium oxide), silver compounds (especially thiosulfite silver complexes, and silver nitrate), iodine, sodium silicon fluoride, and the like. Examples of the organic natural extract-based antifungal agents include, but are not limited to, hinokithiol, Phyllostachys pubescens extracts, creosote oil, and the like.

In the present invention, measurement of the expression level of the gene may be performed using various methods known in the art. For example, the measurement may be performed using RT-PCR (Sambrook et al, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRCpress), hybridization using cDNA microarray (Sambrook et al, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001) or in situ hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001). Where the measurement is performed according to RT-PCR protocol, total RNA is isolated from cells treated with a sample, and then single-stranded cDNA is synthesized using dT primer and reverse transcriptase. Subsequently, PCR is performed using the single-stranded cDNA as a template and a gene-specific primer set. The gene-specific primer sets used in the present invention are shown in Tables 2 and 3 below. Next, the PCR amplification product is amplified, and the formed band is analyzed to measure the expression level of the gene.

In the present invention, measurement of the amount or activity of the protein may be performed by various immunoassay methods known in the art. Examples of the immunoassay methods include, but are not limited to, radioimmunoassay, radio-immunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, and sandwich assay. The immunoassay or immunostaining methods are described in various literatures (Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Fla., 1980; Gaastra, W., Enzyme linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999). For example, when radioimmunoassay is used, protein-specific antibodies labeled with radioisotopes (e.g., C14, I125, P32 and S35) may be used.

When ELISA is used in one embodiment of the present invention, it comprises the steps of: (i) coating an extract of sample-treated cells on the surface of a solid substrate; (ii) incubating the cell extract with a kinase protein-specific or labeled protein-specific antibody as a primary antibody; (iii) incubating the resultant of step (ii) with an enzyme-conjugated secondary antibody; and (iv) measuring the activity of the enzyme. Suitable examples of the solid substrate include hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metals or gels. Most preferably, the solid substrate is a microtiter plate. The enzyme conjugated to the secondary antibody includes an enzyme that catalyzes a color development reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction, but is not limited. Examples of the enzyme include alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as the enzyme conjugated to the secondary antibody, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF (enhanced chemifluorescence) may be used as substrates for color development reactions. When horseradish peroxidase is the enzyme, chloronaphthol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ABTS (2,2′-azine-di[3-ethylbenzthiazoline sulfonate]) and o-phenylenediamine (OPD) may be used as substrates. The final measurement of the activity or signal of the enzyme in the ELISA assay may be performed according to various conventional methods known in the art. When biotin is used as a label, the signal can be easily detected with streptavidin, and when luciferase is used as a label, the signal can be easily detected with luciferin.

In one embodiment, the present invention provides an antifungal pharmaceutical composition comprising an agent (inhibitor or activator) for a fungal pathogenicity-regulating kinase protein. In another embodiment, the fungus is Cryptococcus neoformans.

In one embodiment, the present invention provides an antifungal pharmaceutical composition comprising an agent (inhibitor or activator) for a gene encoding a fungal pathogenicity-regulating kinase protein. In another embodiment, the fungus is Cryptococcus neoformans.

In the present invention, the pharmaceutical composition may be a composition for treating meningoencephalitis or cryptococcosis, but is not limited.

In the present invention, the agent may be an antibody. In one embodiment, the inhibitor may be an inhibitor that inhibits the activity of the protein by binding to the protein, thereby blocking signaling of the protein. For example, it may be a peptide or compound that binds to the protein. This peptide or compound may be selected by a screening method including protein structure analysis or the like and designed by a generally known method. In addition, when the inhibitor is a polyclonal antibody or monoclonal antibody against the protein, it may be produced using a generally known antibody production method.

As used herein, the team “antibody” may be a synthetic antibody, a monoclonal antibody, a polyclonal antibody, a recombinantly produced antibody, an intrabody, a multispecific antibody (including bi-specific antibody), a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fv (scFv) (including bi-specific scFv), a BiTE molecule, a single-chain antibody, a Fab fragments, a F(ab′) fragment, a disulfide-linked Fv (sdFv), or an epitope-binding fragment of any of the above. The antibody in the present invention may be any of an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule. Furthermore, the antibody may be of any isotype. In addition, the antibody in the present invention may be a full-length antibody comprising variable and constant regions, or an antigen-binding fragment thereof, such as a single-chain antibody or a Fab or Fab′2 fragment. The antibody in the present invention may also be conjugated or linked to a therapeutic agent, such as a cytotoxin or a radioactive isotope.

In the present invention, the agent for the gene may be an antisense oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising the same, but is not limited thereto.

In the present invention, the inhibitor may be an inhibitor that blocks signaling by inhibiting expression of the gene, or interferes with transcription of the gene by binding to the gene, or interferes with translation of mRNA by binding to mRNA transcribed from the gene. In one embodiment, the inhibitor may be, for example, a peptide, a nucleic acid, a compound or the like, which binds to the gene, and it may be selected through a cell-based screening method and may be designed using a generally known method. For example, the inhibitor for the gene may be an antisense oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising the same, which may be constructed using a generally known method.

As used herein, the team “antisense oligonucleotide” means DNA, RNA, or a derivative thereof, which has a nucleic acid sequence complementary to the sequence of specific mRNA. The antisense oligonucleotide binds to a complementary sequence in mRNA and acts to inhibit the translation of the mRNA to a protein. In one embodiment, the length of the antisense oligonucleotide is 6 to 100 nucleotides, preferably 8 to 60 nucleotides, more preferably 10 to 40 nucleotides. In one embodiment of the present invention, the antisense oligonucleotide may be modified at one or more nucleotide, sugar or backbone positions in order to enhance their effect (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The nucleic acid backbone may be modified with a phosphorothioate linkage, a phosphotriester linkage, a methyl phosphonate linkage, a short-chain alkyl intersugar linkage, a cycloalkyl intersugar linkage, a short-chain heteroatomic intersugar linkage, a heterocyclic intersugar linkage or the like. The antisense oligonucleotide may also include one or more substituted sugar moieties. The antisense oligonucleotide may include modified nucleotides. The modified nucleotides include hypoxanthine, 6-methyladenine, 5-Me pyrimidine (particularly, 5-methylcytosine, 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentiobiosyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothimine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, and the like. In addition, the antisense oligonucleotide in the present invention may be chemically linked to one or more moieties or conjugates in order to enhance its activity or cellular uptake. In one embodiment of the present invention, the moiety may be a lipophilic moiety such as a cholesterol moiety, a cholesteryl moiety, cholic acid, thioether, thiocholesterol, an aliphatic chain, phospholipid, polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, or hexylamino-carbonyl-oxycholesterol moiety, but is not limited thereto. Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides, are well known in the field to which the present invention pertain (see U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). In one embodiment of the present invention, the modified nucleic acid may increase resistance to nuclease and increase the binding affinity between antisense nucleic acid and the target mRNA. In one embodiment, the antisense oligonucleotide may generally be synthesized in vitro and administered in vivo, or synthesized in vivo. In an example of synthesizing the antisense oligonucleotide in vitro, RNA polymerase I is used. In an example of synthesizing the antisense RNA in vivo, a vector having origin of recognition region (MCS) in opposite orientation is used to induce transcription of antisense RNA. The antisense RNA preferably includes a translation stop codon for inhibiting translation to peptide.

As used herein, the team “siRNA” means is a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). The siRNA can inhibit expression of a target gene, and thus provide an effective gene knock-down method or gene therapy method. In the present invention, the siRNA molecule may consist of a sense RNA strand (having a sequence corresponding to mRNA) and an antisense RNA strand (having a sequence complementary to mRNA) and foam a duplex structure. In the present invention, the siRNA molecule may have a single-strand structure comprising self-complementary sense and antisense strands. In one embodiment of the present invention, the siRNA is not restricted to a RNA duplex of which two strands are completely paired, and it may comprise non-paired portion such as mismatched portion with non-complementary bases and bulge with no opposite bases. In one embodiment of the present invention, the overall length of the siRNA may be 10-100 nucleotides, preferably 15-80 nucleotides, more preferably 20-70 nucleotides. In the present invention, the siRNA may comprise either blunt or cohesive end, as long as it can silence gene expression. The cohesive end may have a 3′-end overhanging structure or a 5′-end overhanging structure. In the present invention, the siRNA molecule may have a structure in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands. In this case, the siRNA molecule famed by expression of the nucleotide sequence forms a hairpin structure by intramolecular hybridization, resulting in the formation of a stem-and-loop structure.

As used herein, the term “shRNA” refers to short hairpin RNA. When an oligo DNA that connects a 3-10-nucleotide linker between the sense and complementary nonsense strands of the target gene siRNA sequence is synthesized and then cloned into a plasmid vector, or when shRNA is inserted and expressed in retrovirus, lentivirus or adenovirus, a looped hairpin shRNA is produced and converted by an intracellular dicer to siRNA that exhibits the RNAi effect. The shRNA exhibits the RNAi effect over a longer period of time than the siRNA.

As used herein, the term “miRNA (microRNA)” refers to an 18-25-nt single-stranded RNA molecule which controls gene expression in eukaryotic organisms. It is known that the miRNA binds complementarily to the target mRNA, acts as a posttranscriptional gene suppressor, and functions to suppress translation and induce mRNA destabilization.

As used herein, the term “vector” refers to a gene structure comprising a foreign DNA inserted into a genome encoding a polypeptide, and includes a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a virus vector.

In one embodiment of the present invention, the pharmaceutical composition may be administered in combination with at least one azole-based antifungal agent selected from the group consisting of fluconazole, itraconazole, voriconazole and ketoconazole, or may be administered in combination with at least one non-azole-based antifungal agent selected from the group consisting of amphotericin B, natamycin, rimocidin, nystatin, flucytosine and fludioxonil.

In the present invention, the antifungal pharmaceutical composition may comprise a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient. This adjuvant may be an excipient, a disintegrant, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a flavoring agent, a solubilizing agent or the like.

The antifungal pharmaceutical composition according to the present invention may comprise, in addition to the active ingredient, at least one pharmaceutically acceptable carrier. In one embodiment, when the pharmaceutical composition is formulated as a liquid solution, a carrier may be used, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, malto-dextrin solution, glycerol, ethanol, or a mixture of two or more thereof, which is sterile and physiologically suitable. If necessary, other conventional additives may be added, including antioxidants, buffers, bacteriostatic agents or the like.

In one embodiment of the present invention, the antifungal pharmaceutical composition may be formulated as injectable formulations such as aqueous solutions, suspensions, emulsions or the like, pills, capsules, granules or tablets, by use of a diluent, a dispersing agent, a surfactant, a binder or a lubricant. Furthermore, the composition may preferably be formulated using a suitable method as disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton Pa., depending on each disease or components. In one embodiment of the present invention, the pharmaceutical composition may be formulated in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, injectable liquid formulations, or sustained-release formulations of the active ingredient, or the like. The pharmaceutical composition of the present invention may be administered in a conventional manner by an intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, intrarectal, oral, intraocular or intradermal route.

In the present invention, the effective amount of the active ingredient in the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease. Thus, the effective amount may be adjusted depending on various factors, including the kind of disease, the severity of the disease, the kinds and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, the patient's age, weight, general health state, sex and diet, the period of administration, the route of administration, the secretion rate of the composition, treatment time, and concurrently used drugs.

Advantageous Effects

According to the present invention, novel antifungal agent candidates can be effectively screened using kinases. In addition, using an antifungal pharmaceutical composition comprising an agent (antagonist or antagonist) for kinase according to the present invention, fungal infection can be effectively prevented, treated and/or diagnosed.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the phylogenetic correlation among protein kinases in Cryptococcus neoformans, and FIG. 2 shows a comparison of major kinases in Cryptococcus neoformans, C. albicans and A. fumigatus. Regarding FIG. 1, protein sequence-based alignment was performed using ClustalX2 (University College Dublin). Using this alignment data, the phylogenetic tree was illustrated by Interactive Tree Of Life (http://itol.embl.de) (Letunic, I. & Bork, P. Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39, W475-478, doi:10.1093/nar/gkr201 (2011)). Among the 183 kinases found in C. neoformans, the present inventors constructed 114 gene-deletion kinases, and the kinases named based on the nomenclature rules for S. cerevisiae genes. The different colour codes represent the different classes of protein kinases predicted by Kinomer 1.0 (http://www.compbio.dundee.ac.uk/kinomer) (Martin, D. M., Miranda-Saavedra, D. & Barton, G. J. Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar/gkn834 (2009)). Red marked genes indicate the 60 pathogenicity-related kinases, and the distribution of these kinases for total kinases and various classes. FIG. 2 is a Pie-chart for the kinase classes predicted by Kinomer 1.0 to reveal the relative portion of protein kinase classes in human infectious fungal pathogens, C. neoformans, Candida albicans and Aspergillus fumigatus.

FIG. 3 shows phenotypic clustering of protein kinases in Cryptococcus neoformans. The phenotypes were scored by seven grades (−3: strongly sensitive/reduced, −2: moderately sensitive/reduced, −1: weakly sensitive/reduced, 0: wild-type like, +1: weakly resistant/increased, +2: moderately resistant/increased, +3: strongly resistant/increased). The excel file containing the phenotype scores of each kinase mutant was loaded by Gene-E software (http://www.broadinstitute.org/cancer/software/GENE-E/) and then the kinase phenome clustering was drawn using one minus Pearson correlation. The abbreviations used in FIG. 3 have the following meanings: [T25: 25° C., T30: 30° C., T37: 37° C., T39: 39° C., CAP: capsule production; MEL: melanin production; URE: urease production; MAT: mating filamentation, HPX: hydrogen peroxide, TBH: tert-butyl hydroperoxide, MD: menadione, DIA: diamide, MMS: methyl methanesulfonate, HU: hydroxyurea, 5FC: 5-flucytosine, AMB: amphotericin B, FCZ: fluconazole, FDX: fludioxonil, TM: tunicamycin, DTT: dithiothreitol, CDS: cadmium sulfate, SDS: sodium dodecyl sulfate, CR: Congo red, CFW: calcofluor white, KCR: YPD+KCl, NCR: YPD+NaCl, SBR: YPD+sorbitol, KCS: YP+KCl, NCS: YP+NaCl, SBS: YP+sorbitol].

FIG. 4 shows the phenotypic traits of ga183 custom-character mutant and snf1Δ mutant. FIG. 4a shows the results of comparing the phenotypic traits between a wild-type strain and snf1Δ and ga183Δ mutants under various stress conditions, and indicates that in 1 μg/ml fludioxonil (FDX), the snf1Δ and ga183Δ mutants showed increased susceptibility compared to the wild-type strain, and in 0.65 mM tert-butyl hydroperoxide (tBOOH), the snf1Δ and ga183Δ mutants showed increased resistance compared to the wild-type strain. FIG. 4b shows the results of comparing carbon source utilization between a wild-type strain and snf1Δ and ga183Δ mutants. An experiment was performed under the conditions of 2% glucose, 2% galactose, 3% glycerol, 3% ethanol, 2% maltose, 2% sucrose, 2% sodium acetate, and 1% potassium acetate, and the experimental results indicated that the snf1Δ and ga183Δ mutants required ethanol, sodium acetate and potassium acetate as carbon sources.

FIG. 5 shows the results of an experiment performed to examine whether Fpk1 regulates Ypk1-dependent phenotypes in the pathogenicity of Cryptococcus neoformans. (a) A scheme for the replacement of the FPK1 promoter with histone H3 promoter to construct an FPK1-overexpressing strain. (b) The FPK1 overexpressing strain was analyzed by Southern blot analysis, and YSB3986 and YSB3981 strains were produced by overexpressing FPK1 using a ypk1Δ mutant as a parent strain. (c) Overexpression of FPK1 was verified by Northern blot analysis. rRNA was used as a loading control. (d) WT strain (H99S), ypk1Δ (YSB1736) mutant, and FPK1 overexpression strains (YSB3986 and YSB3981) were cultured in YPD liquid medium for 16 hours, spotted on YPD medium, and incubated at the indicated temperature to observe the degree of growth. (e and f) The strains were tested on YPD medium containing 1.5 M NaCl, 0.04% sodium dodecyl sulphate, 1 μg/ml fluorodioxonil, 1 μg/ml amphotericin B, 3 mM hydrogen peroxide, 3 mg/ml calcofluor white, 100 mM hydroxyurea, 2 mM diamide, 300 μg/ml flucytosine and 5 mg/ml fluconazole. Cells were further incubated at 30° C. for 3 days and photographed. (g) The regulatory model for Ypk1 and Fpk1 kinases in C. neoformans, which can be proposed based on the experimental results.

FIGS. 6, 7 and 8 show the results of identifying pathogenic kinases by insect killing assay. Each mutant was grown for 16 hours in liquid YPD medium, washed three times with PBS buffer, and then inoculated into G. mellonella larva using 4,000 mutant cells per larva (15 larvae per group). The infected larvae were incubated at 37° C. and monitored for their survival each day. Statistical analysis of the experimental results was performed using the Log-rank (Mantel-Cox) test. FIGS. 6, 7 and 8a show the survival data of two independent mutants for each kinase. FIG. 8b shows the results of two repeated experiments for kinases from which only one mutant was produced.

FIGS. 9 and 10 shows the results of a signature-tag mutagenesis (STM)-based murine model virulence test. In the STM study, ste50Δ and hx11Δ strains were used as virulent and non-virulent control strains. STM scores were measured by using qPCR analysis using the STM-specific primers listed in Table 2 below for three-independent biological replicates. (a-d) All the kinase mutants were divided into four sets. The genes of each set consisted of two-independent mutants, and when one mutant was present, two independent experiments were performed.

FIG. 11 summarizes the pathogenicity-related kinases in Cryptococcus neoformans. STM scores were calculated by the quantitative PCR method, arranged numerically and coloured in gradient scales (FIG. 11a). Red marked letters show the novel infectivity-related kinases revealed by this analysis. Gene names for the 25 kinases that were co-identified by both insect killing and STM assays were depicted below the STM zero line. The P-value between control and mutant strains was determined by one-way analysis of variance (ANOVA) employing Bonferroni correlation with three mice per each STM set. Each set was repeated twice using independent strains. For single strain mutants, two independent experiments were repeatedly performed using each single strain. In the STM study, the roles of a total of 54 kinases in the infectivity of C. neoformans were analyzed. Referring to FIGS. 5 to 8, a total of 6 kinases were not shown to be involved in pathogenicity regulation in the murine model infectivity test, but were shown to be pathogenicity-related kinases by the wax moth killing assay (FIG. 11b). For bub1 and kin4 single mutant strains, the experiment was repeated twice.

FIG. 12 shows the pleiotropic roles of Ipk1 in Cryptococcus neoformans. Using WT (wild-type) and ipk1Δ mutants (YSB2157 and YSB2158), various experiments were performed. In FIG. 12a, ipk1Δ mutants (YSB2157 and YSB2158) showed attenuated virulence in the insect-based in vivo virulence assay. In this assay, WT and PBS were used as controls. In FIG. 12b, ipk1Δ mutants showed increased capsule production. Cells, incubated overnight, were placed on a DME plate at 37° C. for 2 days. 50 μl of 1.5×10⁸cells were packed into each capillary tube, and the packed cell volume was monitored every day. After 3 days when the cells were precipitated by gravity, the packed cell volume in the total volume was calculated and normalized to WT. The P value of each strain was less than 0.05. (*) Error bars indicate SEM. In FIG. 12c, ipk1Δ mutants show melanin-deficient phenotypes. Melanin production was assayed on Niger seed plates containing 0.2% glucose after 3 days. In FIG. 12d, ipk1Δ deletion mutants show defects in urease production. Urease production was assayed on Christensen's agar media at 30° C. after 2 days. In FIG. 12e, ipk1Δ mutants display severe defects in mating. Mating was assayed on V8 media (pH 5, per L: V8 juice 50 ml (Campbell), KH₂PO₄(Bioshop, PPM302) 0.5 g, agar (Bioshop, AGR001.500) 40 g) plate for 9 days. FIGS. 12f and 12g are micrographs obtained from 10-fold diluted spot analysis (10²to 10⁵-fold dilution). Growth rate was measured under various growth conditions indicated on the photographs. For analysis of chemical susceptibility, YPD medium was treated with the following chemicals: HU; 100 mM hydroxyurea as DNA damage reagent, TM; 0.3 μg/ml tunicamycin as ER (endoplasmic reticulum) stress inducing reagent, CFW; 3 mg/ml calcofluor white as cell wall damage reagent, SDS; 0.03% sodium dodecyl sulfate for membrane stability testing, CDS; 30 M CdSO₄as heavy metal stress reagent, HPX; 3 mM hydrogen peroxide as oxidizing reagent, 1M NaCl for osmotic shock, and 0.9 ml/mg AmpB (amphotericin B), 14 μg/ml FCZ (fluconazole), 300 μg/ml 5-FC (flucytosine), and 1 μg/ml FDX (fludioxonil) for analysis of antifungal agent susceptibility.

FIG. 13 shows the results of experiments using cdc7d, cbk1Δ and kic1Δ mutants. (a-c) cdc7Δ mutants (YSB2911, YSB2912), met1Δ mutants (YSB3063, YSB3611) and cka1 (YSB3051, YSB3052) were grown overnight in YPD medium, diluted 10-fold serially, and spotted on solid YPD medium and a YPD medium containing 100 mM hydroxyurea (HU), 0.06% methyl methanesulphonate (MMS), 1 μg/ml amphotericin B (AmpB), 1 μg/ml fludioxonil (FDX), 3 mM hydrogen peroxide (HPX) and 300 μg/ml flucytosine (5-FC). The spotted cells were further incubated at 30° C. or the indicated temperatures for 3 days and then photographed. (d) Wild-type and kic1Δ (YSB2915, YSB2916), cbk1Δ (YSB2941, YSB2942) and cka1Δ (YSB3051, YSB3052) mutants were incubated in YPD medium for 16 hours or more, and then fixed with 10% paraformaldehyde for 15 minutes and washed twice with PBS solution. The fixed cells were stained with 10 μg/ml Hoechst solution (Hoechst 33342, Invitrogen) for 30 minutes, and then observed with a fluorescence microscope (Nikon eclipse Ti microscope).

FIG. 14 shows the results of experiments on bud32Δ mutants. (a) Wild-type and bud32Δ mutants (YSB1968, YSB1969) were incubated overnight in YPD medium, diluted 10-fold serially, and then spotted on YPD medium containing the following chemicals, and observed for their growth rate under various growth conditions: 1.5 M NaCl, 1.5 M KCl, 2 M sorbitol, 1 μg/ml amphotericin B (AmpB), 14 μg/ml fluconazole (FCZ), 1 μg/ml fludioxonil (FDX), 300 μg/ml flucytosine, 100 mM hydroxyurea (HU), 0.04% methyl methanesulphonate (MMS), 3 mM hydrogen peroxide (HPX), 0.7 mM tert-butyl hydroperoxide (tBOOH), 2 mM diamide (DIA), 0.02 mM menadione (MD), and 0.03% sodium dodecyl sulphate (SDS). The cells spotted on the YPD medium containing these chemicals were further incubated at 30° C., and then photographed. (b) Melanin production of wild-type and bud32Δ mutants was assayed on Niger seed plates containing 0.1% glucose, and urease production was assayed after incubation on Christensen's agar media at 30° C. To examine capsule production, cells incubated overnight were placed on a DME plate at 37° C. for 2 days. 50 μl of 1.5×10⁸cells were packed into each capillary tube, and after 3 days, the packed cell volume was monitored every day by gravity. The packed cell volume in the total volume was calculated and normalized to WT. The results were analyzed by one-way analysis of variance (ANOVA) employing Bonferroni correlation, and the analysis was repeated three times. (c) To examine the mating efficacy, wild-type and bud32Δ mutants were spotted onto V8 mating medium and then incubated at room temperature in the dark for 9 days. (d) WT and bud32Δ mutants grown at 30° C. to the logarithmic phase and then were treated with or without fluconazole (FCZ) for 90 min. Total RNA was extracted from each sample, and the expression level of ERG11 was analyzed by Northern blotting.

FIG. 15 shows the results of experiments on arg5, 6Δ mutants and met3Δ. (a, b) Wild-type (H99S), arg5, 6Δ mutants (YSB2408, YSB2409, YSB2410) and met3Δ mutants (YSB3329, YSB3330) were incubated overnight in YPD medium and then washed with PBS. The washed cells were diluted 10-fold serially and spotted on solid synthesis complete medium. [SC; yeast nitrogen base without amino acids (Difco) supplemented with the indicated concentration of the following amino acids and nucleotides: 30 mg/l L-isoleucine, 0.15 g/l L-valine, 20 mg/l adenine sulphate, 20 mg/l L-histidine-HCl, 0.1 g/l L-leucine, 30 mg/l L-lysine, 50 mg/l L-phenylalanine, 20 mg/l L-tryptophan, 30 mg/l uracil, 0.4 g/l L-serine, 0.1 g/l glutamic acid, 0.2 g/l L-threonine, 0.1 g/l L-aspartate, 20 mg/l L-arginine, 20 mg/l L-cysteine, and 20 mg/l L-methionine]. SC-arg (a), SC-met and SC-met-cys (b) media indicate the SC medium lacking arginine, methionine and/or cysteine supplements. (b) A schematic view showing methionine and cysteine biosynthesis pathways. (c) Wild-type, arg5, 6Δ mutants and met3Δ mutants were incubated overnight in YPD medium, diluted 10-fold serially, and then spotted on YPD medium containing the following chemicals, and observed for their growth rate under various growth conditions: 1 μg/ml amphotericin B (AmpB), 14 μg/ml fluconazole (FCZ), 1 μg/ml fludioxonil (FDX), and 3 mM hydrogen peroxide (HPX). The spotted cells were incubated at 30° C. or indicated temperature for 3 days, and then photographed.

FIG. 16 shows retrograde vacuole trafficking that controls the pathogenicity of Cryptococcus neoformans. Retrograde vacuole trafficking controls the pathogenicity of Cryptococcus neoformans. Various tests were performed using WT and vps15Δ mutants [YSB1500, YSB1501]. In FIG. 16a, Vps15 is required for virulence of C. neoformans. WT and PBS were used as positive and negative virulence controls, respectively. In FIG. 16b, vps15Δ mutants display enlarged vacuole morphology. Scale bars indicate 10 μm. In FIG. 16c, vps15Δ mutants show significant growth defects under ER stresses. Overnight cultured cells were spotted on the YPD medium containing 15 mM dithiothreitol (DTT) or 0.3 μg/ml tunicamycin (TM), further incubated at 30° C. for 3 days, and photographed. In FIG. 16d, vps15Δ mutants show significant growth defects at high temperature and under cell membrane/wall stresses. Overnight cultured cells were spotted on the YPD medium and further incubated at the indicated temperature or spotted on the YPD medium containing 0.03% SDS or 5 mg/ml calcofluor white (CFW) and further incubated at 30° C. Plates were photographed after 3 days. In FIG. 16e, Vps15 is not involved in the regulation of the calcineurin pathway in C. neoformans. For quantitative RT-PCR (qRT-PCR), RNA was extracted from three biological replicates with three technical replicates of WT and vps15Δ mutants. CNA1, CNB1, CRZ1, UTR2 expression levels were normalized by ACT1 expression levels as controls. Data were collected from the three replicates. Error bars represent SEM (standard error of means). In FIG. 16f, Vps15 negatively regulates the HXL1 splicing. For RT-PCR, RNA was extracted from WT and vps15Δ mutants and cDNA was synthesized. HXL1 and ACT1-specific primer pairs were used for RT-PCR (Table 3). This experiment was repeated twice and one representative experiment is presented.

FIG. 17 shows the results of experiments on vrk1Δ mutants. FIG. 17a shows the results of spotting WT and vrk1Δ strains on YPD medium and on YPD medium containing 2.5 mM hydrogen peroxide (HPX), 600 μg/ml flucytosine (5-FC) or 1 μg/ml fludioxonil (FDX). The strains were incubated at 30° C. for 3 days and photographed. FIG. 17b shows the results of relative quantification of the packed cell volume. Three independent measurements shows a significant difference between WT and vrk1Δ strains (***; 0.0004 and **; 0.0038, s.e.m). FIG. 17c shows relative quantification of Vrk1-mediated phosphorylation. Peptide samples were analyzed three times on average, and peptides were obtained from two independent experiments. The data is the mean±s.e.m of two independent experiments. Student's unpaired t-test was applied for determination of statistical significance. ***P<0.001, **P<0.01, *P<0.05. PSMs represent peptide spectrum matching.

BEST MODE

In one embodiment of the present invention, there is provided a method for screening an antifungal agent, comprising the steps of: (a) bringing a sample to be analyzed into contact with a cell containing a pathogenicity-regulating kinase protein or a gene encoding the protein; (b) measuring the amount or activity of the protein or the expression level of the gene; and (c) determining that the sample is an antifungal agent, when the amount or activity of the protein or the expression level of the gene is measured to be down-regulated or up-regulated.

In the method for screening the antifungal agent, the pathogenicity-regulating kinase protein may be one or more selected from the group consisting of BUD32, ATG1, CDC28, KIC1, MEC1, KIN4, MKK1/2, BCK1, SNF1, SSK2, PKAT, GSK3, CBK1, KIC1, SCH9, RIM15, HOG1, YAK1, IPK1, CDC7, SSN3, CKA1, MEC1, ARG5, 6P, MET3, VPS15 and VRK1.

In another embodiment of the present invention, the cell used in screening of the antifungal agent is a Cryptococcus neoformans cell, and the antifungal agent is an antifungal agent for treating meningoencephalitis or cryptococcosis.

In another embodiment of the present invention, there is provided an antifungal pharmaceutical composition comprising an antagonist or inhibitor of the Cryptococcus neoformans pathogenicity-regulating kinase protein or an antagonist or inhibitor of the gene encoding the protein. In this regard, the pathogenicity-regulating kinase protein may be one or more selected from the group consisting of BUD32, ATG1, CDC28, KIC1, MEC1, KIN4, MKK1/2, BCK1, SNF1, SSK2, PKA1, GSK3, CBK1, KIN1, SCH9, RIM15, HOG1, YAK1, IPK1, CDC7, SSN3, CKA1, MEC1, ARG5, 6P, MET3, VPS15 and VRK1.

In still another embodiment of the present invention, the antifungal pharmaceutical composition is for treating meningoencephalitis or cryptococcosis, and the antagonist or inhibitor may be a small molecule; an antibody against the protein; or an antisense oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising one or more of these, against the gene.

In yet another embodiment of the present invention, the antifungal pharmaceutical composition is an antifungal pharmaceutical composition to be administered in combination with an azole-based or non-azole-based antifungal agent. The azole-based antifungal agent may be at least one selected from the group consisting of fluconazole, itraconazole, voriconazole and ketoconazole. In addition, the non-azole-based antifungal agent may be at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, nystatin and fludioxonil.

Mode for Invention

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.

Animal care and all experiments were conducted in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee (IACUC) of Yonsei University. The Yonsei University IACUC approved all of the vertebrate studies.

EXAMPLES
Example 1
Identification of Protein Kinases in Cryptococcus neoformans

To select the putative kinase genes in the genome of C. neoformans var. grubii (H99 strain), two approaches were used. The first approach used was Kinome v. 1.0 database (www.compbio.dundee.ac.uk/kinomer/) which systematically predicts and classifies eukaryotic protein kinases based on a highly sensitive and accurate hidden Markov model (HMM)-based method (Martin, D. M., Miranda-Saavedra, D. & Barton, G. J. Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar/gkn834, 2009). Through the Kinome database, 97 putative kinases in the genome of serotype D C. neoformans (JEC21 strain) were predicted. The ID of each JEC21 kinase gene was mapped with the H99 strain based on the most recent genome annotation (version 7), 95 putative kinases were queried. However, it was shown that this Kinome list was incomplete, because it failed to present all histidine kinases and some known kinases such as Hog1. For this reason, the present inventors surveyed a curated annotation of kinases in the H99 genome database provided by the Broad Institute (www.broadinstitute.org/annotation/genome/cryptococcus_neoformans) and the JEC21 genome database within the database of the National Center for Biotechnology Information. For each gene that had a kinase-related annotation, the present inventors performed protein domain analyses using Pfam (http://pfam.xfam.org/) to confirm the presence of kinase domains and to exclude the genes with annotations such as phosphatases or kinase regulators. Through this analysis, 88 additional putative kinases genes were queried. As a result, 183 putative kinase genes in C. neoformans were retrieved. The phylogenetic relationship thereof is shown in FIG. 1.

Eukaryotic protein kinase superfamilies are further classified into six conventional protein kinase groups (ePKs) and three atypical groups (aPKs) (Miranda-Saavedra, D. & Barton, G. J. Classification and functional annotation of eukaryotic protein kinases. Proteins 68, 893-914, doi:10.1002/prot.21444, 2007). ePKs include the AGC group (including cyclic nucleotide and calcium-phospholipid-dependent kinases, ribosome S6-phosphoprylated kinases, G protein-linked kinases and all similar analogues of these sets), CAMKs (calmodulin-regulated kinases); the CK1 group (casein kinase 1, and similar analogues), the CMGC group (including cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases and CDK-like kinases), the RGC group (receptor guanylate cyclase), STEs (including many kinase functions in the MAP kinase cascade), TKs (tyrosine kinases) and TKLs (tyrosine kinase-like kinases) (FIGS. 1 and 2). The aPKs include the alpha-kinase group, PIKK (phosphatidylinositol 3-kinase-related kinase group), RIO and PHDK (pyruvate dehydrogenase kinase group). To classify 183 C. neoformans protein kinases based on these criteria, the present inventors queried their amino acid sequences in the Kinomer database. Some of the previously classified kinases (Martin, D. M., Miranda-Saavedra, D. & Barton, G. J. Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar/gkn834, 2009) were classified otherwise (14 out of 95), presumably due to sequence differences between JEC21 and H99. Most of other kinases identified by annotation did not correspond to the previous category (82 out of 88), and were classified as “others”. Therefore, it was found that the C. neoformans genome consists of 89 ePKs (18 AGC, 22 CAMK, 2 CK1, 24 CMGC, 2 PDHK, 18 STE, 3 TKL), 10 aPKs (2 PDHK, 6 PIKK, 2 RIO), and 84 “others” (FIG. 1). The others include 7 histidine kinases (FIGS. 1 and 2). Based on prediction by the HMMER sequence profiles of Superfamily (version 1.73) (Wilson, D. et al. SUPERFAMILY—sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res 37, D380-386, doi:10.1093/nar/gkn762 (2009)), it was shown that two human fungal pathogens, C. albicans and A. fumigatus, have 188 and 269 protein kinases, respectively. Among pathogenic fungal protein kinases, CMGC (12-13%), CAMK (12-18%), STE (6-10%) and AGC (6-10%) kinases appear to be the most common clades (FIGS. 1 and 2).

Given that most eukaryotic genomes are predicted to contain kinase at a ratio of about 1-2% of the genome, the protein kinase ratio of C. neoformans (˜2.6%) was higher than expected. This indicates that C. neoformans has both saprobic and parasitic life cycles in which pathogenic yeast is in contact with more diverse environmental signals and host signals. Nevertheless, it is still necessary to explain whether all these predicted kinases have biologically significant kinase activity. The phylogenetic comparison of 183 putative kinases in C. neoformans with those in other strains and higher eukaryotes suggest that kinases much more evolutionarily conserved than transcription factors (TFs) in strains and other eukaryotes. In conclusion, the kinome network appears to be evolutionarily conserved in at least sequence similarity among fungi, which is in sharp contrast to evolutionary distribution of TF networks.

Example 2
Construction of Kinase Gene-Deletion Mutant Library in C. neoformans

To gain insights into the biological functions of Cryptococcus kinome networks and the complexity thereof, the present inventors constructed gene-deletion mutants for each kinase and functionally characterized them. Among the kinases analyzed here, mutants for 22 kinases (TCO1, TCO2, TCO3, TCO4, TCO5, TCO7, SSK2, PBS2, HOG1, BCK1, MKK1/2, MPK1, STE11, STE7, CPK1, PKA1, PKA2, HRK1, PKP1, IRE1, SCH9, and YPK1) were already functionally characterized in part by the present inventor. (Bahn, Y. S., Geunes-Boyer, S. & Heitman, J. Ssk2 mitogen-activated protein kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans. Eukaryot. Cell 6, 2278-2289 (2007); Bahn, Y. S., Hicks, J. K., Giles, S. S., Cox, G. M. & Heitman, J. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade. Eukaryot. Cell 3, 1476-1491 (2004); Bahn, Y. S., Kojima, K., Cox, G. M. & Heitman, J. Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans. Mol. Biol. Cell 16, 2285-2300 (2005); Bahn, Y. S., Kojima, K., Cox, G. M. & Heitman, J. A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans. Mol. Biol. Cell. 17, 3122-3135 (2006); Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767 (2015); Kim, M. S., Kim, S. Y., Yoon, J. K., Lee, Y. W. & Bahn, Y. S. An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem. Biophys. Res. Commun. 390, 983-988, doi:S0006-291X(09)02080-4 [pii]10.1016/j.bbrc.2009.10.089 (2009); Kim, S. Y. et al. Hrk1 plays both Hog1-dependent and -independent roles in controlling stress response and antifungal drug resistance in Cryptococcus neoformans. PLoS One 6, e18769, doi:doi:10.1371/journal.pone.0018769 (2011); Kojima, K., Bahn, Y. S. & Heitman, J. Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans. Microbiology 152, 591-604 (2006); Maeng, S. et al. Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans. Eukaryot. Cell 9, 360-378, doi:EC.00309-09 [pii];10.1128/EC.00309-09 (2010); Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177 (2011)).

For the remaining 161 kinases, the present inventors constructed gene-deletion mutants by using large-scale homologous recombination and by analyzing their in vitro and in vivo phenotypic traits. The constructed mutant was deposited (accession number: KCCM 51297).

In order to perform a large-scale virulence test in mouse hosts, dominant nourseothricin-resistance markers (NATs) containing a series of signature tags (Table 1) were employed. Southern blot analysis was performed to verify both the accurate gene deletion and the absence of any ectopic integration of each gene-disruption cassette. Table 1 below shows 26 kinase gene-deletion strains.

TABLE 1

CNAG_Num.
GENE NAME
YSE#
GENOTYPE

CNAG_00047
PKP1
558, 608
MATα pkp1Δ::NAT-STM#224

CNAG_00106
TCO5
286, 287
MATα tco5Δ::NAT-STM#125

CNAG_00130
HRK1
270, 271
MATα hrk1Δ::NAT-STM#58

CNAG_00363
TCO6
2469, 2554
MATα tco6Δ::NAT-STM#58

CNAG_00396
PKA1
188, 189
MATα pka1Δ::NAT-STM#191

CNAG_00405
KIC1
2915, 2916
MATα kic1Δ::NAT-STM#201

CNAG_00415
CDC2801
2370, 3699
MATα cdc2801Δ::NAT-STM#191

CNAG_00636
CDC7
2911, 2912
MATα cdc7Δ::NAT-STM#213

CNAG_00745
HRK1/NPH1
1438, 1439
MATα hrk1/mph1Δ::NAT-STM#210

CNAG_00769
PBS2
123, 124
MATα pbs2Δ::NAT-STM#213

CNAG_00782
SPS1
3229, 3325
MATα sps1Δ::NAT-STM#288

CNAG_00826
DAK2
1912, 1913
MATα dak2Δ::NAT-STM#282

CNAG_01062
PSK201
1989, 1990
MATα psk201Δ::NAT-STM#191

CNAG_01155
GUT1
1241, 2761
MATα gut1Δ::NAT-STM#242

CNAG_01162
MAK322
3824, 3825
MATα mak322Δ::NAT-STM#159

CNAG_01165
LCB5
3789, 3790
MATα lcb5Δ::NAT-STM#213

CNAG_01209
FAB1
3172
MATα fab1Δ::NAT-STM#169

CNAG_01294
IPK1
2157, 2158
MATα ipk1Δ::NAT-STM#184

CNAG_01333
ALK1
1571, 1573
MATα alk1Δ::NAT-STM#122

CNAG_01523
HOG1
64, 65
MATα hog1Δ::NAT-STM#177

CNAG_01123
PSK202
3922, 3924
MATα psk202Δ::NAT-STM#208

CNAG_01704
IRK6
3830, 3831
MATα irk6Δ::NAT-STM#5

CNAG_01730
STE7
342, 343
MATα ste7Δ::NAT-STM#225

CNAG_01850
TCO1
278, 279
MATα yco1Δ::NAT-STM#102

CNAG_01905
KSP1
1807, 1808, 1809
MATα ksp1Δ::NAT-STM#159

CNAG_01938
KIN1
3930, 3931
MATα kin1Δ::NAT-STM#6

CNAG_01988
TCO3
284, 285
MATα tco3Δ::NAT-STM#119

CNAG_02233
MEC1
3063, 3611
MATα mec1Δ::NAT-STM#204

CNAG_02296
RBK1
1510, 1511
MATα rbk1Δ::NAT-STM#219

CNAG_02357
MKK2
330, 331
MATα mkk2Δ::NAT-STM#224

CNAG_02389
YKP101
1885, 1886
MATα ypk101Δ::NAT-STM#242

CNAG_02511
CPK1
127, 128
MATα cpk1Δ::NAT-STM#184

CNAG_02531
CPK2
373, 374
MATα cpk2Δ::NAT-STM#122

CNAG_02542
IRK2
1904, 1905
MATα irk2Δ::NAT-STM#232

CNAG_02551
DAK3
1940, 1941
MATα dak3Δ::NAT-STM#295

CNAG_02675
HSL101
1800, 1801
MATα hsl101Δ::NAT-STM#146

CNAG_02680
VPS15
1500, 1501
MATα vps15Δ::NAT-STM#123

CNAG_02712
BUD32
1968, 1969
MATα bud32Δ::NAT-STM#295

CNAG_02799
DAK202A
2487, 2489
MATα dak202aΔ::NAT-STM#119

CNAG_02802
ARG2
1503, 1504
MATα arg2Δ::NAT-STM#125

CNAG_02820
PKH201
1234, 1235, 1236
MATα pkh201Δ::NAT-STM#219

CNAG_02859
POS5
3714, 3715
MATα pos5Δ::NAT-STM#58

CNAG_02947
SCY1
2793, 2794
MATα scy1Δ::NAT-STM#150

CNAG_03024
RIM15
1216, 1217
MATα rim15Δ::NAT-STM#191

CNAG_03048
IRK3
1486, 1487
MATα irk3Δ::NAT-STM#273

CNAG_03167
CHK1
1825, 1828
MATα chk1Δ::NAT-STM#205

CNAG_03184
BUB1
3398
MATα bub1Δ::NAT-STM#201

CNAG_03216
SNF101
1575, 1576
MATα snf101Δ::NAT-STM#146

CNAG_03258
TPK202A
2443, 2444
MATα psk202aΔ::NAT-STM#208

CNAG_03290
KIC102
3211, 3212
MATα kic102Δ::NAT-STM#201

CNAG_03355
TCO4
417, 418
MATα tco4Δ::NAT-STM#123

CNAG_03367
URK1
1266, 1267
MATα urk1Δ::NAT-STM#43

CNAG_03369
SWE102
1564, 1565
MATα swe102Δ::NAT-STM#169

CNAG_03567
CBK1
2941, 2942
MATα cbk1Δ::NAT-STM#232

CNAG_03592
THI20
3219, 3220
MATα THI20Δ::NAT-STM#231

CNAG_03670
IRE1
552, 554
MATα ire1Δ::NAT-STM#224

CNAG_03811
IRK5
2952, 2953
MATα irk5Δ::NAT-STM#213

CNAG_03843
ARK1
1725, 1726
MATα ark1Δ::NAT-STM#43

CNAG_03946
GAL302
2852, 2853
MATα gal302Δ::NAT-STM#218

CNAG_04040
FPK1
2948, 2949
MATα fpk1Δ::NAT-STM#211

CNAG_04108
PKP2
2439, 2440
MATα pkp2Δ::NAT-STM#295

CNAG_04162
PKA2
194, 195
MATα pka2Δ::NAT-STM#205

CNAG_04197
YAK1
2040, 2096, 4139
MATα yak1Δ::NAT-STM#184

CNAG_04215
MET3
3329, 3330
MATα met3Δ::NAT-STM#205

CNAG_04221
FBP26
3669
MATα fbp26Δ::NAT-STM#146

CNAG_04230
THI6
1468, 1469
MATα thi6Δ::NAT-STM#290

CNAG_04282
MPK2
3236, 3238
MATα mpk2Δ::NAT-STM#102

CNAG_04316
UTR1
2892, 2893
MATα utr1Δ::NAT-STM#5

CNAG_04408
CKI1
1804, 1805
MATα cki1Δ::NAT-STM#218

CNAG_04433
YAK103
3736, 3737
MATα YAK103Δ::NAT-STM#231

CNAG_04514
MPK1
3814, 3816
MATα mpk1Δ::NAT-STM#240

CNAG_04631
RIK1
1579, 1580
MATα CNAG_04631Δ::NAT-STM#150

CNAG_04678
YPK1
1736, 1737
MATα ypk1Δ::NAT-STM#58

CNAG_04755
BCK1
273, 274
MATα bck1Δ::NAT-STM#43

CNAG_04821
PAN3
2809, 2810
MATα pan3Δ::NAT-STM#204

CNAG_04927
YFH702
2826, 3716
MATα yfh702Δ::NAT-STM#220

CNAG_05005
ATG1
1935, 1936
MATα atg1Δ::NAT-STM#288

CNAG_05063
SSK2
264, 265
MATα ssk2Δ::NAT-STM#210

CNAG_05097
CKY1
1245, 1246
MATα CNAG_05097Δ::NAT-STM#282

CNAG_05216
RAD53
3785, 3786
MATα rad53Δ::NAT-STM#184

CNAG_05220
TLK1
3153, 3188
MATα tlk1Δ::NAT-STM#116

CNAG_05243
XKS1
2851
MATα xks1Δ::NAT-STM#125

CNAG_05439
CMK1
1883, 1901, 2902
MATα cmk1Δ::NAT-STM#227

CNAG_05558
KIN4
2955
MATα kin4Δ::NAT-STM#225

CNAG_05590
TCO2
281, 282
MATα tco2Δ::NAT-STM#116

CNAG_05600
IGI1
1514, 1515
MATα CNAG_05600Δ::NAT-STM#230

CNAG_05694
CKA1
3051, 3052, 3053
MATα cka1Δ::NAT-STM#6

CNAG_05753
ARG5.6
2408, 2409, 2410
MATα arg5/6Δ::NAT-STM#220

CNAG_05771
TEL1
3844, 3845
MATα tel1Δ::NAT-STM#225

CNAG_05965
IRK4
2806, 2808
MATα irk4Δ::NAT-STM#211

CNAG_06033
MAK32
3240, 3241
MATα mak32Δ::NAT-STM#169

CNAG_06051
GAL1
2829, 2830
MATα gal1Δ::NAT-STM#224

CNAG_06086
SSN3
3038, 3039
MATα ssn3Δ::NAT-STM#219

CNAG_06161
VRK1
2216, 2217
MATα vrk1Δ::NAT-STM#23

CNAG_06193
CRK1
1709, 1710
MATα crk1Δ::NAT-STM#43

CNAG_06278
TCO7
348
MATα tco7Δ::NAT-STM#209

CNAG_06301
SCH9
619, 620
MATα sch9Δ::NAT-STM#169

CNAG_06310
IRK7
2136, 2137
MATα irk7Δ::NAT-STM#208

CNAG_06366
HRR2502
2053
MATα hrr2502Δ::NAT-STM#125

CNAG_06552
SNF1
2372, 2373
MATα snf1Δ::NAT-STM#204

CNAG_06553
GAL83
2415, 2416
MATα gal83Δ::NAT-STM#288

CNAG_06568
SKS1
1410, 1411
MATα sks1Δ::NAT-STM#211

CNAG_06632
ABC1
2072, 2797
MATα CNAG_06632Δ::NAT-STM#119

CNAG_06671
YKL1
3926, 3927
MATα CNAG_06671Δ::NAT-STM#122

CNAG_06697
MPS1
3632, 3633
MATα mps1Δ::NAT-STM#116

CNAG_06730
GSK3
2038, 2039
MATα gsk3Δ::NAT-STM#123

CNAG_06809
IKS1
1310, 2119
MATα iks1Δ::NAT-STM#116

CNAG_06980
STE11
313, 314
MATα ste11Δ::NAT-STM#242

CNAG_07359
IRK1
1950, 1951
MATα irk1Δ::NAT-STM#5

CNAG_07580
TRM7
3056, 3057
MATα trm7Δ::NAT-STM#102

CNAG_07667
SAT4
3612
MATα sat4Δ::NAT-STM#212

CNAG_07744
PIK1
1493, 1494
MATα pik1Δ::NAT-STM#227

CNAG_07779
TDA10
2663, 3223
MATα tda10Δ::NAT-STM#102

CNAG_08022
PHO85
3702, 3703
MATα pho85Δ::NAT-STM#218

*CNAG: Abbreviation for Cryptococcus neoformans serotype A genome database, which is the H99 genomic database gene number provided by the Broad Institute.

For gene-deletion through homologous recombination, gene-disruption cassettes containing the nourseothricin-resistance marker (NAT; nourseothricin acetyl transferase) with indicated signature-tagged sequences were generated by using conventional overlap PCR or NAT split marker/double-joint (DJ) PCR strategies (Davidson, R. C. et al. A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607-2615 (2002); Kim, M. S., Kim, S. Y., Jung, K. W. & Bahn, Y. S. Targeted gene disruption in Cryptococcus neoformans using double-joint PCR with split dominant selectable markers. Methods Mol Biol 845, 67-84, doi:10.1007/978-1-61779-539-8_5 (2012) (Table 1). To validate a mutant phenotype and to exclude any unlinked mutational effects, more than two independent deletion strains were constructed for each kinase mutant (see Table 1). When two independent kinase mutants exhibited inconsistent phenotypes (inter-isolate inconsistency), more than three mutants were constructed. As a result, the present inventors successfully generated 220 gene deletion mutants representing 114 kinases (including those that were previously reported) (Table 1). For 106 kinases, two or more independent mutants were constructed. Some kinases that had been previously reported by others were independently deleted here with unique signature-tagged markers to perform parallel in vitro and in vivo phenotypic analysis. When two independent kinase mutants exhibited inconsistent phenotypes (known as inter-isolate inconsistency), the present inventors attempted to generate more than three mutants.

For the remaining 69 kinases, the present inventors were not able to generate mutants even after repeated attempts. In many cases, the present inventors either could not isolate a viable transformant, or observed the retention of a wild-type allele along with the disrupted allele. The success level for mutant construction of the kinases (114 out of 183 (62%)) was lower than that for transcription factors (TFs) that the present inventors previously reported (155 out of 178 (87%)) (Jung, K. W. et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757, 2015). This is probably because among fungi, kinases are generally much more evolutionarily conserved than TFs, and a greater number of essential or growth-related genes appeared to exist. In fact, 24 (35%) of the kinases are orthologous to kinases that are essential for the growth of Saccharomyces cerevisiae. Notably, 8 genes (RAD53, CDC28, CDC7, CBK1, UTR1, MPS1, PIK1, and TOR2) that are known to be essential in S. cerevisiae were successfully deleted in C. neoformans, suggesting the presence of functional divergence in some protein kinases between ascomycete and basidiomycete fungi.

In the first round of PCR, the 5′- and 3′-flanking regions for the targeted kinase genes were amplified with primer pairs L1/L2 and R1/R2, respectively, by using H99S genomic DNA as a template. For the overlap PCR, the whole NAT marker was amplified with the primers M13Fe (M13 forward extended) and M13Re (M13 reverse extended) by using a pNAT-STM plasmid (obtained from the Joeseph Heitman Laboratory at Duke University in USA) containing the NAT gene with each unique signature-tagged sequence. For the split marker/DJ-PCR, the split 5′- and 3′-regions of the NAT marker were amplified with primer pairs M13Fe/NSL and M13Re/NSR, respectively, with the plasmid pNAT-STM. In the second round of overlap PCR, the kinase gene-disruption cassettes were amplified with primers L1 and R2 by using the combined first round PCR products as templates. In the second round of split marker/DJ-PCR, the 5′- and 3′-regions of NAT-split gene-disruption cassettes were amplified with primer pairs L1/NSL and R2/NSR, respectively, by using combined corresponding first round PCR products as templates. For transformation, the H99S strain (obtained from the Joeseph Heitman Laboratory at Duke University in USA) was cultured overnight at 30° C. in the 50 ml yeast extract-peptone-dextrose (YPD) medium [Yeast extract (Becton, Dickison and company #212750), Peptone (Becton, Dickison and company #211677), Glucose (Duchefa,#G0802)], pelleted and re-suspended in 5 ml of distilled water. Approximately 200 μl of the cell suspension was spread on YPD solid medium containing 1M sorbitol and further incubated at 30° C. for 3hours. The PCR-amplified gene disruption cassettes were coated onto 600 μg of 0.6 μm gold microcarrier beads (PDS-100, Bio-Rad) and biolistically introduced into the cells by using particle delivery system (PDS-100, Bio-Rad). The transformed cells were further incubated at 30° C. for recovery of cell membrane integrity and were scraped after 3 hours. The scraped cells were transferred to the selection medium (YPD solid plate containing 100 μg/ml nourseothricin; YPD+NAT). Stable nourseothricin-resistant (NATr) transformants were selected through more than two passages on the YPD+NAT plates. All NAT^rstrains were confirmed by diagnostic PCR with each screening primer listed in Table 2 below. To verify accurate gene deletion, Southern blot analysis was finally performed (Jung, K. W., Kim, S. Y., Okagaki, L. H., Nielsen, K. & Bahn, Y. S. Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet. Biol. 48, 154-165, doi:S1087-1845(10)00191-X [pii] 10.1016/j.fgb.2010.10.006 (2011). Table 2 below lists primers used in the construction of the kinase mutant library.

TABLE 2

H99 locus

tag
Cn

(Broad
gene
Primer

o.
ID)
name
name
Primer description
Primer sequence (5′-3′)

1
CNAG_00047
PKP1
L1
CNAG_00047 5′

AATGAAGTTCCTGCGACAG

flanking region

primer 1

L2
CNAG_00047 5′

GCTCACTGGCCGTCGTTTTACAA

flanking region

TGGGATGAGAACGCAC

primer 2

R1
CNAG_00047 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

CATTTTCCAGCATCAGC

primer 1

R2
CNAG_00047 3′

GGTGTGGAACATCTTTTGAG

flanking region

primer 2

SO
CNAG_00047

CCTCTGACAGCCACATACTG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_00047

CTGGTTCATCTTGGGTGTC

Southern blot probe

primer 1

PO2
CNAG_00047

TCTGAGCATACCACTCCTTTAC

Southern blot probe

primer 2

STM
NAT#224 STM

AACCTTTAAATGGGTAGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

2
CNAG_00106
TCO5
L1
CNAG_00106 5′

TACACGAGATTGGCTGGCAACC

flanking region

primer 1

L2
CNAG_00106 5′

CTGGCCGTCGTTTTACAAGTGAA

flanking region

CGCCACACCGATGAG

primer 2

R1
CNAG_00106 3′

GTCATAGCTGTTTCCTGTCTCCC

flanking region

GAGGATGTCTTAG

primer 1

R2
CNAG_00106 3′

TGCCAAAGCGTGTAAGTG

flanking region

primer 2

SO
CNAG_00106

ATGGGAAAGGTCAGTAGCACCG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_00106

TCGTCTTTTCTTGGTCCAG

Southern blot probe

primer 1

PO2
CNAG_00106

TGAGGGCGTAGTTGATAATG

Southern blot probe

primer 2

STM
NAT#125 STM

CGCTACAGCCAGCGCGCGCAAG

primer

CG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

3
CNAG_00130
HRK1
L1
CNAG_00130 5

TTCCAGTCAACCGAGTAGC

flanking region

primer 1

L2
CNAG_00130 5′

CTGGCCGTCGTTTTACCTGTATT

flanking region

CATCATTGCGGC

primer 2

R1
CNAG_00130 3′

GTCATAGCTGTTTCCTGCGTCAA

flanking region

ATCCAAGAACATCGTG

primer 1

R2
CNAG_00130 3′
GCCTTCATCGTCGTTAGAC

flanking region

primer 2

SO
CNAG_00130

AAGACGACCACATCTCAGAG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_00130

AGGACTCTGCTCCATCAAG

Southern blot probe

primer 1

PO2
CNAG_00130

GAAAGAGCCTCAGAAAAGTAGG

Southern blot probe

primer 2

STM
NAT#58 STM

CGCAAAATCACTAGCCCTATAGC

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

4
CNAG_00266

L1
CNAG_00266 5′
GGTCGTATCTCTCTFTCAAGC

flanking region

primer 1

L2
CNAG_00266 5′
TCACTGGCCGTCGTTTTACTTG

flanking region
ACGAGTTGTTCAGGGG

primer 2

R1
CNAG_00266 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GATGTGGATGAGAAGGTAGC

primer 1

R2
CNAG_00266 3′
GTGCCGACGAGAAGATAAC

flanking region

primer 2

SO
CNAG_00266
AAGGGATAATGGATGACCAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00266
TCAGTGAGATTCAAGGATGC

Southern blot probe

primer

STM
NAT#213 STM
CTGGGGATTTTGATGTGTCTAT

primer
GT

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

5
CNAG_00363
TCO6
L1
CNAG_00363 5′

GAGAGAATAACAAAAGGGCG

flanking region

primer 1

L2
CNAG_00363 5′

TCACTGGCCGTCGTTTTACAC

flanking region

GAGGGTTAGAGTTGG

primer 2

R1
CNAG_00363 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GCGTCTTTGTAACCCG

primer 1

R2
CNAG_00363 3′

GCAGGTATCTTACACTCCGTTG

flanking region

primer 2

SO
CNAG_00363

ATTAGACACACGACCTGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00363

TGAGGATACTGGTTGACGC

Southern blot probe

primer

STM
NAT#58 STM

CGCAAAATCACTAGCCCTATAGC

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

6
CNAG_00388

L1
CNAG_00388 5′
TTTTGAGCGGGGAAACAC

flanking region

primer 1

L2
CNAG_00388 5′
TCACTGGCCGTCGTTTTACGGG

flanking region
TCTCGTCTGTATTTTCG

primer 2

R1
CNAG_00388 3′
CATGGTCATAGCTGTTTTCCTGG

flanking region
ATACCCAGGATTCCACTG

primer 1

R2
CNAG_00388 3′
ACCATTATCGTCGCCTTCG

flanking region

primer 2

SO
CNAG_00388
CAATCCCAATGGCTTTCAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00388
CGGGTCAAGATGAAAATGTTC

Southern blot probe
GTC

primer

STM
NAT#208 STM
TGGTCGCGGGAGATCGTGGTT

primer
T

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

7
CNAG_00396
PKA1
L1
CNAG_00396 5′

AAACGACTGTGTAATGCGAG

flanking region

primer 1

L2
CNAG_90396 5′

CTGGCCGTCGTTTTACGGAGCC

flanking region

AGAATAAAGGAGTTG

primer 2

R1
CNAG_00396 3′

GTCATAGCTGTTTCCTGGCACTA

flanking region

AATGGGTGAGCAC

primer 1

R2
CNAG_00396 3′

CGATTTGTCCAGTGATTCAGTGA

flanking region

C

primer 2

SO
CAT4G_00396

GTTGGAAGTAGCAGTGTCTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00396

TGTCGGAGGAGAATGAACG

Southern blot probe

primer

STM
NAT#191 STM

ATATGGATGTTTTTAGCGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

8
CNAG_00405
KIC1
L1
CNAG_00405 5′

AAGATGAGCGTTGCGAAG

flanking region

primer 1

L2
CNAG_00405 5′

TCACTGGCCGTCGTTTTACGCGT

flanking region

GGTGCTAAGAACAAC

primer 2

R1
CNAG_00405 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

GGTAGACTCCCAGAATGC

primer 1

R2
CNAG_00405 3′

TAATGTGTCAACTGCCGC

flanking region

primer 2

SO
CNAG_00405

TTGGTTTCAAGGGGGAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00405

AAAGTGGACCGTTTGGAG

Southern blot probe

primer

STM
NAT#201 STM

CACCCTCTATCTCGAGAAAGCTC

primer

C

STM
STU common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

9
CNAG_00415
CDC2801
L1
CNAG_00415 5′

CGCATTCTGGACAAAAGC

flanking region

primer 1

L2
CNAG_00415 5′

TCACTGGCCGTCGTTTTACTTTG

flanking region

CCGTATCTTCCTGG

primer 2

R1
CNAG_00415 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

ATGTATCTAATCCCTCCG

primer 1

R2
CNAG_00415 3′

AGATTCGGTGCTTTGTGTC

flanking region

primer 2

SO
CNAG_00415

TTGGTCTGGGAACCTTTAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00415

AATGTGCTACTGCCGACAG

Southern blot probe

primer

STM
NAT#191 STM

ATATGGATGTTTTTAGCGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

10
CNAG_00556

L1
CNAG_00556 5′
GAACCGAAAAGGGCATTC

flanking region

primer 1

L2
CNAG_00556 5′
TCACTGGCCGTCGTTTTACTGG

flanking region
AGCAGGTGGTTCTAAG

primer 2

R1
CNAG_00556 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
CAGGAGAGAGGAATGAAAC

primer 1

R2
CNAG_00556 3′
CCACCGTCCATTACTTACTG

flanking region

primer 2

SO
CNAG_00556
TGTCAACCCGCTCAAACAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00556
AGAGAAGTCCTTGCGATTG

Southern blot probe

primer

STM
NAT#290 STM
ACCGACAGCTCGAACAAGCAA

primer
GAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

11
CNAG_00636
CDC7
L1
CNAG_00636 5′

GCTGGAAGCGTGATGATAC

flanking region

primer 1

L2
CNAG_00636 5′

TCACTGGCCGTCGTTTTACTGTG

flanking region

TAGGAGGGGAGATGAG

primer 2

R1
CNAG_00636 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GGACATCCACCAGAGAGG

primer 1

R2
CNAG_00636 3′

CAAATGGGTGTCTCAGAGC

flanking region

primer 2

SO
CNAG_00636

TGAGTGATGCCTTACGCTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00636

CCCTGTAGACTTACCTTCCC

Southern blot probe

primer

STM
NAT#213 STM

CTGGGGATTTTGATGTGTCTATG

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

12
CNAG_00683

L1
CNAG_00683 5′
GAAAACGAGTCCTGGATAGTT

flanking region
C

primer 1

L2
CNAG_00683 5′
TCACTGGCCGTCGTTTTACATG

flanking region
GTTGGATGGGTAGGAG

primer 2

R1
CNAG_00683 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
CTGCCAACAGACATCAAC

primer 1

R2
CNAG_00683 3′
AGAAAAACTCGGACACCTG

flanking region

primer 2

SO
CNAG_00683
TGTAAAAAACAGAGGAGCCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00683
TTCAGAGTCATCCCACGGTG

Southern blot probe

primer

STM
NAT#273 STM
GAGATCTTTCGGGAGGTCTGG

primer
ATT

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

13
CNAG_00745
HRK11
L1
CNAG_00745 5′

GCAAAAATGGGGAAGATAGG

NPH1

flanking region

primer 1

L2
CN4G_00745 5′

TCACTGGCCGTCGTTTTACTTCC

flanking region

CCAAAATCACTCCC

primer 2

R1
CNAG_00745 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GAGATGAGTGGGTGAAG

primer 1

R2
CNAG_00745 3′

TGTGTCAGACCTGTTATCGTTTC

flanking region

primer 2

SO
CNAG_00745

CTCAACCACTCTCTTACGGA

diagnostic screening

primer, pairing with

PO
CNAG_00745

CGAGGTTAGGAGGAAAGGTC

Southern blot probe

primer

STM
NAT#210 STM

CTAGAGCCCGCCACAACGCT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

14
CNAG_00769
PBS2
L1
CNAG_00769 5′

AGGAAGGTGGAGTGTGTG

flanking region

primer 1

L2
CNAG_00769 5′

CTGGCCGTCGTTTTACATGCGAG

flanking region

GAAGAAAGGTCG

primer 2

R1
CNAG_00769 3′

GTCATAGCTGTTTCCTGAACCGA

flanking region

CGACCGACTTATGC

primer 1

R2
CNAG_00769 3′

GTAAGGTAGTCGCAACAACG

flanking region

primer 2

SO
CNAG_00769

CGATACCCTTCTTGCCTGTAG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_00769

AACACGACAGGAAATCCG

Southern blot probe

primer 1

PO2
CNAG_00769

TGGAAGGTTACAAGCCGAC

Southern blot probe

primer 2

STM
NAT#213 STM

CTGGGGATTTTGATGTGTCTATG

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

15
CNAG_00782
SPS1
L1
CNAG_00782 5′

CCCGATGAAAGTAATGGC

flanking region

primer 1

L2
CNAG_00782 5′

TCACTGGCCGTCGTTTTACAATG

flanking region

TCCTCTCTTCTGCTCTC

primer 2

R1
CNAG_00782 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

GACTGCGAAGAAAGGC

primer 1

R2
CNAG_00782 3′

CTTACATCCAGACATCCCAC

flanking region

primer 2

SO
CNAG_00782

GGGTGAGCAACAAGAAATG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00782

CTCCTCCTTTCTTTTATGCC

Southern blot probe

primer

STM
NAT#288 STM

CTATCCAACTAGACCTCTAGCTA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

16
CNAG_00826
DAK2
L1
CNAG_00826 5′

AGTTTGAATGAAGGGGCG

flanking region

primer 1

L2
CNAG_00826 5′

TCACTGGCCGTCGTTTTACGGAA

flanking region

GATGTGTCGGTCTGTC

primer 2

R1
CNAG_00826 3′

CATGGTCATAGCTGTTTCCTGCG

flanking region

GAAGGTATTCTCAAGGC

primer 1

R2
CNAG_00826 3′

GCTGTTCAGTTTCCTCTCTATG

flanking region

primer 2

SO
CNAG_00826

ACAGCGATGTGGGGATAAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_00826

CATACTTTCCTCGGGATTTC

Southern blot probe

primer

STM
NAT#282 STM

TCTCTATAGCAAAACCAATC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

17
CNAG_00877

L1
CNAG_00877 5′
TCCACACACGAATGGTATC

flanking region

primer 1

L2
CNAG_00877 5′
TCACTGGCCGTCGTTTTACTTG

flanking region
TCAGCAAGGGAATGGGCAGTG

primer 2

R1
CNAG_00877 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
GGATGATTTGAGGGATAG

primer 1

R2
CNAG_00877 3′
ATTGAAACTACCAGTGGCACC

flanking region
CCG

primer 2

SO
CNAG_00877
CCAATACGGTGCTTATGTGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_00877
CGCAGAGTAGGTTGTGTTG

Southern blot probe

primer

STM
NAT#204 STM
GATCTCTCGCGCTTGGGGGA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

18
CNAG_01061

L1
CNAG_01061 5′
AAAAGGGGTGGGTCAAAG

flanking region

primer 1

L2
CNAG_01061 5′
TCACTGGCCGTCGTTTTACGGG

flanking region
TATTGGGTTTCCTCTG

primer 2

R1
CNAG_01061 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CCATTAGCATTCGGAGAG

primer 1

R2
CNAG_01061 3′
GAAGTATCAGAGGAGTCCCG

flanking region

primer 2

SO
CNAG_01061
CGTGGTCACTTATGTCCTTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01061
AAAAGTGCGAAGGGAGGTC

Southern blot probe

primer

STM
NAT#220 STM
CAGATCTCGAACGATACCCA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

19
CNAG_01062
PSK201
L1
CNAG_01062 5′

GTCCACTTTATTTTCGGGC

flanking region

primer 1

L2
CNAG_01062 5′

TCACTGGCCGTCGTTTTACGAGG

flanking region

AGTAATGACCGTGACC

primer 2

R1
CNAG_01062 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GTAAAAAGGGGTGGGTC

primer 1

R2
CNAG_01062 3′

GGTATTGGGTTTCCTCTGTG

flanking region

primer 2

SO
CNAG_01062

GATTAGTATTCCTGTGCCACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01062

GGAAATGTAGGGGGTAGACG

Southern blot probe

primer

STM
NAT#191 STM

ATATGGATGTTTTTAGCGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

20
CNAG_01155
GUT1
L1
CNAG_01155 5′

AATCGTTCCCTTCCTAAGC

flanking region

primer 1

L2
CNAG_01155 5′

TCACTGGCCGTCGTTTTACAAAC

flanking region

CGAGACCTCTGAAGG

primer 2

R1
CNAG_01155 3′

CATGGTCATAGCTGTTTCCTGGG

flanking region

AGAAAGCCAGACTGAAG

primer 1

R2
CNAG_01155 3′

ATGGTAGTTTTGCGGGTG

flanking region

primer 2

SO
CNAG_01155

CAGAGAAGTTGACTGGGATG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01155

GTTCATCGCTTCAACCAG

Southern blot probe

primer

STM
NAT#242 STM

GTAGCGATAGGGGTGTCGCTTT

primer

AG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

21
CNAG_01162
MAK322
L1
CNAG_01162 5′
GACCGCAGTAGAACTTACACC

flanking region

primer 1

L2
CNAG_01162 5′

TCACTGGCCGTCGTTTTACGAGG

flanking region

AAATGTTGAAGGTGTG

primer 2

R1
CNAG_01162 3′

CATGGTCATAGCTGTTTCCTGCG

flanking region

GAAGGAAAGAGTTTAGACG

primer 1

R2
CNAG_01162 3′

ATCAGGCAACCGCATAAC

flanking region

primer 2

SO
CNAG_01162

ATGCTGCCAGAACACTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01162

TCCTCCCAAATAAGTGCC

Southern blot probe

primer

STM
NAT#159 STM

ACGCACCAGACACACAACCAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

22
CNAG_01165
LCB5
L1
CNAG_01165 5′

CCCAAATCTCGTTCGTTG

flanking region

primer 1

L2
CNAG_01165 5′

TCACTGGCCGTCGTTTTACTTGT

flanking region

GTGGCTGTAGAGGTG

primer 2

R1
CNAG_01165 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

CATCGCACATAACTTTC

primer 1

R2
CNAG_01165 3′

ATTCTGAAGGCGTAAGTCG

flanking region

primer 2

SO
CNAG_01165

AAAAGGGTCGTAAGATGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01165

ACGCCGAATAGGTTTGTG

Southern blot probe

primer

STM
NAT#213 STM

CTGGGGATTTTGATGTGTCTATG

primer

T

STM
STM common

GCATGCCCTGCCCGTAAGAATTC

common
primer

G

23
CNAG_01209
FAB1
L1
CNAG_01209 5′

TTTCTGATGGGAGGGAGTG

flanking region

primer 1

L2
CNAG_01209 5′

TCACTGGCCGTCGTTTTACGCGT

flanking region

GGTATGGATAGACAAG

primer 2

R1
CNAG_01209 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

AAGATTTGGGGGCTGG

primer 1

R2
CNAG_01209 3′

GCTGAAGGTGAGCGATAAG

flanking region

primer 2

SO
CNAG_01209

AGTCAGTGTCCAAACTTCTGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01209

AAAGGGAATCCAGGAACG

Southern blot probe

primer

STM
NAT#169 STM

ACATCTATATCACTATCCCGAAC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

24
CNAG_01250

L1
CNAG_01250 5′
GCTTTTTCGTTGGAGGTG

flanking region

primer 1

L2
CNAG_01250 5′
TCACTGGCCGTCGTTTTACTGC

flanking region
TCTGTCATCTTCCAGC

primer 2

R1
CNAG_01250 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
TAGCGTGTTACCACAGGC

primer 1

R2
CNAG_01250 3′
CGTCCTCAAAATACAACTCG

flanking region

primer 2

SO
CNAG_01250
TGGTAAATCCTCGTGCTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01250
GCGAAAGTAACCCAGATGC

Southern blot probe

primer

STM
NAT#227 STM
TCGTGGTTTAGAGGGAGCGC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

25
CNAG_01285

L1
CNAG_01285 5′
CAATAACCCATTACCACTGC

flanking region

primer 1

L2
CNAG_01285 5′
TCACTGGCCGTCGTTTTACTTG

flanking region
TTGGCAAGACCACTG

primer 2

R1
CNAG_01285 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
TTTCTCCTGAAGCCACTG

primer 1

R2
CNAG_01285 3′
TTAGAGGCGGTAGTTACGG

flanking region

primer 2

SO
CNAG_01282
TTACGATACTTGGCTGAAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01285
AGCATTTTGGCTGTAGGC

Southern blot probe

primer

STM
NAT#240 STM
GGTGTTGGATCGGGGTGGAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

26
CNAG_01294
IPK1
L1
CNAG_01294 5′

GGAAAAGAGAAGAGCACGG

flanking region

primer 1

L2
CNAG_01294 5′

TCACTGGCCGTCGTTTTACCATC

flanking region

AACCATAGCAAGCAAC

primer 2

R1
CNAG_01294 3′

CATGGTCATAGCTGTTTCCTGGG

flanking region

CTGGTCAAAGAATGGAC

primer 1

R2
CNAG_01294 3′

TGGTAGGATGTGTTGTGGAG

flanking region

primer 2

SO
CNAG_01294

TTTGCTCTCTTCGCCAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01294

CGCATTCTCATCTTATCCC

Southern blot probe

primer

STM
NAT#184 STM

ATATATGGCTCGAGCTAGATAGA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

27
CNAG_01333
ALK1
L1
CNAG_01333 5′

GCATTTTCATTGCTGGTCAC

flanking region

primer 1

L2
CNAG_01333 5′

TCACTGGCCGTCGTTTTACACGG

flanking region

AAGGAGGAGATAACTAAC

primer 2

R1
CNAG_01333 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

GTTGTATGGCGAGGATG

primer 1

R2
CNAG_01333 3′

GTCCTGTGAATCGGGAGAT

flanking region

primer 2

SO
CNAG_01333

TGTTTCACCAGAGTCAGCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01333

ACGGGAGTGTTGTATGAGC

Southern blot probe

primer

STM
NAT#122 STM

ACAGCTCCAAACCTCGCTAAACA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

28
CNAG_01364

L1
CNAG_01364 5′
TCGCTCGCCTTGATTTGAC

flanking region

primer 1

L2
CNAG_01364 5′
TCACTGGCCGTCGTTTTACAAG

flanking region
TGGCTGTTGTGGAGGTCTG

primer 2

R1
CNAG_01364 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TGCGGTGATACCTTGCCAG

primer 1

R2
CNAG_01364 3′
TCCCCCGTTACCTTTATG

flanking region

primer 2

SO
CNAG_01364
CAGCCAATCTTTTCCCTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01364
TTTTCGCCAGCCACCTTCAG

Southern blot probe

primer

STM
NAT#5 STM
TGCTAGAGGGCGGGAGAGTT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

29
CNAG_01523
HOG1
L1
CNAG_01523 5′

TGTGGTAGGTGCGTTATCG

flanking region

primer 1

L2
CNAG_01523 5′

CTGGCCGTCGTTTACAGAAAGC

flanking region

CCATCCATCAG

primer 2

R1
CNAG_01523 3′

GTCATAGCTGTTTCCTGTCTTGG

flanking region

TAAGTCTCTGTGCC

primer 1

R2
CNAG_01523 3′

TACTCAACCCCATACTCACTCCC

flanking region

G

primer 2

SO
CNAG_01523

TGAAGACAAAAGGCGTGGG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_01523

TCACAGAGCGTTGATTACG

Southern blot probe

primer 1

PO2
CNAG_01523

CAGGCTCATCGGTAGGATCA

Southern blot probe

primer 2

STM
NAT#177 STM

CACCAACTCCCCATCTCCAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

30
CNAG_01612
PSK202
L1
CNAG_01612 5′

ACGCTTGTTTCTTCGTCC

flanking region

primer 1

L2
CNAG_01612 5′

TCACTGGCCGTCGTTTCGTCC

flanking region

GATGATAAAGTGAGG

primer 2

R1
CNAG_01612 3′

CATGGTCATAGCTGTTTCCTGTC

flanking region

TTCCCCTTTCTGATGG

primer 1

R2
CNAG_01612 3′

CCGACCAAAAACAGGTTC

flanking region

primer 2

SO
CNAG_01612

AACTGGCATTGAAGGTGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01612

GACAAGCATTGGGAAACC

Southern blot probe

primer

STM
NAT#208 STM

TGGTCGCGGGAGATCGTGGTTT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

31
CNAG_01664

L1
CNAG_01664 5′
CCTACATCCAGGACAAACG

flanking region

primer 1

L2
CNAG_01664 5′
TCACTGGCCGTCGTTTTACCAC

flanking region
CTTCTCCGACCTTTTC

primer 2

R1
CNAG_01664 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CCGCATAAAGAAAAGCC

primer 1

R2
CNAG_01664 3′
AAAGCGAGGTTGAAGAGGG

flanking region

primer 2

SO
CNAG_01664
CGTCGTAGTGGGTGTAGATG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01664
AGGACAACAAGTCTGGGATAGC

Southern blot probe

primer

STM
NAT#218 STM
CTCCACATCCATCGCTCCAA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

32
CNAG_01687

L1
CNAG_01687 5′
GCTCCTAAATACCTGCCACTC

flanking region

primer 1

L2
CNAG_01687 5′
TCACTGGCCGTCGTTTTACCTC

flanking region
ATCCGCAGAAATGTATC

primer 2

R1
CNAG_01687 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GTTCGCTTATGGTCTATGG

primer 1

R2
CNAG_01687 3′
TTGCGACCTTTTTCTCGG

flanking region

primer 2

SO
CNAG_01687
TGTTAGAAAAGCCTGTGACG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01687
CCCAAGATAGTCTCGTTTGC

Southern blot probe

primer

STM
NAT#290 STM
ACCGACAGCTCGAACAAGCAA

primer
GAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

33
CNAG_01704
IRK6
L1
CNAG_01704 5′

GGTCAACTTTCCCTTGTCG

flanking region

primer 1

L2
CNAG_01704 5′

TCACTGGCCGTCGTTTTACTTGA

flanking region

GAGAGCGTGATAAAGC

primer 2

R1
CNAG_01704 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

ACATTGACCTTCCTGTAAC

primer 1

R2
CNAG_01704 3′

GCCCTAAACAAACTAACTCTGTC

flanking region

C

primer 2

SO
CNAG_01704

AGCCTCCTCTTTCCTTACAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01704

GCTGGTGCCTCTTTTGATTC

Southern blot probe

primer

STM
NAT#5 STM primer

TGCTAGAGGGCGGGAGAGTT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

34
CNAG_01730
STE7
L1
CNAG_01730 5′

TTGTAAGGCTCTCATTCGC

flanking region

primer 1

L2
CNAG_01730 5′

CTGGCCGTCGTTTTACTGAAGGC

flanking region

AAAACTGGTGC

primer 2

R1
CNAG_01730 3′

GTCATAGCTGTTTCCTGCCTTAC

flanking region

CGTGCTTTTCTGC

primer 1

R2
CNAG_01730 3′

TTACTTCCGCCCAACGACAC

flanking region

primer 2

SO
CNAG_01730

TCCTCGCTCACAAAATGGGC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_01730

CCAATAGACATCAAGCCGTC

Southern blot probe

primer 1

PO2
CNAG_01730

AAACAGAGAAGAGAAGGGACC

Southern blot probe

primer 2

STM
NAT#225 STM

CCATAGAACTAGCTAAAGCA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

35
CNAG_01820

L1
CNAG_01820 5′
TCAGAAGCAGACAAGGCGTC

flanking region

primer 1

L2
CNAG_01820 5′
TCACTGGCCGTCGTTTTACTTT

flanking region
TGGGGAGGAAGTGCTGAGG

primer 2

R1
CNAG_01820 3′
CATGGTCATAGCTGTTTTCCTGG

flanking region
TTGGTCATTTGTGCGAC

primer 1

R2
CNAG_01820 3′
GGCATTATGAGCAAATCGG

flanking region

primer 2

SO
CNAG_01820
TAGCAGAAGGAGAGGACGGTT

diagnostic screening
C

primer, pairing with

B79

PO
CNAG_01820
CCTTGACGATGTTGGTCTG

Southern blot probe

primer

STM
NAT#6STM
ATAGCTACCACACGATAGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

36
CNAG_01845

L1
CNAG_01845 5′
GAATAATCAGCAGCGGTG

flanking region

primer 1

L2
CNAG_01845 5′
TCACTGGCCGTCGTTTTACGTT

flanking region
CGTTGTTGGTTGTCG

primer 2

R1
CNAG_01845 3′
CATGGTCATAGCTGTTTTCCTGG

flanking region
GGAGCCAATAATGTGGAG

primer 1

R2
CNAG_01845 3′
TCTTCATCCTTCCCTTGC

flanking region

primer 2

SO
CNAG_91845
TAAGGGCAAAAGGGTCAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01845
TTTTTAGCGTCCGTCTCG

Southern blot probe

primer

STM
NAT#205 STM
TATCCCCCTCTCCGCTCTCTAG

primer
CA

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

37
CNAG_01850
TCO1
L1
CNAG_01850 5′

GTTTCTGCTTCCACCTCAC

flanking region

primer 1

L2
CNAG_01850 5′

CTGGCCGTCGTTTTACTTTACAC

flanking region

ACACGGGCGATGTCCTG

primer 2

R1
CNAG_01850 3′

GTCATAGCTGTTTCCTGACTGAG

flanking region

CAAATCGGCGTAGG

primer 1

R2
CNAG_01850 3′

AAGTGAGGGGCATTACAGG

flanking region

primer 2

SO
CNAG_01850

CGACACAATACTCTAACTGCG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_01850

CTTTCGTCTTTGCCACAC

Southern blot probe

primer 1

PO2
CNAG_01850

AATCACCCTTTGCTACGG

Southern blot probe

primer 2

STM
NAT#102 STM

CCATAGCGATATCTACCCCAATC

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

38
CNAG_01905
KSP1
L1
CNAG_01905 5′

CGATTTTGTCTGGGCTCTC

flanking region

primer 1

L2
CNAG_01905 5′

TCACTGGCCGTCGTTTTACAAGA

flanking region

TGATTCGGGCACAG

primer 2

R1
CNAG_01905 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

CTCTTTCTCAATCATCG

primer 1

R2
CNAG_01905 3′

ACAACATCTTCGCCAACG

flanking region

primer 2

SO
CNAG_01905

TACCGACTCGCAATACACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01905

ATACCTTTGTGGCTTCGC

Southern blot probe

primer

STM
NAT#159 STM

ACGCACCAGACACACAACCAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

39
CNAG_01907

L1
CNAG_01907 5′
GCATTCTCTCAACTCGCTC

flanking region

primer 1

L2
CNAG_01907 5′
TCACTGGCCGTCGTTTTACTCG

flanking region
TAGCCTCTGTCTCTATCCC

primer 2

R1
CNAG_01907 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
GTTTCAGCCAATACCAGG

primer 1

R2
CNAG_01907 3′
TGAACCCCTTTGACCCATCC

flanking region

primer 2

SO
CNAG_01907
CCTCTTCTGTATGCTGCGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_01907
TCTGGAATGGAGGCTTTC

Southern blot probe

primer

STM
NAT#282 STM
TCTCTATAGCAAAACCAATC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

40
CNAG_01938
KIN1
L1
CNAG_01938 5′

AGAGACAAAGGTGAGGTCG

flanking region

primer 1

L2
CNAG_01938 5′

TCACTGGCCGTCGTTTTACCACG

flanking region

GGATAATGTTGACG

primer 2

R1
CNAG_01938 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

AGTATCAAATGCTGGC

primer 1

R2
CNAG_01938 3′

AGATAATAAGGGTGCGGC

flanking region

primer 2

SO
CNAG_01938

TGAGGTGGAGGCTTGTCTAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01938

GGACTTCTTTGGTTGGGAG

Southern blot probe

primer

STM
NAT#6 STM primer

ATAGCTACCACACGATAGCT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

41
CNAG_01988
TCO3
L1
CNAG_01988 5′

CCCAGAAAAGAAGGTTGG

flanking region

primer 1

L2
CNAG_01988 5′

CTGGCCGTCGTTTTACTTGTGGT

flanking region

TTGTGGGTAGCGTGG

primer 2

R1
CNAG_01988 3′

GTCATAGCTGTTTCCTGGGCATC

flanking region

ATTGCTCATTCTTGTG

primer 1

R2
CNAG_01988 3′

AAAAGGTGAAATAGGGGCGGCG

flanking region

primer 2

SO
CNAG_01988

TGTTTCTCAATGAAGTGTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_01988

ATGGGGAGGTCTATGCGTTAGC

Southern blot probe

primer 1

PO2
CNAG_01988

ATGGGGAGGTCTATGCGTTAGC

Southern blot probe

primer 2

STM
NAT#119 STM

CTCCCCACATAAAGAGAGCTAAA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

42
CNAG_02007

L1
CNAG_02007 5′
GAGCAGCGAAATAACCAAG

flanking region

primer 1

L2
CNAG_02007 5′
TCACTGGCCGTCGTTTTACCAG

flanking region
TAGCGAGGTGACAGATG

primer 2

R1
CNAG_02007 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CGATTGGACACTTACCAC

primer 1

R2
CNAG_02007 3′
AGCCCGAGTTCTTTTTAGAC

flanking region

primer 2

SO
CNAG_02007
AGAAATAGCGTTGCCACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02007
GCTTGTTTGGTAGATAGTCAG

Southern blot probe
C

primer

STM
NAT#232 STM
CTTTAAAGGTGGTTTGTG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

43
CNAG_02028

L1
CNAG_02028 5′
AAATCCGCAGGGGAAAAC

flanking region

primer 1

L2
CNAG_02028 5′
TCACTGGCCGTCGTTTTACTGG

flanking region
GAAAAGGATGGACAGG

primer 2

R1
CNAG_02028 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
CTCCGTCCTCAAAGAAAAATA

primer 1
CC

R2
CNAG_02028 3′
TTCCGTTTCCAATCGCAAG

flanking region

primer 2

SO
CNAG_02028
TTTTGCCCTTGCCCTGTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02028
ATCTTGCTCATACCGAACC

Southern blot probe

primer

STM
NAT#225 STM
CCATAGAACTAGCTAAAGCA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

44
CNAG_02194

L1
CNAG_02194 5′
TTGGTCCTCTGCGAAAAC

flanking region

primer 1

L2
CNAG_02194 5′
TCACTGGCCGTCGTTTTACGCT

flanking region
GTTGCTGAGAGTTTGTG

primer 2

R1
CNAG_02194 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
CAAACCCGAAGGTGAAG

primer 1

R2
CNAG_02194 3′
ACGACTTATTCCCCATCCC

flanking region

primer 2

SO
CNAG_02194
CACCTCGTTTGATGAATGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02194
CTCTCTCCTTCTCGTATCTGG

Southern blot probe

primer

STM
NAT#273 STM
GAGATCTTTCGGGAGGTCTGG

primer
ATT

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

45
CNAG_02202

L1
CNAG_02202 5′
AACAACCGAAACCAGCGAC

flanking region

primer 1

L2
CNAG_02202 5′
TCACTGGCCGTCGTTTTACGGA

flanking region
AGGTGATGTTTGTGGC

primer 2

R1
CNAG_02202 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
GCCGACAATGGTCTTATC

primer 1

R2
CNAG_02202 3′
TCCTGGTCATCGTGCTAACC

flanking region

primer 2

SO
CNAG_02202
CTTATGCCACTCCTAACCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02202
GCCGAGATACCTGTAAAGTCC

Southern blot probe

primer

STM
NAT#6 STM
ATAGCTACCACACGATAGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

46
CNAG_02233
MEC1
L1
CNAG_02233 5′

TTCCTCATCCACGATACTTC

flanking region

primer 1

L2
CNAG_02233 5′

TCACTGGCCGTCGTTTTACGACA

flanking region

GAGGTTTGAGGATGC

primer 2

R1
CNAG_02233 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

TTGTCCACGACCCTCTC

primer 1

R2
CNAG_02233 3′

TCATTGCCACCTCCACCAAG

flanking region

primer 2

SO
CNAG_02233

CTGATTGAAGGAACTTACCTCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02233

GGAGAAGTTCACGAAGGTCTG

Southern blot probe

primer

STM
NAT#204 STM

GATCTCTCGCGCTTGGGGGA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

47
CNAG_02285

L1
CNAG_02285 5′
TCCTCTGTTCTTGTCGTGG

flanking region

primer 1

L2
CNAG_02285 5′
TCACTGGCCGTCGTTTTACCTG

flanking region
CTCAGTGGTAGACATTTTG

primer 2

R1
CNAG_02285 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TCTCAGGCTTGGCTCTAC

primer 1

R2
CNAG_02285 3′
CGCCCTGTGATGATAATAACC

flanking region
TTC

primer 2

SO
CNAG_02285
TGGACAAAGGGACACTTACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02285
TGACAACACCAACGATGG

Southern blot probe

primer

STM
NAT#150 STM
ACATACACCCCCATCCCCCC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

48
CNAG_02296
RBK1
L1
CNAG_02296 5′

TCACTCATCACCAGGTAACG

flanking region

primer 1

L2
CNAG_02296 5′

TCACTGGCCGTCGTTTTACAGAA

flanking region

ACTGGAAAGCAGACG

primer 2

R1
CNAG_02296 3′

CATGGTCATAGCTGTTTCCTGCT

flanking region

TGCTTAGGAAAATCACCC

primer 1

R2
CNAG_02296 3′

GCACAAGAAAACCAGTCCAG

flanking region

primer 2

SO
CNAG_02296

GCTCGGTATGTTTATCACCTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02296

GAGTGTGGAAGAGAGAGGAAC

Southern, blot probe

primer

STM
NAT#219 STM

CCCTAAAACCCTACAGCAAT

primer

ST41
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

49
CNAG_02357
MKK2
L1
CNAG_02357 5′

GCGTCATTTCCCAATCAC

flanking region

primer 1

L2
CNAG_02357 5′

CTGGCCGTCGTTTTACTCGGTGT

flanking region

CTTCAGTTCAGAG

primer 2

R1
CNAG_02357 3′

GTCATAGCTGTTTCCTGACCCTA

flanking region

CCCTTGGCAACTAC

primer 1

R2
CNAG_02357 3′

CCCTTTGTTTGTTGCTGAC

flanking region

primer 2

SO
CNAG_02357

TTTTGCCCACTCCCCCTTTACCA

diagnostic screening

C

primer, pairing with

B79

PO1
CNAG_02357

GCAAAGTCACATACACGGC

Southern blot probe

primer 1

PO2
CNAG_02357

GATGTCCGAGTGATAACCTG

Southern blot probe

primer 2

STM
NAT#224 STM

AACCTTTAAATGGGTAGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

50
CNAG_02389
YPK101
L1
CNAG_02389 5′

TACCTGCCGACAAATGAC

flanking region

primer 1

L2
CNAG_02389 5′

TCACTGGCCGTCGTTTTACACAT

flanking region

AGCGGCTGCTTTTC

primer 2

R1
CNAG_02389 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GGGGTTCTAAAAGACG

primer 1

R2
CNAG_02389 3′

ACCATCATCTCTGCGTTG

flanking region

primer 2

SO
CNAG_02389

AACCGCAAGTAGGGCATAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02389

TGAGCAAAAAAGGCGAGC

Southern blot probe

primer

STM
NAT#242 STM

GTAGCGATAGGGGTGTCGCTTT

primer

AG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

51
CNAG_02459

L1
CNAG_02459 5′
TCTCGGGGTCTTCAATCTC

flanking region

primer 1

L2
CNAG_02459 5′
TCACTGGCCGTCGTTTTACGTG

flanking region
CGGATTCGTTATTTGG

primer 2

R1
CNAG_02459 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AAGAGGGTTAGGTTTGGC

primer 1

R2
CNAG_02459 3′
GCCACTTCCGTATCAAAAG

flanking region

primer 2

SO
CNAG_02459
GCACTGCTGCTTGAAATC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02459
ATAGATTCTGATGCGGCG

Southern blot probe

primer

STM
NAT#122 STM
ACAGCTCCAAACCTCGCTAAA

primer
CAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

52
CNAG_02511
CPK1
L1
CNAG_02511 5′

CTGTAGAAGATGTGAGTTTGGG

flanking region

primer 1

L2
CNAG_02511 5′

CTGGCCGTCGTTTACTGATTGA

flanking region

TGAGAGATACGGG

primer 2

R1
CNAG_02511 3′

GTCATAGCTGTTTCCTGGGCGG

flanking region

AGAAATAGAGGTTG

primer 1

R2
CNAG_02511 3′

CGCACAAGAAGTAAGAGGTG

flanking region

primer 2

SO
CNAG_02511

GGCTATGGACCGTATTCAC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_02511

TATCTCACAAGCCACTCCC

Southern blot probe

primer 1

PO2
CNAG_02511

ATGCTGCTCACCGTTAGTC

Southern blot probe

primer 2

STM
NAT#184 STM

ATATATGGCTCGAGCTAGATAGA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

53
CNAG_02531
CPK2
L1
CNAG_02531 5′

ATGTGCTTGGTTTGCCCGAG

flanking region

primer 1

L2
CNAG_02531 5′

CTGGCCGTCGTTTTACAACCTGA

flanking region

CTTTGCGAGGAGC

primer 2

R1
CNAG_02531 3′

GTCATAGCTGTTTCCTGGGAAGA

flanking region

GTTGAAGAGGCTG

primer 1

R2
CNAG_02531 3′

ACTGTGGCTGTTGTTCAGGC

flanking region

primer 2

SO
CNAG_02531

CCAAGGGAAGTCTACCAATAC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_02531

GGGGAAAGATTAGTGCGTC

Southern blot probe

primer 1

PO2
CNAG_02531

GTGCGTAGATGAACGAGTG

Southern blot probe

primer2

STM
NAT#122 STM

ACAGCTCCAAACCTCGCTAAACA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

54
CNAG_02542
IRK2
L1
CNAG_02542 5′

TGTGCTGGTATCTGATGAGC

flanking region

primer 1

L2
CN4G_02542 5′

TCACTGGCCGTCGTTTTACGTGA

flanking region

GCGGCTTTGAAAATG

primer 2

R1
CNAG_02542 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

GGCTATCTTTGTGTATGC

primer 1

R2
CNAG_02542 3′

CCCTTTGCTCACTTTCATACC

flanking region

primer 2

SO
CNAG_02542

TTTTTCGGGTCTGACGAC

diagnostic screening

primer, pairing with

G79

PO
CNAG_02542

CTGTTCACCAAGTTCCCTAATC

Southern blot probe

primer

STM
NAT#232 STM

CTTTAAAGGTGGTTTGTG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

55
CNAG_02551
DAK3
L1
CNAG_02551 5′

ATCTAATCCTCCCTGTCCAC

flanking region

primer 1

L2
CNAG_02551 5′

TCACTGGCCGTCGTTTTACGCGT

flanking region

GATTTCAGGTTCAG

primer 2

R1
CNAG_02551 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GCGTGGTTTCCTGTAAG

primer 1

R2
CNAG_02551 3′

GGTCATAACTCAGAGGGGTC

flanking region

primer 2

SO
CNAG_02551

GAGAGCGAAGCAATAGGAAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02551

AAGCAATCTCCAGACTCCC

Southern blot probe

primer

STM
NAT#295 STM

ACACCTACATCAAACCCTCCC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

56
CNAG_02675
HSL101
L1
CNAG_02675 5′

CAATGCCGTCATCATCAAAC

flanking region

primer 1

L2
CNAG_02675 5′

TCACTGGCCGTCGTTTTACAAGG

flanking region

GCGAACAGGATAATAC

primer 2

R1
CNAG_02675 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

TAATGTGAGAGCAGCAATAC

primer 1

R2
CNAG_02675 3′

TATGTGGCAGAAACCGTG

flanking region

primer 2

SO
CNAG_02675

GCTGTCTTGTTTGCGTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02675

AGGAGTAGTTATCACTTCGGG

Southern blot probe

primer

STM
NAT#146 STM

ACTAGCCCCCCCTCACCACCT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

57
CNAG_02680
VPS15
L1
CNAG_02680 5′

AGGACCTTCATCAGGACGAC

flanking region

primer 1

L2
CNAG_02680 5′

TCACTGGCCGTCGTTTTACAAAC

flanking region

TACCTCCCCCGTTAC

primer 2

R1
CNAG_02680 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

AAATGTATGGATTCGCC

primer 1

R2
CNAG_02680 3′

CTGCGAATCTCGTCTAAGG

flanking region

primer 2

SO
CNAG_02680

TTGAAAGGTCCCACCAGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02680

GGGAGGAAGTGAGGACTATG

Southern blot probe

primer

STM
NAT#123 STM

CTATCGACCAACCAACACAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

58
CNAG_02686

L1
CNAG_02686 5′
CACACTTTGCTCTTGTCTGAG

flanking region

primer 1

L2
CNAG_02686 5′
TCACTGGCCGTCGTTTTACATG

flanking region
GAGATGCGATAAGCG

primer 2

R1
CNAG_02686 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GAATCCTCCCTCAACGAG

primer 1

R2
CNAG_02686 3′
AAAGACGACGCCTACTCTGC

flanking region

primer 2

SO
CNAG_02686
TGTTCCTCTTCCCTGACAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02686
CACAATCAAAGCGTTAGGG

Southern blot probe

primer

STM
NAT#191 STM
ATATGGATGTTTTTAGCGAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

59
CNAG_02712
BUD32
L1
CNAG_02712 5′

ATAGGGGATGACCTTGGAG

flanking region

primer 1

L2
CNAG_02712 5′

TCACTGGCCGTCGTTTTACTGAT

flanking region

GCCAAAGACCAGTG

primer 2

R1
CNAG_02712 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

GAAGAGGAAGGAAGAGAGAC

primer 1

R2
CNAG_02712 3′

GAGCGATAATAGCCACCAC

flanking region

primer 2

SO
CNAG_02712

GGGCAATCTTTCTTCGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02712

CTCGTTCTCTGGTTCTTCTG

Southern blot probe

primer

STM
NAT#296 STM

CGCCCGCCCTCACTATCCAC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

60
CNAG_02787

L1
CNAG_02787 5′
AACCCCTTGTGTCCCCAAAC

flanking region

primer 1

L2
CNAG_02787 5′
TCACTGGCCGTCGTTTTACTGA

flanking region
GCAGGCGGATACGATAC

primer 2

R1
CNAG_02787 3′
ATGGTCATAGCTGTTTCCTTGC

flanking region
AAAAAGGACAGAAGAAGAGG

primer 1

R2
CNAG_02787 3′
TTCTCCCATTTCTCCACCC

flanking region

primer 2

SO
CNAG_02787
AGCAGAGCCAGATGGTAGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02787
TTCCACTTGGCAACTGTCC

Southern blot probe

primer

STM
NAT#227 STM
TCGTGGTTTAGAGGGAGCGC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

61
CNAG_02799
DAK202A
L1
CNAG_02799 5′

TTGATACTTTGGGTCTGGG

flanking region

primer 1

L2
CNAG_02799 5′

TCACTGGCCGTCGTTTTACCGG

flanking region

GAGCCATTATTGGTAAG

primer 2

R1
CNAG_02799 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

TTGGATGGCTTGCGAGGG

primer 1

R2
CNAG_02799 3′

CCATACAATGACCTGSGAC

flanking region

primer 2

SO
CNAG_02799

AACCATCAACTGCCCTCAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02799

GGTAGTATCGGTGATTTGAGTGA

Southern blot probe

G

primer

STM
NAT#119 STM

CTCCCCACATAAAGAGAGCTAAA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

62
CNAG_02802
ARG2
L1
CNAG_02802 5′

CCAGCAGTTAGGGATTCAG

flanking region

primer 1

L2
CNAG_02802 5′

TCACTGGCCGTCGTTTTACCATC

flanking region

GTAGAGTCGTTATTACCG

primer 2

R1
CNAG_02802 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

TTGGAGTCCTATCGCC

primer 1

R2
CNAG_02802 3′

ATGTCAATGGTAGCCCACC

flanking region

primer 2

SO
CNAG_02802

TTTGTTGTTGCCTGACCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02802

GTCGCTCAAAGTGTCTTCTC

Southern blot probe

primer

STM
NAT#125 STM

CGCTACAGCCAGCGCGCGCAAG

primer

CG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

63
CNAG_02820
PAR201
L1
CNAG_02820 5′

CCCTCGCCAGAATCAATAC

flanking region

primer 1

L2
CNAG_02820 5′

TGACTGGCCGTCGTTTTACGAGA

flanking region

GGATGTTGAGGTTGC

primer 2

R1
CNAG_02820 3′

CATGGTCATAGCTGTTTCCGTT

flanking region

GGGATTAGGGCGTATC

primer 1

R2
CNAG_02820 3′

TCTGCCTCTACAAACCACTG

flanking region

primer 2

SO
CNAG_02820

GGAGAGACAGGGGATAAAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02820

ATACCTCCCTTCTCCCAAC

Southern blot probe

primer

STM
NAT_190 219 STM

CCCTAAAACCCTACAGCAAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

64
CNAG_02847

L1
CNAG_02847 5′
AGACCGATAAAAACAGGACC

flanking region

primer 1

L2
CNAG_02847 5′
TCACTGGCCGTCGTTTTACAAC

flanking region
AATGAAGGCACCTCG

primer 2

R1
CNAG_02847 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
GAACATTCAAACGGAGAC

primer 1

R2
CNAG_02847 3′
ACCAGTTGACAAAGGTATCG

flanking region

primer 2

SO
CNAG_02847
AAGAATACTCCAGAAGGGACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02847
GCTTCTGGGGATAAGGTGAG

Southern blot probe

primer

STM
NAT#296 STM
CGCCCGCCCTCACTATCCAC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

65
CNAG_02859
POS5
L1
CNAG_02859 5′

TACACGACAGTAACTCCCTCCG

flanking region

primer 1

L2
CNAG_02859 5′

TGACTGGCCGTCGTTTTAGGAAA

flanking region

TAACACACACGCTGC

primer 2

R1
CNAG_02859 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

AAAGTGGCTGGGTGAAG

primer 1

R2
CNAG_02859 3′

AAAGAACTTGAGAAGACCCG

flanking region

primer 2

SO
CNAG_02859

AGCAACGAGTCCACATACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02859

TACACACCTCCAGTTTGACCTCG

Southern blot probe

C

primer

STM
NAT#58 STM

CGCAAAATCACTAGCGCTATAGC

primer

G

STM
STM common

GCATGCGCTGCCGCTAAGAATTC

common
primer

G

66
CNAG_02866

L1
CNAG_02866 5′
GAAGATAGTCAATCCGCAAG

flanking region

primer 1

L2
CNAG_02866 5′
TCACTGGCCGTCGTTTTACATC

flanking region
TACCACTATTCTCCTGGC

primer 2

R1
CNAG_02866 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CTGATTGTTCTTGACATTCCG

primer 1

R2
CNAG_02866 3′
AAGGAGGATGAAGGAAGGC

flanking region

primer 2

SO
CNAG_02866
ACAGGAACCTCCGTAACAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02866
ATTGGTGAAGGTCTGGGCAGT

Southern blot probe
TCG

primer

STM
NAT#102 STM
CCATAGCGATATCTACCCCAA

primer
TCT

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

67
CNAG_02897

L1
CNAG_02897 5′
GATGTAGCGGATTGTTTGAC

flanking region

primer 1

L2
CNAG_02897 5′
TCACTGGCCGTCGTTTTACTCC

flanking region
TTCTGCCTGGGTGTTTC

primer 2

R1
CNAG_02897 5′
CATGGTCATAGCTGTTTCCTGG

flanking region
ATTTGGTGTTTGCTAACGG

primer 1

R2
CNAG_02897 3′
CTCCATCCAGCAACTCTATG

flanking region

primer 2

SO
CNAG_02897
AGGAAGCAACGCTGACTGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02897
TGGTTGTAATGGCACCGTC

Southern blot probe

primer

STM
NAT#122 STM
ACAGCTCCAAACCTCGCTAAA

primer
CAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

68
CNAG_02915
PKH202
L1
CNAG_02915 5′

TGGTGGAAATGGACTGTG

flanking region

primer 1

L2
CNAG_02915 5′

TCACTGGCCGTCGTTTTACCAGC

flanking region

CTCGGGTTTTTTTG

primer 2

R1
CNAG_02915 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

CACGAAAAGCACGAAG

primer 1

R2
CNAG_02915 3′

TCCTTGGACAACTGGTAGC

flanking region

primer 2

SO
CNAG_02915

AGGTGGGATTGCTCAAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02915

TGAAGGCGTGCTCAAATG

Southern blot probe

primer

STM
NAT#177 STM

CACCAACTCCCCATCTCCAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

69
CNAG_02947
SCY1
L1
CNAG_02947 5′

CGTCACCAACAAGTCACAG

flanking region

primer 1

L2
CNAG_02947 5′

TCACTGGCCGTCGTTTTACGAGA

flanking region

AGAGGTTTGAGGCTG

primer 2

R1
CNAG_02947 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

CCTGTCTGGGAGAAGAGC

primer 1

R2
CNAG_02947 3′

TTCCAAGACTTCCCCAAC

flanking region

primer 2

SO
CNAG_02947

CCATTACCTTTATGTCCCCAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_02947

TTGCCCATTCCTGTCTTAG

Southern blot probe

primer

STM
NAT#150 STM

ACATACACCCCCATCCCCCC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

70
CNAG_02962

L1
CNAG_02962 5′
CAAGGCGTTCTTCTTTGG

flanking region

primer 1

L2
CNAG_02962 5′
TCACTGGCCGTCGTTTTACGTC

flanking region
GTGATAATGGCGTTTG

primer 2

R1
CNAG_02962 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CTAAAAGATTGACTCCGAGG

primer 1

R2
CNAG_02962 3′
GAATAGGTCGTGAATGGATGT

flanking region
C

primer 2

SO
CNAG_02962
CTGATAAAAGAGCAGAGAGG

diagnostic screening
G

primer, pairing with

B79

PO
CNAG_02962
GGTGGCTATCAAAGTTGTTAG

Southern blot probe
G

primer

STM
NAT#242 STM
GTAGCGATAGGGGTGTCGCTT

primer
TAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

71
CNAG_02976

L1
CNAG_02976 5′
GCAAAGTGAAGAAGGCGAG

flanking region

primer 1

L2
CNAG_02976 5′
TCACTGGCCGTCCTTTTTACTTG

flanking region
GTGACGGTCCCTTCAAG

primer 2

R1
CNAG_02976 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AATCCTTGCTGGGGGAAGC

primer 1

R2
CNAG_02976 3′
CGATTCATCTCCATAACCAGT

flanking region
G

primer 2

SO
CNAG_02976
GGCATAATGAAACCAGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_02976
CGCAAAAACTCGTCATAGG

Southern blot probe

primer

STM
NAT#169 STM
ACATCTATATCACTATCCCGA

primer
ACC

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

72
CNAG_03024
RIM15
L1
CNAG_03024 5′

CTGAGTGCGATGATTGTTTG

flanking region

primer 1

L2
CNAG_03024 5′

GCTCACTGGCCGTCGTTTTACTT

flanking region

TCCTGACTTTGGGTGC

primer 2

R1
CNAG_03024 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

GAGGACAGATTCTATGGC

primer 1

R2
CNAG_03024 3′

CAGAGAATAAGGTCCCCTCC

flanking region

primer 2

SO
CNAG_03024

TCAAGGGATAGAAGTTCGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03024

GAGATAAACAGAGCCAAACG

Southern blot probe

primer

STM
NAT#191 STM

ATATGGATGTTTTTAGCGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

73
CNAG_03048
IRK3
L1
CNAG_03048 5′

GATTGAGTTTCGGTTGGG

flanking region

primer 1

L2
CNAG_03048 5′

TCACTGGCCGTCGTTTTACCTAA

flanking region

AAACGGAGCGGAAG

primer 2

R1
CNAG_03048 3′

ATGGTCATAGCTGTTTCCTGCGA

flanking region

ACTTCTCAAGCAACG

primer 1

R2
CNAG_03048 3′

ATACAACCCCCATACTCCC

flanking region

primer 2

SO
CNAG_03048

AAAGGGATTCGGGCTTAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03048

CCAGGGGTTGATGTCATAG

Southern blot probe

primer

STM
NAT#273 STM

GAGATCTTTCGGGAGGTCTGGA

primer

TT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

74
CNAG_03137

L1
CNAG_03137 5′
CAAGGAGGTCAACCCTACAG

flanking region

primer 1

L2
CNAG_03137 5′
TCACTGGCCGTCGTTTTACAGG

flanking region
CGTCTTCTGTCCATAG

primer 2

R1
CNAG_03137 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
GTCGTCCTCTTTTTGTGC

primer 1

R2
CNAG_03137 3′
AGGACTTGTCGGTCTTCAG

flanking region

primer 2

SO
CNAG_03137
GGTAAGTTGCTTTATCCCCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03137
GCTGTGAGCAGTTGATACG

Southern blot probe

primer

STM
NAT#211 STM
GCGGTCGCTTTATAGCGATT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

75
CNAG_03167
CHK1
L1
CNAG_03167 5′

GTATCTCCATCCCACACATC

flanking region

primer 1

L2
CNAG_03167 5′

TCACTGGCCGTCGTTTTACTTGA

flanking region

CAGAGAGGGGCTTAC

primer 2

R1
CNAG_03167 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

ACATTGGAGGGCGTTG

primer 1

R2
CNAG_03167 3′

CTGACAACAAGCAGCCTATC

flanking region

primer 2

SO
CNAG_03167

ATACCACCACAAACGCCTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03167

GGACTACTTTCCGAAGGTTC

Southern blot probe

primer

STM
NAT#205 STM

TATCCCCCTCTCCGCTCTCTAGC

primer

A

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

76
CNAG_03171

L1
CNAG_03171 5′
CGTCCAACCATCAATCAC

flanking region

primer 1

L2
CNAG_03171 5′
TCACTGGCCGTCGTTTTACACC

flanking region
TTGGTAGGAGTGTGGAG

primer 2

R1
CNAG_03171 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
AGTTGCGATTCTGTGGG

primer 1

R2
CNAG_03171 3′
TAGGGACGAGTATCAGGAGCA

flanking region
G

primer 2

SO
CNAG_03171
TCCTCTGTTCTTGTCGTGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03171
TAAGCCTCGTAGAGCCAAG

Southern blot probe

primer

STM
NAT#159 STM
ACGCACCAGACACACAACCAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

77
CNAG_03184
BUB1
L1
CNAG_03184 5′

CAACGCCATTGAGGAAAG

flanking region

primer 1

L2
CNAG_03184 5′

TCACTGGCCGTCGTTTTACGCCT

flanking region

GATGTTCTCTTTCTGAG

primer 2

R1
CNAG_03184 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GCGACTTTGAGGGATGGC

primer 1

R2
CNAG_03184 3′

ATCCCAGAACAGTGGCAGAC

flanking region

primer 2

SO
CNAG_03184

GGAGGATACATCAGGTGAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03184

AACAGCACTTTGGGGTAAC

Southern blot probe

primer

STM
NAT#201 STM

CACCCTCTATCTCGAGAAAGCTC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

78
CNAG_03216
SNF101
L1
CNAG_03216 5′

GGAGATGAAGGGAATGAGTC

flanking region

primer 1

L2
CNAG_03216 5′

TCACTGGCCGTCGTTTTACCGAC

flanking region

GCAAGAGGATAACAAC

primer 2

R1
CNAG_03216 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GCAGGAGATGAGGGATAG

primer 1

R2
CNAG_03216 3′

CTGCTCTTGTTTAGCCACC

flanking region

primer 2

SO
CNAG_03216

TCCGACTCTGATAACGACTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03216

AAAGCCTCCTCTTCCAACC

Southern blot probe

primer

STM
NAT#146 STM

ACTAGCCCCCCCTCACCACCT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

79
CNAG_03258
TPK202A
L1
CNAG_03258 5′

AGGGACTGAATCCAAAGGG

flanking region

primer 1

L2
CNAG_03258 5′

TCACTGGCCGTCGTTTTACTTCT

flanking region

CGTCTTCGGCAAGGCAAGTG

primer 2

R1
CNAG_03258 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GGACAAGGGCTAATGG

primer 1

R2
CNAG_03258 3′

AAGGCTGGACTTTGTTGGGGAC

flanking region

primer 2

SO
CNAG_03258

GATTGCGAAGATGTGAACTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03258

TTTCCCTGTTGCCATCTC

Southern blot probe

primer

STM
NAT#208 STM

TGGTCGCGGGAGATCGTGGTTT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

80
CNAG_03290
KIC102
L1
CNAG_03290 5′

CGCTGACTTGGAGTATGTG

flanking region

primer 1

L2
CNAG_03290 5′

TCACTGGCCGTCGTTTTACAAGT

flanking region

CTGCGGAAAGGTTC

primer 2

R1
CNAG_03290 3′

CATGGTCATAGCTGTTTCCTGTC

flanking region

ACCTCTGCTTTTGTCTTG

primer 1

R2
CNAG_03290 3′

CCGACAAGGATGAAACAAAGAT

flanking region

GG

primer 2

SO
CNAG_03290

TGGATGTCTTAGAAGGGAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03290

GGAAGACAAGAACAAACGG

Southern blot probe

primer

STM
NAT#201 STM

CACCCTCTATCTCGAGAAAGCTC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

81
CNAG_03355
TCO4
L1
CNAG_03355 5′

AATGCCATAGGACACCTCTGACC

flanking region

C

primer 1

L2
CNAG_03355 5′

CTGGCCGTCGTTTTACTGTGACT

flanking region

ATGGTAAGCACCG

primer 2

R1
CNAG_03355 3′

GTCATAGCTGTTTCCTGAATGCC

flanking region

ATAGGACACCTCTGACCC

primer 1

R2
CNAG_03355 3′

TGTGACTATGGTAAGCACCG

flanking region

primer 2

SO
CNAG_03355

GTTGCTTGGTTTTTCTTCGG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_03355

AAACGGCAGCATTGACTAC

Southern blot probe

primer 1

PO2
CNAG_03355

TATGTAAGCAGCCTGTTCG

Southern blot probe

primer 2

STM
NAT#123 STM

CTATCGACCAACCAACACAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

82
CNAG_03358

L1
CNAG_03358 5′
GCAGAATCGTGAAACATTACC

flanking region
C

primer 1

L2
CNAG_03358 5′
TCACTGGCCGTCGTTTTACTCA

flanking region
TTGAGGAGGTAGGGAGG

primer 2

R1
CNAG_03358 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GAAAGGTGTCGGGGATAG

primer 1

R2
CNAG_03358 3′
ACGGAGAAGCAGGAACATC

flanking region

primer 2

SO
CNAG_03358
CAGACAATCGCAGAGTGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03358
CTCTCGGAACTTCTTGACG

Southern blot probe

primer

STM
NAT#230 STM
ATGTAGGTAGGGTGATAGGT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

83
CNAG_03367
URK1
L1
CNAG_03367 5′

ACCCTTCTTTTTGGTCCC

flanking region

primer 1

L2
CNAG_03367 5′

TCACTGGCCGTCGTTTTACTTGG

flanking region

TTTTTGCTCTGCGGC

primer 2

R1
CNAG_03367 3′

CATGGTCATAGCTGTTTCCTGGT

flanking region

TTGCTGTTGGATTCGC

primer 1

R2
CNAG_03367 3′

ATTTCCCCGCATTTGCCAC

flanking, region

primer 2

SO
CNAG_03367

TCGCACATTCTTGTCAGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03367

GATGATGGAAAGAGTAGACCG

Southern blot probe

primer

STM
NAT#43 STM

CCAGCTACCAATCACGCTAC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

84
CNAG_03369
SWE102
L1
CNAG_03369 5′

TGCTACGCTAAGACTGGACTAC

flanking region

primer 1

L2
CNAG_03369 5′

TCACTGGCCGTCGTTTTACGGAG

flanking region

CGTGGTTGAAAGAAC

primer 2

R1
CNAG_03369 3′

CATGGTCATAGCTGTTTCCTGAC

flanking region

GAACTTGTGCTCTCTGC

primer 1

R2
CNAG_03369 3′

ACAGTTTCCTGACGAGAATG

flanking region

primer 2

SO
CNAG_03369

GCCGATACATTTTGGGTAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03369

TGGATGGTGAGGAGTTGAG

Southern blot probe

primer

STM
NAT#169 STM

ACATCTATATCACTATCCCGAAC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

85
CNAG_03567
CBK1
L1
CNAG_03567 5′

CAACCGATTTGCCAAGAG

flanking region

primer 1

L2
CNAG_03567 5′

TCACTGGCCGTCGTTTTACTTGT

flanking region

TGTCCCTGGATTGG

primer 2

R1
CNAG_03567 3′

CATGGTCATAGCTGTTTCCTGTA

flanking region

AGGAGTGCGATGGATG

primer 1

R2
CNAG_03567 3′

CGTTTTTCATCCTGCGAG

flanking region

primer 2

SO
CNAG_03567

TCATTCCCACCATTCACG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03567

TCTGACTTCACCGAATGC

Southern blot probe

primer

STM
NAT#232 STM

CTTTAAAGGTGGTTTGTG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

86
CNAG_03592
THI20
L1
CNAG_03592 5′

TTGTGAGCAGGTTTCCGTG

flanking region

primer 1

L2
CNAG_03592 5′

TCACTGGCCGTCGTTTTACTACC

flanking region

TGAATACCAGCACCACCG

primer 2

R1
CNAG_03592 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

ATAGTGGCAGGACCTTGC

primer 1

R2
CNAG_03592 3′

TTACATCGCCGCTGTTTCC

flanking region

primer 2

SO
CNAG_03592

TGTCTCTGGTGTCTGGTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03592

GAAAGCAGTAGCGATAGCAG

Southern blot probe

primer

STM
NAT#231 STM

GAGAGATCCCAACATCACGC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

87
CNAG_03670
IRE1
L1
CNAG_03670 5′

GCCCCATCATCATAATCAC

flanking region

primer 1

L2
CNAG_03670 5′

GCTCACTGGCCGTCGTTTTACAC

flanking region

TATGTGTCCATCTGAGGC

primer 2

R1
CNAG_03670 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

TGAGTTGAGGGAGGAAAG

primer 1

R2
CNAG_03670 3′

GAAGAAGAGCGTCAAGAAGG

flanking region

primer 2

SO
CNAG_03670

AGGAATACGAGGTTTATCGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03670

AGCATTAGGGGTGTAGGTG

Southern blot probe

primer

STM
NAT#224 STM

AACCTTTAAATGGGTAGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

88
CNAG_03701

L1
CNAG_03701 5′
AGCGTATTCTTCAGGGCTC

flanking region

primer 1

L2
CNAG_03701 5′
TCACTGGCCGTCGTTTTACAAG

flanking region
AAGGGAGAGTGGTTGTGACGG

primer 2

R1
CNAG_03701 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GAAGTGTTTTCCCGTCCC

primer 1

R2
CNAG_03701 3′
TAAAGGAGTGTTGGACCCC

flanking region

primer 2

SO
CNAG_03701
ACAAACCTCACTGTGCCTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_03701
CAATACCGACTGAGACACACT

Southern blot probe
C

primer

STM
NAT#125 STM
CGCTACAGCCAGCGCGCGCAA

primer
GCG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

89
CNAG_03791

L1
CNAG_03791 5′
GAAGCATCCTCAAAAGGG

flanking region

primer 1

L2
CNAG_03791 5′
TCACTGGCCGTCGTTTTACTGG

flanking region
CTGGAGATTTGAAAGAG

primer 2

R1
CNAG_03791 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
TTTTGGAAGTAAACGGGG

primer 1

R2
CNAG_03791 3′
GCAACTCGTCAAAGACCTG

flanking region

primer 2

SO
CNAG_03791
CGACTTCTTCAGCAATGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03791
TATTCCAGTCCGAGTAGCG

Southern blot probe

primer

STM
NAT#210 STM
CTAGAGCCCGCCACAACGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

90
CNAG_03796

L1
CNAG_03796 5′
AGGTCGGAAGATTTTGCG

flanking region

primer 1

L2
CNAG_03796 5′
TCACTGGCCGTCGTTTTACTAG

flanking region
GGTCGTTTGTGTTATCC

primer 2

R1
CNAG_03796 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
TTTTGGCTTTGGGTCAG

primer 1

R2
CNAG_03796 3′
TGAGCAGTAGTGTATTGGGTG

flanking region

primer 2

SO
CNAG_03796
AATCTCCTCTTGGGCTCAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03796
ATACCACAGCACCCACAAG

Southern blot probe

primer

STM
NAT#240 STM
GGTGTTGGATCGGGGTGGAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

91
CNAG_03811
IRK5
L1
CNAG_03811 5′

TCTTTAGCGTTTGACCCTG

flanking region

primer 1

L2
CNAG_03811 5′

TCACTGGCCGTCGTTTTACTTCC

flanking region

AACACTCCGTAGCAG

primer 2

R1
CNAG_03811 3′

CATGGTCATAGCTGTTTCCTGCT

flanking region

GATGGAAGATGTTGAAGC

primer 1

R2
CNAG_03811 3′

GTCGCATCTTTTTGCTGG

flanking region

primer 2

SO
CNAG_03811

TCACAATCATTCTGACCAGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03811

CCGCAAAGGTAAAGTTCG

Southern blot probe

primer

STM
NAT#213 STM

CTGGGGATTTTGATGTGTCTATG

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

92
CNAG_03821

L1
CNAG_03821 5′
GGGTCATTTTCACCGAATC

flanking region

primer 1

L2
CNAG_03821 5′
TCACTGGCCGTCGTTTTACCTT

flanking region
TGTGTGCCGTTCTAAAC

primer 2

R1
CNAG_03821 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CCAGATGGTCATTTCTTC

primer 1

R2
CNAG_03821 3′
GGAAATAGAAACAGCGGTG

flanking region

primer 2

SO
CNAG_03821
ACCAGGTCTTCCTCCATTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03821
TGAGAGATTCTTGTTCCGAG

Southern blot probe

primer

STM
NAT#177 STM
CACCAACTCCCCATCTCCAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

93
CNAG_03843
ARK1
L1
CNAG_03843 5′

CAATAGGCGTGAACAAGC

flanking region

primer 1

L2
CNAG_03843 5′

TCACTGGCCGTCGTTTTACGGGA

flanking region

TACTGGTGTTTTTGG

primer 2

R1
CNAG_03843 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

GTCAACAATGCGTCAG

primer 1

R2
CNAG_03843 3′

GAAAGGAAGGAGCGAAAG

flanking region

primer 2

SO
CNAG_03843

ATAGAGCGGGAGGAAATG

diagnostic screening

primer, pairing with

B79

PO
CNAG_03843

TGGGTGGGAGTGATTTCTG

Southern blot probe

primer

STM
NAT#43 STM

CCAGCTACCAATCACGCTAC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

94
CNAG_03946
GAL302
L1
CNAG_03946 5′

AAAACTCACATCCGCTGC

flanking region

primer 1

L2
CNAG_03946 5′

TCACTGGCCGTCGTTTTACGCAG

flanking region

AGAGTTGAAGACGGTG

primer 2

R1
CNAG_03946 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

TGGAGGTGAGTTCTGTAATC

primer 1

R2
CNAG_03946 3′

CCCTATTCCTTTCCTTGTTC

flanking region

primer 2

SO
CNAG_03946

AGACCAATGTAGACCCTATGTG

diagnostic screening

primer, pairing with

B379

PO
CNAG_03946

ACAAGCACATCCATTCCTAC

Southern blot probe

primer

STM
NAT#218 STM

CTCCACATCCATCGCTCCAA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

95
CNAG_04040
FPK1
L1
CNAG_04040 5′

ATCGTCTCAGCCTCAACAG

flanking region

primer 1

L2
CNAG_04040 5′

TCACTGGCCGTCGTTTTACTCTT

flanking region

CCACTTTGACGGTG

primer 2

R1
CNAG_04040 3′

CATGGTCATAGCTGTTTCCTGTC

flanking region

CGTTTGGGGAGTTTAG

primer 1

R2
CNAG_04040 3′

GGCTATCTTCTTGGCTTGC

flanking region

primer 2

SO
CNAG_04040

CCTTTGGGTTTTTGGGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04040

ATTAGTCTGCCCAAACGG

Southern blot probe

primer

STM
NAT#211 STM

GCGGTCGCTTTATAGCGATT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

C

OEL2
CNAG_04040 5′

CACTCGAATCCTGCATGCGGGA

flaking region for

TGTTTGTGTGACTGAG

overexpression

construction

OER1
CNAG_04040 5′

CCACAACACATCTATCACATGTC

coding region for

GTCTCTCGCGTCACC

overexpression

construction

NP1
CNAG_04040

TTCAAACTCGGGAGGACAG

Northern blot probe

primer

96
CNAG_04083

L1
CNAG_04083 5′
TTCCTCCATCTTCGCATC

flanking region

primer 1

L2
CNAG_04083 5′
TCACTGGCCGTCGTTTTACTCG

flanking region
TGCCCTTTTTGGTAG

primer 2

R1
CNAG_04083 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AAGAAAGAACACCCCTCC

primer 1

R2
CNAG_04083 3′
AACAGGTTGCGATTGTGC

flanking region

primer 2

SO
CNAG_04083
GCCGTTATGGGTGAAAGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04083
GAAAGGGAGAAGAGTGAAGG

Southern blot probe

primer

STM
NAT#210 STM
CTAGAGCCCGCCACAACGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

97
CNAG_04108
PKP2
L1
CNAG_04108 5′

AAAAGAGGAGGGAGAAGGG

flanking region

primer 1

L2
CNAG_04108 5′

TCACTGGCCGTCGTTTTACTGAA

flanking region

GTATCCACACACCCC

primer 2

R1
CNAG_04108 3′

CATGGTCATAGCTGTTTCCTGCG

flanking region

TCTTTGAGTTAGGTGCTG

primer 1

R2
CNAG_04108 3′

TGATTGGGGAAGCGTTAG

flanking region

primer 2

SO
CNAG_04108

TGTCGGTTTTTGTGGTTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04108

TTAGCCTCTTGCCAACTCC

Southern blot probe

primer

STM
NAT#295 STM

ACACCTACATCAAACCCTCCC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

98
CNAG_04118

L1
CNAG_04118 5′
TCAGCGAGATGATAGGTCG

flanking region

primer 1

L2
CNAG_04118 5′
TCACTGGCCGTCGTTTTACCCG

flanking region
CTATCTCTATCTCTGTCC

primer 2

R1
CNAG_04118 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
ACAAGATAAAGATTGGCGG

primer 1

R2
CNAG_04118 3′
CGCCATCTCCTTTCTATCG

flanking region

primer 2

SO
CNAG_04118
CAAAAGAGAATCCTGGAGACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04118
GGAGAATGAGTCAAATGCTG

Southern blot probe

primer

STM
NAT#212 STM
AGAGCGATCGCGTTATAGAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

99
CNAG_04148

L1
CNAG_04148 5′
GAAGCCCTTGGTATTTTCC

flanking region

primer 1

L2
CNAG_04148 5′
TCACTGGCCGTCGTTTTACCCT

flanking region
CGTAGCCCAAGAAATG

primer 2

R1
CNAG_04148 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
CGTATTGGGTGAATGGC

primer 1

R2
CNAG_04148 3′
TGCTGATACCCTGTTTCG

flanking region

primer 2

SO
CNAG_04148
CGATGATAGGTCCGAAATC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04148
AGACCAAACATCCCAAGC

Southern blot probe

primer

STM
NAT#224 STM
AACCTTTAAATGGGTAGAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

100
CNAG_04156

L1
CNAG_04156 5′
TTCTCCTCCTTCTTTATGCC

flanking region

primer 1

L2
CNAG_04156 5′
TCACTGGCCGTCGTTTTACAGA

flanking region
CAAGAGGGTTTACCTGC

primer 2

R1
CNAG_04156 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
TTACTGAGGCTGCGTTCC

primer 1

R2
CNAG_04156 3′
GCGGATAGAAGCACTGAAAC

flanking region

primer 2

SO
CNAG_04156
GTCCATCGGTAACAAGTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04156
GTGGTAAGCACGGCTAATC

Southern blot probe

primer

STM
NAT#177 STM
CACCAACTCCCCATCTCCAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

101
CNAG_04162
PKA2
L1
CNAG_04162 5′

AATAACACACCAGCCGCTCTGAC

flanking region

C

primer 1

L2
CNAG_04162 5′

CTGGCCGTCGTTTTACTGATGGT

flanking region

GATGGATGTGC

primer 2

R1
CNAG_04162 3′

GTCATAGCTGTTTCCTGCGGCAG

flanking region

TAGAGATAGCACAG

primer 1

R2
CNAG_04162 3′

GGAGTGGTGGAGAATGTTC

flanking region

primer 2

SO
CNAG_04162

TACCTGCTGCTATGACCCTACG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04162

CCACTTGCTTCAACCTCAC

Southern blot probe

primer

STM
NAT#205 STM

TATCCCCCTCTCCGCTCTCTAGC

primer

A

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

102
CNAG_04191

L1
CNAG_04191 5′
CAAGTGGTGTCGCATTTC

flanking region

primer 1

L2
CNAG_04191 5′
TCACTGGCCGTCGTTTTACCGC

flanking region
AACCTGTTTAGTCAGAC

primer 2

R1
CNAG_04191 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CAAAAGAAGAGCAAGGC

primer 1

R2
CNAG_04191 3′
GGGCTAAGAAGTTTGATGTTC

flanking region
C

primer 2

SO
CNAG_04191
ATGAGGGTTTTCAGCACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04191
GGGAAGGAGTGACAAAGATA

Southern blot probe
G

primer

STM
NAT#159 STM
ACGCACCAGACACACAACCAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

103
CNAG_04197
YAK1
L1
CNAG_04197 5′

GTGTGTCATTGGGTTTTGC

flanking region

primer 1

L2
CNAG_04197 5′

TCACTGGCCGTCGTTTTACAATG

flanking region

AATCTGCGGGAGTC

primer 2

R1
CNAG_04197 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

AAGTTGACTCGGCATCG

primer 1

R2
CNAG_04197 3′

GCTTCGTCATCAAACAGTTC

flanking region

primer 2

SO
CNAG_04197

GGTGATTTTTCATCGCCC

diagnostic screening

primer, pairing with

PO
CNAG_04197

CAGCGATGGCTCCTCTATC

Southern blot probe

primer

STM
NAT#184 STM

ATATATGGCTCGAGCTAGATAGA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

104
CNAG_04215
MET3
L1
CNAG_04215 5′

CTCACAAATGAAAGCAGCAG

flanking region

primer 1

L2
CNAG_04215 5′

TCACTGGCCGTCGTTTTACGAGA

flanking region

AGAGAATCGTGAAGAGC

primer 2

R1
CNAG_04215 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

TTGTAGCGTTGTAGATGG

primer 1

R2
CNAG_04215 3′

GCGTTGTTTATTCACAGGAG

flanking region

primer 2

SO
CNAG_04215

CTGTTCTTTGTGTCTTTGCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04215

TCTTTCGGATAACGGCGTG

Southern blot probe

primer

STM
NAT#205 STM

TATCCCCCTCTCCGCTCTCTAGC

primer

A

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

105
CNAG_04221
FBP26
L1
CNAG_04221 5′

TGGAGGTCAGTAATCGGTCG

flanking region

primer 1

L2
CNAG_04221 5′

TCACTGGCCGTCGTTTTACGGAT

flanking region

TGGATGGATGTGAAC

primer 2

R1
CNAG_04221 3′

CATGGTCATAGCTGTTTCCTGTC

flanking region

CGATGTATGCTCTGGTC

primer 1

R2
CNAG_04221 3′

TGTTTCTCCCCTTGTCACC

flanking region

primer 2

SO
CNAG_04221

TGGAAATGAGTTCTCTTGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04221

TCCTAAAATCCCGCTCTGC

Southern blot probe

primer

STM
NAT#146 STM

ACTAGCCCCCCCTCACCACCT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

106
CNAG_04230
THI6
L1
CNAG_04230 5′

TCATCACCAGTAACGAAAGG

flanking region

primer 1

L2
CNAG_04230 5′

TCACTGGCCGTCGTTTTACAGGC

flanking region

TCAACAAAACCGAG

primer 2

R1
CNAG_04230 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

GACTCGGACCCATTCAG

primer 1

R2
CNAG_04230 3′

TGGTGAGTCTTTGCGAAG

flanking region

primer 2

SO
CNAG_04230

TGACCCGAGGTAGAGAATC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04230

ATCAAGAATCTCGCCCAC

Southern blot probe

primer

STM
NAT#290 STM

ACCGACAGCTCGAACAAGCAAG

primer

AG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

107
CNAG_04272

L1
CNAG_04272 5′
GCCTGAAAAGAAGGAAACC

flanking region

primer 1

L2
CNAG_04272 5′
TCACTGGCCGTCGTTTTACCCT

flanking region
TCCTAATGTCTTTCCAGTC

primer 2

R1
CNAG_04272 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AGGAAGTGGAAGCGTTC

primer 1

R2
CNAG_04272 3′
TCGTCTTCGCCAAACTCTGC

flanking region

primer 2

SO
CNAG_04272
GAACGCCGAAACAAAACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04272
CTTGGGAGGAAAATCAGC

Southern blot probe

primer

STM
NAT#212 STM
AGAGCGATCGCGTTATAGAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

108
CNAG_04282
MPK2
L1
CNAG_04282 5′

ATGGCAGCAAGCGTAACTC

flanking region

primer 1

L2
CNAG_04282 5′

TCACTGGCCGTCGTTTTACGTTT

flanking region

TATGCCCGTTGTGTTG

primer 2

R1
CNAG_04282 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

CAAAGTCAGTCTGGTAACC

primer 1

R2
CNAG_04282 3′

ATACATCTTCGTAGCCCCG

flanking region

primer 2

SO
CNAG_04282

TCCAAATAGACCAAGCCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04282

CGTTGAGTGTTTGGTAGCC

Southern blot probe

primer

STM
NAT#102 STM

CCATAGCGATATCTACCCCAATC

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

109
CNAG_04314

L1
CNAG_04314 5′
CCATTCGTAGCCCTTATCTG

flanking region

primer 1

L2
CNAG_04314 5′
TCACTGGCCGTCGTTTTACACG

flanking region
GAGTCTGGTTTTCAGG

primer 2

R1
CNAG_04314 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TTGATGGAAGGAGTCGC

primer 1

R2
CNAG_04314 3′
AAGAGGGCATCACTAAGGC

flanking region

primer 2

SO
CNAG_04314
ATTGGACTGGACCATAGCC

diagnostic screening

primer, pairing with

V79

PO
CNAG_04314
GATAAAGACAGAACTCAGCAC

Southern blot probe
C

primer

STM
NAT#231 STM
GAGAGATCCCAACATCACGC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

110
CNAG_04316
UTR1
L1
CNAG_04316 5′

GGTGATTGCCTGTTGTTG

flanking region

primer 1

L2
CNAG_04316 5′

TCACTGGCCGTCGTTTTACAGAC

flanking region

GAAGGAGGAGGAGTAG

primer 2

R1
CNAG_04316 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

AGTGGTTCAGAGGAATAAG

primer 1

R2
CNAG_04316 3′

ACTTGCCCATACTGGAGGTC

flanking region

primer 2

SO
CNAG_04316

CAGGATGTAGTGGAGACTGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04316

CCAGTAACCCATCACCTATTAG

Southern blot probe

primer

STM
NAT#5 STM primer

TGCTAGAGGGCGGGAGAGTT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

111
CNAG_04335

L1
CNAG_04335 5′
CGATAGAGTAGTAGTTTTAGG

flanking region
GGG

primer 1

L2
CNAG_04335 5′
TCACTGGCCGTCGTTTTACCTT

flanking region
ACGAGTCCATCTTCGC

primer 2

R1
CNAG_04335 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
ACCGATTCCAGTTACAGC

primer 1

R2
CNAG_04335 3′
AGATGGACGAGGTGGTGATG

flanking region

primer 2

SO
CNAG_04335
TGATGTGCTCTACTGGAAGCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04335
TCATCAATGTCAGGCTGGG

Southern blot probe

primer

STM
NAT#146 STM
ACTAGCCCCCCCTCACCACCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

112
CNAG_04347

L1
CNAG_04347 5′
GAGTTTGAGCGGTCATTG

flanking region

primer 1

L2
CNAG_04347 5′
TCACTGGCCGTCGTTTTACAGG

flanking region
TCCTCAAGGTATGGAGC

primer 2

R1
CNAG_04347 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CCCTCAATGTTATCCACG

primer 1

R2
CNAG_04347 3′
GTAGCGAGAGCGATTCATC

flanking region

primer 2

SO
CNAG_04347
TCCAGGGAACAGTGAGTAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04347
TTCAATGATGCCCGAGCAG

Southern blot probe

primer

STM
NAT#210 STM
CTAGAGCCCGCCACAACGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

113
CNAG_04408
CKI1
L1
CNAG_04408 5′

CGTCATTTCTGGGATAGACTG

flanking region

primer 1

L2
CNAG_04408 5′

TCACTGGCCGTCGTTTTACTCCT

flanking region

TCTATGCCTGGGTAGC

primer 2

R1
CNAG_04408 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

ACGCAAGGATGTCCCAGCAG

primer 1

R2
CNAG_04408 3′

TGCTTGTAGGCAATGGCTGG

flanking region

primer 2

SO
CNAG_04408

GATTTCATCCGCCTGTTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04408

ATCTTCCGCTGCTTCAGAC

Southern blot probe

primer

STM
NAT#218 STM

CTCCACATCCATCGCTCCAA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

114
CNAG_04433
YAK103
L1
CNAG_04433 5′

AGCCTGTGAGTTGTGCGTTG

flanking region

primer 1

L2
CNAG_04433 5′

TCACTGGCCGTCGTTTTACGGTT

flanking region

TTCCTGCTATCACGC

primer 2

R1
CNAG_04433 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

CCTCAAAACTCAGCATTG

primer 1

R2
CNAG_04433 3′

AAGAAACCTCTCCATTCCC

flanking region

primer 2

SO
CNAG_04433

AATACCTTGTTGGCGAGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04433

CATCAGGAGGTTTACCACC

Southern blot probe

primer

STM
NAT#231 STM

GAGAGATCCCAACATCACGC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

115
CNAG_04514
MPK1
L1
CNAG_04514 5′

TTTGCTTGCTCCTCTTCTC

flanking region

primer 1

L2
CNAG_04514 5′

TCACTGGCCGTCGTTTTACGAGA

flanking region

AGTAGAGGCAGTGACG

primer 2

R1
CNAG_04514 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

GGAGAAACAGTTGGAGAG

primer 1

R2
CNAG_04514 3′

TTCAGCAGGTCAATCAGG

flanking region

primer 2

SO
CNAG_04514

CGACTCACGATGTAACTTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04514

ACCTCAACTCTCTCAGACACC

Southern blot probe

primer

STM
NAT#240 STM

GGTGTTGGATCGGGGTGGAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

116
CNAG_04577

L1
CNAG_04577 5′
AGGTTTGAGCCATCTGAAC

flanking region

primer 1

L2
CNAG_04577 5′
TCACTGGCCGTCGTTTTACAAA

flanking region
GGGCATAACCAGTGAC

primer 2

R1
CNAG_04577 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
TTGGAGTATGGGAGATGC

primer 1

R2
CNAG_04577 3′
GTCTTTTCTTTCCCACTTGG

flanking region

primer 2

SO
CNAG_04577
GAGATGGGTAATGGTGATGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04577
GCTTGTAACCACGCTCTATC

Southern blot probe

primer

STM
NAT#282 STM
TCTCTATAGCAAAACCAATC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

117
CNAG_04631
RIK1
L1
CNAG_04631 5′

TCATCAGTTTCGTCCAGC

flanking region

primer 1

L2
CNAG_04631 5′

TCACTGGCCGTCGTTTTACATAA

flanking region

CGGGATTGGGGTTG

primer 2

R1
CNAG_04631 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

GCTGATGAGGTCAAGG

primer 1

R2
CNAG_04631 3′

ATCTCACTGCCCTATTCCC

flanking region

primer 2

SO
CNAG_04631

TTCCACTCCTTCTCCCTCTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_04631

CAGGAAGGCTAAAACCACAG

Southern blot probe

primer

STM
NAT#150 STM

ACATACACCCCCATCCCCCC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

118
CNAG_04678
YPK1
L1
CNAG_04678 5′

CGACTATGGGTTCGTTACTGG

flanking region

primer 1

L2
CNAG_04678 5′

TCACTGGCCGTCGTTTTACTGTC

flanking region

TATGCGTTTTCCGAC

primer 2

R1
CNAG_04678 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GTGTAGAATGGCAGAGC

primer 1

R2
CNAG_04678 3′

GCACCGTGGAGGTAGTAATG

flanking region

primer 2

SO
CNAG_04678

TACCCATCATTCCCTGCTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04678

ACACCGTATCAGCACAAGC

Southern blot probe

primer

STM
NAT#58 STM

CGCAAAATCACTAGCCCTATAGC

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

119
CNAG_04755
BCK7
L1
CNAG_04755 5′

GCTGTTGGTTCTCTCTTGC

flanking region

primer 1

L2
CNAG_04755 5′

CTGGCCGTCGTTTTACGGTTTGC

flanking region

GATGAATAGTCC

primer 2

R1
CNAG_04755 3′

GTCATAGCTGTTTCCTGTTCCGA

flanking region

ACGCTCATACTCC

primer 1

R2
CNAG_04755 3′

TTCCTTCGTTTGTCCGTCG

flanking region

primer 2

SO
CNAG_04755

CAGGCTTTTTTTCTGGCTAC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_04755

TACCTCCTTCATTCCTGCCGTC

Southern blot probe

primer 1

PO2
CNAG_04755

GCTTCGTTATCAGTCGTCAC

Southern blot probe

primer 2

STM
NAT#43 STM

CCAGCTACCAATCACGCTAC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

120
CNAG_04821
PAN3
L1
CNAG_04821 5′

CTCTTACAGACGGTTCTTTAGG

flanking region

primer 1

L2
CNAG_04821 5′

TCACTGGCCGTCGTTTTACTCTC

flanking region

CTTTGCCTTCTCCGAG

primer 2

R1
CNAG_04821 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

AATGCGGGCAATAACC

primer 1

R2
CNAG_04821 3′

GCCAAAAAGCAAAAAGTGGAGC

flanking region

primer 2

SO
CNAG_04821

GCAGGAAGAACAAGGTGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04821

GGAACGAGAGAGTGATACACG

Southern blot probe

primer

STM
NAT#204 STM

GATCTCTCGCGCTTGGGGGA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

121
CNAG_04843

Ll
CNAG_04843 5′
CAATCAAACAAGCGACCTC

flanking region

primer 1

L2
CNAG_04843 5′
TCACTGGCCGTCGTTTTACGAA

flanking region
GATTTCTCAACAAGCGG

primer 2

R1
CNAG_04843 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
ACAGCATAGAGAGGGTGTG

primer 1

R2
CNAG_04843 3′
TCCTCCACCATTTCAGACG

flanking region

primer 2

SO
CNAG_04843
GGGGAGCAAACTCTTGAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_04843
CATCTCATCCGTTCTCTGC

Southern blot probe

primer

STM
NAT#116 STM
GCACCCAAGAGCTCCATCTC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

122
CNAG_04927
YFH702
L1
CNAG_04927 5′

GGCATAACTTTCAACGGC

flanking region

primer 1

L2
CNAG_04927 5′

TCACTGGCCGTCGTTTTACAGTC

flanking region

TCCACGACATCTTCTG

primer 2

R1
CNAG_04927 3′

CATGGTCATAGCTGTTTCCTGTA

flanking region

TGCCAGTGGTCAGGTTC

primer 1

R2
CNAG_04927 3′

TCGTATTTGACTTCCCTGG

flanking region

primer 2

SO
CNAG_04927

TGTTTTGAGAGTCCTTCGG

diagnostic screening

primer, pairing with

B79

PO
CMG_04927

TGTCTTTGTGCGTTATGGG

Southern blot probe

primer

STM
NAT#220 STM

CAGATCTCGAACGATACCCA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

123
CNAG_05005
ATG1
L1
CNAG_05005 5′

CGCAGAACAGTCCTACACAAC

flanking region

primer 1

L2
CNAG_05005 5′

TCACTGGCCGTCGTTTTACCTCC

flanking region

TTGCGAGTTTGAGTC

primer 2

R1
CNAG_05005 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

CTGAGAAAAAAGTTGGC

primer 1

R2
CNAG_05005 3′

CGGGAGGAAAACTTGTTC

flanking region

primer 2

SO
CNAG_05005

GATTCACACAAGAGAGCGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05005

TTCCCCTCCTCATTTGTC

Southern blot probe

primer

STM
NAT#288 STM

CTATCCAACTAGACCTCTAGCTA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

124
CNAG_05063
SSK2
L1
CNAG_05063 5′

CCTATCTTATTTTTGCGGGG

flanking region

primer 1

L2
CNAG_05063 5′

CTGGCCGTCGTTTTACTCCTCTT

flanking region

TGTGCCGTATTC

primer 2

R1
CNAG_05063 5′

GTCATAGCTGTTTCCTGATGTTG

flanking region

GAGCAGATGGTG

primer 2

R2
CNAG_05063 3′

CGACTCGTCAACCAAGTTAC

flanking region

primer 2

SO
CNAG_05063

CTAAGGATAGGATGTGGAAGG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_05063

AAGGACGACGAGAGTGAGTAG

Southern blot probe

primer 1

PO2
CNAG_05063

TCCAAACGAACCTTGACAG

Southern blot probe

primer 2

STM
NAT#210 STM

CTAGAGCCCGCCACAACGCT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

125
CNAG_05097
CKY1
L1
CNAG_05097 5′

TGTTCTTCCTTGATGCTCTC

flanking region

primer 1

L2
CNAG_05097 5′

TCACTGGCCGTCGTTTTACGCAG

flanking region

ATACGGAGAAGTCAGAC

primer 2

R1
CNAG_05097 3′

CATGGTCATAGCTGTTTCCTGAG

flanking region

AACATCCCTGTCCTTGC

primer 1

R2
CNAG_05097 3′

ATTATGGGAGAGGCGATG

flanking region

primer 2

SO
CNAG_05097

ATCTTTGTCGGTGTCAGCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05097

AGTCCATCACTCCTTCGG

Southern blot probe

primer

STM
NAT#282 STM

TCTCTATAGCAAAACCAATC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

126
CNAG_05104

L1
CNAG_05104 5′
GCTTTTTGACGAGACAACTG

flanking region

primer 1

L2
CNAG_05104 5′
TCACTGGCCGTCGTTTTACGAT

flanking region
AAAACCCGAGGACATTC

primer 2

R1
CNAG_05104 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
GTTGCTTCCGTATCTGTTG

primer 1

R2
CNAG_05104 3′
AGCAAGTGAAAGAAGGGC

flanking region

primer 2

SO
CNAG_05104
TATCAGGGCTTGGGTGTAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05104
TCTGATAGGGAGCCATACG

Southern blot probe

primer

STM
NAT#208 STM
TGGTCGCGGGAGATCGTGGTT

primer
T

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

127
CNAG_05125

L1
CNAG_05125 5′
TGGTTTTGGCTGCTTCTG

flanking region

primer 1

L2
CNAG_05125 5′
TCACTGGCCGTCGTTTTACGTG

flanking region
AGCAGGTGTTAGAGTGC

primer 2

R1
CNAG_05125 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
AGGACAGTTTATTGGGG

primer 1

R2
CNAG_05125 3′
CACCCAGTAAATACCATCCTG

flanking region

primer 2

SO
CNAG_05125
AGGTTCAAGCGTGATGTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05125
CGCTGACAACACAGATAAGAG

Southern blot probe

primer

STM
NAT#219 STM
CCCTAAAACCCTACAGCAAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

128
CNAG_05200

L1
CNAG_05200 5′
TCCGACAACGAGATTGAAC

flanking region

primer 1

L2
CNAG_05200 5′
TCACTGGCCGTCGTTTTACTCT

flanking region
CCATCTTGACACATTCC

primer 2

R1
CNAG_05200 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GTTTACACCTTACCTCCCAC

primer 1

R2
CNAG_05200 3′
GGAATGGGCAAATGCTAC

flanking region

primer 2

SO
CNAG_05200
TATCCCCACCAAGAAGTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05200
ACAGACCCGTTCCAATGTC

Southern blot probe

primer

STM
NAT#224 STM
AACCTTTAAATGGGTAGAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

129
CNAG_05216
RAD53
L1
CNAG_05216 5′

CCTTGGCTGACACTTTACC

flanking region

primer 1

L2
CNAG_05216 5′

TCACTGGCCGTCGTTTTACCTGT

flanking region

GTGTTTTGGGTTTGG

primer 2

R1
CNAG_05216 3′

CATGGTCATAGCTGTTTCCTGTC

flanking region

CATTATGAAGGAGTCGG

primer 1

R2
CNAG_05216 3′

GTAGACCCTCTTCTTCCTCG

flanking region

primer 2

SO
CNAG_05216

TAGGAGCGATTGCTGAAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05216

ACCAATCAATCAGCCGAC

Southern blot probe

primer

STM
NAT#184 STM

ATATATGGCTCGAGCTAGATAGA

primer

G

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

130
CNAG_05220
TLK1
L1
CNAG_05220 5′

ATCGCTTCTCGTTTGACC

flanking region

primer 1

L2
CNAG_05220 5′

TCACTGGCCGTCGTTTTACATCA

flanking region

ACGACCATCTGGGAC

primer 2

R1
CNAG_05220 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

GCTACTGCTGTGTATTGC

primer 1

R2
CNAG_05220 3′

GCGGTAAAGGTGGAAAGTC

flanking region

primer 2

SO
CNAG_05220

CTTTGAAACCGACCATAGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05220

GGACCGAGACACTACTCACAAC

Southern blot probe

primer

STM
NAT#116 STM

GCACCCAAGAGCTCCATCTC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

131
CNAG_05243
XKS1
L1
CNAG_05243 5′

GCACGAATAAATGCCTGC

flanking region

primer 1

L2
CNAG_05243 5′

TCACTGGCCGTCGTTTTACCTGA

flanking region

GCAAAGGACTTACCTG

primer 2

R1
CNAG_05243 3′

CATGGTCATAGCTGTTTCCTGCG

flanking region

GATTGGAATGCCTGTAG

primer 1

R2
CNAG_05243 3′

GGAGAGTGTTGGAATACGGTAG

flanking region

primer 2

SO
CNAG_05243

AGCCGAAGCCATTTTGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05243

CATCATCACCAGCGATTG

Southern blot probe

primer

STM
NAT#125 STM

CGCTACAGCCAGCGCGCGCAAG

primer

CG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

132
CNAG_05274

L1
CNAG_05274 5′
ATGCTGTTTTGTGGGGGTAGG

flanking region
C

primer 1

L2
CNAG_05274 5′
TCACTGGCCGTCGTTTTACGCT

flanking region
TCTCCGTTTGTTTCG

primer 2

R1
CNAG_05274 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
ATCACAGGGCTTGACGGACTG

primer 1
AG

R2
CNAG_05274 3′
CACTTTTCTTTCTGTCCTCCC

flanking region

primer 2

SO
CNAG_05274
CAACAACGCCAAGAAAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05274
TTGGCGGAACGGATGAATCG

Southern blot probe

primer

STM
NAT#58 STM
CGCAAAATCACTAGCCCTATA

primer
GCG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

133
CNAG_05386

L1
CNAG_05386 5′
TTGCGGAATAAGAAGGGG

flanking region

primer 1

L2
CNAG_05386 5′
TCACTGGCCGTCGTTTTACGTG

flanking region
CTTTATGTGGATTTGGG

primer 2

R1
CNAG_05386 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
CAATCCAAATGAGTGACG

primer 1

R2
CNAG_05386 3′
ACAGGAAGAACAGCAGGAG

flanking region

primer 2

SO
CNAG_05386
GCTATGGGAGTTTTTCCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05386
GCAAATGGGCGTTATTCC

Southern blot probe

primer

STM
NAT#177 STM
CACCAACTCCCCATCTCCAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

134
CNAG_05439
CMK1
L1
CNAG_05439 5′

GGATTGTTAGGTAGGTAGGGG

flanking region

primer 1

L2
CNAG_05439 5′

TCACTGGCCGTCGTTTTACAAGA

flanking region

AGGCGGCTGGATAAG

primer 2

R1
CNAG_05439 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

AGCCCACAATCAAAGTC

primer 1

R2
CNAG_05439 3′

GTGTCATCGTAGGGGTTTC

flanking region

primer 2

SO
CNAG_05439

ATTGCCTATCTGCCTGTGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05439

TCAATGAAACCGCGTGTG

Southern blot probe

primer

STM
NAT#227 STM

TCGTGGTTTAGAGGGAGCGC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

135
CNAG_05484

L1
CNAG_05484 5′
CCAACACCGCCTATTTATC

flanking region

primer 1

L2
CNAG_05484 5′
TCACTGGCCGTCGTTTTACGTG

flanking region
AGTGCCGAGAAAAATG

primer 2

R1
CNAG_05484 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
CTGTGTTGTATGGGACGAG

primer 1

R2
CNAG_05484 3′
TCTCACTCATCTCAAAACGC

flanking region

primer 2

SO
CNAG_05484
TGCTGTTTTAGCCCTTGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05484
AGAGATTGGTGATGGAGCC

Southern blot probe

primer

STM
NAT#205 STM
TATCCCCCTCTCCGCTCTCTAG

primer
CA

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

136
CNAG_05549

L1
CNAG_05549 5′
GGAAGCAGAGGAAGTCTTTAG

flanking region

primer 1

L2
CNAG_05549 5′
TCACTGGCCGTCGTTTTACAGG

flanking region
GTTTTTCCAGACAGC

primer 2

R1
CNAG_05549 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AGAGACCTCCTTCCGACAG

primer 1

R2
CNAG_05549 3′
GATTCGTCCACAACAAAGAC

flanking region

primer 2

SO
CNAG_05549
GACGGCATCAAGGAAAATG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05549
GAGGTGGTGATGTAGAAATAG

Southern blot probe
G

primer

STM
NAT#230 STM
ATGTAGGTAGGGTGATAGGT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

137
CNAG_05558
KIN4
L1
CNAG_05558 5′

ATTCAATGGAGCGGGAGTG

flanking region

primer 1

L2
CNAG_05558 5′

TCACTGGCCGTCGTTTTACCGAA

flanking region

TAAGAATGATGGTGACCG

primer 2

R1
CNAG_05558 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

TGAGTAAGTTCCGCCCC

primer 1

R2
CNAG_05558 3′

AAGGCTGAGGACTGCTACTAC

flanking region

primer 2

SO
CNAG_05558

ATTCTGGTATGAAGCCTCGCAGC

diagnostic screening

C

primer, pairing with

B79

PO
CNAG_05558

TTCCAACTTCAGGTCACG

Southern blot probe

primer

STM
NAT#225 STM

CCATAGAACTAGCTAAAGCA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

138
CNAG_05590
TCO2
L1
CNAG_05590 5′

CAAAACTGGAAGAAGCGAAG

flanking region

primer 1

L2
CNAG_05590 5′

CTGGCCGTCGTTTTACTTGCCAG

flanking region

ATGAAGAGTCACGCC

primer 2

R1
CNAG_05590 3′

GTCATAGCTGTTTCCTGTCCCAT

flanking region

CCTCTGTGATTCCC

primer 1

R2
CNAG_05590 3′

ATTGTGGAGTGGTGGAGTGGAC

flanking region

primer 2

SO
CNAG_05590

TGAGGAGGAAAGTTTTAGCG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_05590

GTTACCGATTCTTGGACCTG

Southern blot probe

primer 1

PO2
CNAG_05590

TGCTTCACCCTTTCAGTCTC

Southern blot probe

primer 2

STM
NAT#116 STM

GCACCCAAGAGCTCCATCTC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

139
CNAG_05600
IGL1
L1
CNAG_05600 5′

TTCTTCTCCTCTATCCCCG

flanking region

primer 1

L2
CNAG_05600 5′

TCACTGGCCGTCGTTTTACGATG

flanking region

ATAGCGATGGTAGCC

primer 2

R1
CNAG_05600 3′

CATGGTCATAGCTGTTTCCTGGG

flanking region

AAGAAGTTTGGGTTCG

primer 1

R2
CNAG_05600 3′

TGGGGAAGAACCAGAAGTAG

flanking region

primer 2

SO
CNAG_05600

TCCCTGTAAGATTCGCCAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05600

TTCTCCATAGGTAGCCACG

Southern blot probe

primer

STM
NAT#230 STM

ATGTAGGTAGGGTGATAGGT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

140
CNAG_05694
CKA1
L1
CNAG_05694 5′

TGTCAAAAGCACACTCAGG

flanking region

primer 1

L2
CNAG_05694 5′

TCACTGGCCGTCGTTTTACTGCG

flanking region

AATAGTTGCTGCTC

primer 2

R1
CNAG_05694 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

GACCTGCCGTGTATTTAG

primer 1

R2
CNAG_05694 3′

AAACATCACTCACCGTTCC

flanking region

primer 2

SO
CNAG_05694

CGACAAGTTGCTGAAGTTTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05694

ACATTTGGAGTCGGTTGG

Southern blot probe

primer

STM
NAT#6 STM primer

ATAGCTACCACACGATAGCT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

141
CNAG_05753
ARG5,6
L1
CNAG_05753 5′

ATTTTCCAGTCGTCCGTC

flanking region

primer 1

L2
CNAG_05753 5′

TCACTGGCCGTCGTTTTACTAAT

flanking region

ACTGAGGGCAGAGCG

primer 2

R1
CNAG_05753 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

CCTTTGACCATCCAGGG

primer 1

R2
CNAG_05753 3′

TTGATGTTTCGCAGCACC

flanking region

primer 2

SO
CNAG_05753

ACCAGTCAGCAACGAAACG

diagnostic screening

primer, pairing with

JOHE12579

PO
CNAG_05753

CGACAGCAAGGGTTTTTG

Southern blot probe

primer

STM
NAT#220 STM

CAGATCTCGAACGATACCCA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

142
CNAG_05771
TEL1
L1
CNAG_05771 5′

ACCCTCCATACATCCTTCC

flanking region

primer 1

L2
CNAG_05771 5′

TCACTGGCCGTCGTTTTACGGCT

flanking region

ATCGTTTCGGTAAGG

primer 2

R1
CNAG_05771 3′

CATGGTCATAGCTGTTTCCTGCA

flanking region

GTATGGATGGGGAGTAATAG

primer 1

R2
CNAG_05771 3′

AACTCCCAAAGATGAGCC

flanking region

primer 2

SO
CNAG_05771

TAGCAGCAAAAGTGAGCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05771

GAAATCGTCAAACTCGTTCC

Southern blot probe

primer

STM
NAT#225 STM

CCATAGAACTAGCTAAAGCA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

143
CNAG_05935

L1
CNAG_05935 5′
GGTCAATCCAGATGCTATCAG

flanking region

primer 1

L2
CNAG_05935 5′
TCACTGGCCGTCGTTTTACTTT

flanking region
GGGTTTGGGTTTGGGCAGC

primer 2

R1
CNAG_05935 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
CCGTGTTGTTCTTTCGTAG

primer 1

R2
CNAG_05935 3′
CAAGGGTGTTGGTATCTACG

flanking region

primer 2

SO
CNAG_05935
CGGAAGATTACTCCTGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05935
TTACTCATACGCAGGACCC

Southern blot probe

primer

STM
NAT#220 STM
CAGATCTCGAACGATACCCA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

144
CNAG_05965
IRK4
L1
CNAG_05965 5′

TCATAGACGATGTTGCCG

flanking region

primer 1

L2
CNAG_05965 5′

TCACTGGCCGTCGTTTTACCAAG

flanking region

ATGGAAGCCAGACTTAC

primer 2

R1
CNAG_05965 3′

CATGGTCATAGCTGTTTCCTGCC

flanking region

ATCTTCCTTCTCCGAAC

primer 1

R2
CNAG_05965 3′

TTTCGGGAGAGTTTTGCG

flanking region

primer 2

SO
CNAG_05965

GCTGTTGTTTCTCACTGTAACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_05965

GATGTATCTGGCAAAGGGTC

Southern blot probe

primer

STM
NAT#211 STM

GCGGTCGCTTTATAGCGATT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

145
CNAG_05970

L1
CNAG_05970 5′
TGAAGCGTGAGTGTAAACG

flanking region

primer 1

L2
CNAG_05970 5′
TCACTGGCCGTCGTTTTACGGG

flanking region
CAAAGGAATGTGATG

primer 2

R1
CNAG_05970 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
TCATTCTTGGATTTCCCTG

primer 1

R2
CNAG_05970 3′
ACAGAAAGGGGTGAAACG

flanking region

primer 2

SO
CNAG_09570
AGACTTGCCCGATTTTGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_05970
TGGCGGTTTATCCTTTCC

Southern blot probe

primer

STM
NAT#212 STM
AGAGCGATCGCGTTATAGAT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

146
CNAG_06001

L1
CNAG_06001 5′
ATCTCCACCTCTTCGCCAACTT

flanking region
CC

primer 1

L2
CNAG_06001 5′
TCACTGGCCGTCGTTTTACCGT

flanking region
CATTTTTTTGGGATACGCC

primer 2

R1
CNAG_06001 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
AGAAGAAGTTGCGGAAGTC

primer 1

R2
CNAG_06001 3′
GGAAGAAAGCGATTTACGG

flanking region

primer 2

SO
CNAG_06001
TTCCTTGCCCTTCCAATCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06001
GGATAAAAGCCTGTCAGTCG

Southern blot probe

primer

STM
NAT#119 STM
CTCCCCACATAAAGAGAGCTA

primer
AAC

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

147
CNAG_06033
MAK32
L1
CNAG_06033 5′

CAAACAACAGATTCCGCC

flanking region

primer 1

L2
CNAG_06033 5′

TCACTGGCCGTCGTTTTACTTCG

flanking region

GATGGACGGATGTAG

primer 2

R1
CNAG_06033 3′

CATGGTCATAGCTGTTTCCTGGG

flanking region

AGATTTCTCTGCCATCC

primer 1

R2
CNAG_06033 3′

AACGCTGGGAAAACTACC

flanking region

primer 2

SO
CNAG_06033

CAGCGTGAAAGTAGCATTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06033

GCTCTTGTCATTCTCGTTCC

Southern blot probe

primer

STM
NAT#169 STM

ACATCTATATCACTATCCCGAAC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common primer

G

148
CNAG_06051
GAL1
L1
CNAG_06051 5′

GCGGTTGAGTGTGTTATTG

flanking region

primer 1

L2
CNAG_06051 5′

TCACTGGCCGTCGTTTTACGCTC

flanking region

CCCTAACACATTGACTC

primer 2

R1
CNAG_06051 3′

CATGGTCATAGCTGTTTCCTGGT

flanking region

CCTGACGCTCTGAMCTG

primer 1

R2
CNAG_06051 3′

GCTATGGGTATGAATCGCC

flanking region

primer 2

SO
CNAG_06051

AGAGACCAGAAGTGAGAGGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06051

GACGCTGACAACAAAAGC

Southern blot probe

primer

STM
NAT#224 STM

AACCTTTAAATGGGTAGAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

149
CNAG_06086
SSN3
L1
CNAG_06086 5′

CGGAGTCTACATTGCTCAGAG

flanking region

primer 1

L2
CNAG_06086 5′

TCACTGGCCGTCGTTTTACAGTA

flanking region

ATCGGTTATCCCACG

primer 2

R1
CNAG_06086 3′

CATGGTCATAGCTGTTTCCTGGA

flanking region

GGATAACGGTGATGCTAAG

primer 1

R2
CNAG_06086 3′

CCACTTGTTTTGCTTGTGC

flanking region

primer 2

SO
CNAG_06086

AGGCACGGGGATTTTTAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06086

ATTTGAACCCACCGACACT

Southern blot probe

primer

STM
NAT#219 STM

CCCTAAAACCCTACAGCAAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

150
CNAG_06161
VRK1
L1
CNAG_06161 5′

TATCGGCAGCGACTCTACTC

flanking region

primer 1

L2
CNAG_06161 5′

TCACTGGCCGTCGTTTTACCGCA

flanking region

ACCATCAACCTAAGC

primer 2

R1
CNAG_06161 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

AGACGCCAAACGCATC

primer 1

R2
CNAG_06161 3′

CCAACCCAACTACTACATACTGC

flanking region

primer 2

SO
CNAG_06161

GAAGAACTGGAAGCATTGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06161

CGAGAAGAGTGAGAAATGGG

Southern blot probe

primer

STM
NAT#123 STM

CTATCGACCAACCAACACAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

151
CNAG_06174

L1
CNAG_06174 5′
GCTCACATCGTAACGGTTG

flanking region

primer 1

L2
CNAG_06174 5′
TCACTGGCCGTCGTTTTACAAT

flanking region
GAGCCGAGAACTTACG

primer 2

R1
CNAG_06174 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TGGAGGGCTTTGTTAGC

primer 1

R2
CNAG_06174 3′
GCTCAACAACAACAGCAAGAG

flanking region

primer 2

SO
CNAG_06174
TCCGATGCTCACGAATAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06174
GTCTCGCACTGTATCAATAAG

Southern blot probe
C

primer

STM
NAT#119 STM
CTCCCCACATAAAGAGAGCTA

primer
AAC

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

152
CNAG_06193
CRK1
L1
CNAG_06193 5′

TCCCCTGCTGTATTCATTG

flanking region

primer 1

L2
CNAG_06193 5′

TCACTGGCCGTCGTTTTACCTTG

flanking region

TGCTAATGTTGTCACG

primer 2

R1
CNAG_06193 3′

CATGGTCATAGCTGTTTCCTGTA

flanking region

ACCAGTCTCATCCTCCAC

primer 1

R2
CNAG_06193 3′

TATTCCAGAGGTAGCGGCGTCA

flanking region

AG

primer 2

SO
CNAG_06193

ATAAGGGGGAAAGACCGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06193

GGTTGCCTTCCATACACTC

Southern blot probe

primer

STM
NAT#43 STM

CCAGCTACCAATCACGCTAC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

153
CNAG_06278
TCO7
L1
CNAG_06278 5′

CCACCTTTCTCATTCGTATG

flanking region

primer 1

L2
CNAG_06278 5′

CTGGCCGTCGTTTTACTCTTCTT

flanking region

CAGATGGTTCCC

primer 2

R1
CNAG_06278 3′

GTCATAGCTGTTTCCTGCACACT

flanking region

CACTCAACGCATC

primer 1

R2
CNAG_06278 3′

CTCCATTTGTTCCATTAGCC

flanking region

primer 2

SO
CNAG_06278

TAAGCCCTCGGAAACACTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06278

CCTTTCTCATTCGTATGGTGTG

Southern blot probe

primer

STM
NAT#209 STM

AGCACAATCTCGCTCTACCCATA

primer

A

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

154
CNAG_06301
SCH9
L1
CNAG_06301 5′

TTCTTCGTGCTGAGAGGAG

flanking region

primer 1

L2
CNAG_06301 5′

GCTCACTGGCCGTCGTTTTACAG

flanking region

ATGTGGCGTAGTCAGCAC

primer 2

R1
CNAG_06301 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

TGAGAATGCGGTGGAC

primer 1

R2
CNAG_06301 3′

GGATGGATGGATGCTCAT

flanking region

primer 2

SO
CNAG_06301

TTCTTCGTGCTGAGAGGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06301

AACCGAAACCCTCAGAACC

Southern blot probe

primer

STM
NAT#169 STM

ACATCTATATCACTATCCCGAAC

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

155
CNAG_06310
IRK7
L1
CNAG_06310 5′

GGTGCTAAAGGATGGTATGG

flanking region

primer 1

L2
CNAG_06310 5′

TCACTGGCCGTCGTTTTACGTTG

flanking region

CTGTTGTTTCTGTAGGTC

primer 2

R1
CNAG_06310 3′

CATGGTCATAGCTGTTTCCTGTT

flanking region

GGTTATCCGCTTACGAC

primer 1

R2
CNAG_06310 3′

GTATGGCTATCAACCTGCTG

flanking region

primer 2

SO
CNAG_06310

CCGACCAAGATGAAAAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06310

GATAGCAACTTTACCCCCC

Southern blot probe

primer

STM
NAT#208 STM

TGGTCGCGGGAGATCGTGGTTT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

156
CNAG_06366
HRR2502
L1
CNAG_06366 5′

TTCTCGTCTTCGCTTTCG

flanking region

primer 1

L2
CNAG_06366 5′

TCACTGGCCGTCGTTTTACGGAG

flanking region

AAGGCATTGCTAAAC

primer 2

R1
CNAG_06366 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

TGTGCCCTCGTAATGG

primer 1

R2
CNAG_06366 3′

TTCGCTGACTTGCTTGAG

flanking region

primer 2

SO
CNAG_06366

TTCCTCGCTTTCAACTCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06366

GTTTCCTTCTTCACCCTACC

Southern blot probe

primer

STM
NAT#125 STM

CGCTACAGCCAGCGCGCGCAAG

primer

CG

STM
STM common
GCATGCCCTGCCCCTAAGAATTC

common
primer
G

157
CNAG_06432

L1
CNAG_06432 5′
CGTCACACAACACTGCTACAG

flanking region

primer 1

L2
CNAG_06432 5′
TCACTGGCCGTCGTTTTACTTG

flanking region
ATTGACGAGGAACCG

primer 2

R1
CNAG_06432 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
GAACTTAGTGGGTCTTGACG

primer 1

R2
CNAG_06432 3′
GCGGTGATGGGTTGTTATC

flanking region

primer 2

SO
CNAG_06432
ACTTGGCGGTAGTCTGAAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06432
ATACCTGGCGGCTAATCAG

Southern blot probe

primer

STM
NAT#224 STM
AACCTTTAAATGGGTAGAG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

158
CNAG_06445

L1
CNAG_06445 5′
GCGATAGGTCAGTAGATTGGG

flanking region

primer 1

L2
CNAG_06445 5′
TCACTGGCCGTCGTTTTACGCT

flanking region
TACATCTGTTGGCACG

primer 2

R1
CNAG_06445 3′
CATGGTCATAGCTTGTTTCCTGC

flanking region
GCCTCACAAGAGTCAAAG

primer 1

R2
CNAG_06445 3′
CAATCAGGACAATCATACGC

flanking region

primer 2

SO
CNAG_06445
GAAGAGGAAATGTCAGGGTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06445
CAGAAAGGAACTCACAGGC

Southern blot probe

primer

STM
NAT#122 STM
ACAGCTCCAAACCTCGCTAAA

primer
CAG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

159
CNAG_06454

L1
CNAG_06454 5′
AACAAAACCGCTGGCAACACC

flanking region
C

primer 1

L2
CNAG_06454 5′
TCACTGGCCGTCGTTTTACTCC

flanking region
AGAGTCTTCTTCAGGCG

primer 2

R1
CNAG_06454 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
ACCAAGATGCCAAAAGC

primer 1

R2
CNAG_06454 3′
AATGGTTGACAAGCGTGCC

flanking region

primer 2

SO
CNAG_06454
ACCCCTTACTGGCGAAAAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06454
GGCAAAACTTACACCTCGC

Southern blot probe

primer

STM
NAT#232 STM
CTTTAAAGGTGGTTTGTG

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

160
CNAG_06489

L1
CNAG_06489 5′
TTCTGGAGACCCATCGTCAG

flanking region

primer 1

L2
CNAG_06489 5′
TCACTGGCCGTCGTTTTACCAA

flanking region
CGCCCTGTTATTTCTTC

primer 2

R1
CNAG_06489 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TGGTCAGATGTGTGTCGG

primer 1

R2
CNAG_06489 3′
CTACTTTGCCGAGTCTCAAG

flanking region

primer 2

SO
CNAG_06489
CAGGACTTGCGTAGCCTATC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06489
TGGGTGATGACGATGAGAC

Southern blot probe

primer

STM
NAT#125 STM
CGCTACAGCCAGCGCGCGCAA

primer
GCG

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

161
CNAG_06490

L1
CNAG_06490 5′
GGAGGGTGTTTTTGAGGTC

flanking region

primer 1

L2
CNAG_06490 5′
TCACTGGCCGTCGTTTTACGGG

flanking region
GACTTTTTTGATGGC

primer 2

R1
CNAG_06490 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
AAGAGGAAGAGGAAGATGAA

primer 1
G

R2
CNAG_06490 3′
TCGTTCTGGTTGTCTGCTC

flanking region

primer 2

SO
CNAG_06490
GGTGAGAAAGTAGCCTTCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06490
CAGGACTTGCGTAGCCTATC

Southern blot probe

primer

STM
NAT#231 STM
GAGAGATCCCAACATCACGC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

162
CNAG_06500

L1
CNAG_06500 5′
GATACAGCGGGCAAAAAG

flanking region

primer 1

L2
CNAG_06500 5′
TCACTGGCCGTCGTTTTACAGA

flanking region
ATGGGATGTGGTCGTC

primer 2

R1
CNAG_06500 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GAACGGGGTTGTGTTTG

primer 1

R2
CNAG_06500 3′
ATACAGACACTCCGATGCG

flanking region

primer 2

SO
CNAG_06500
ATAAAGAGGGTTTGGGGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06500
ATCGCATTTCAAGGGTGG

Southern blot probe

primer

STM
NAT#225 STM
CCATAGAACTAGCTAAAGCA

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

163
CNAG_06552
SNF1
L1
CNAG_06552 5′

CCATCATCCTTCGGTTTTTC

flanking region

primer 1

L2
CNAG_06552 5′

TCACTGGCCGTCGTTTTACAGTT

flanking region

GTTATTGCCAGCGG

primer 2

R1
CNAG_06552 3′

CATGGTCATAGCTGTTTCCTGCT

flanking region

TTTTGGAGATGGCTTGC

primer 1

R2
CNAG_06552 3′

ATACCACGGAAAGGCGTTC

flanking region

primer 2

SO
CNAG_06552

GGATTGTGGTGTTGAAGTCG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06552

ATGCTTGCCTTTCTGGAC

Southern blot probe

primer

STM
NAT#204 STM

GATCTCTCGCGCTTGGGGGA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

164
CNAG_06553
GAL83
L1
CNAG_06553 5′

TGAGCACTTTGAGGTATTGG

flanking region

primer 1

L2
CNAG_06553 5′

TCACTGGCCGTCGTTTTACGTGT

flanking region

GATGTATGGGTGTGTG

primer 2

R1
CNAG_06553 3′

CATGGTCATAGCTGTTTCCTGCA

flanking region

TCTGCTGTGAAACATTGG

primer 1

R2
CNAG_06553 3′

GGAAAGGGGTGAAAATGG

flanking region

primer 2

SO
CNAG_06553

ATGCTTGCCTTTCTGGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06553

TATTGACCAGGAGGAAGGC

Southern blot probe

primer

STM
NAT#288 STM

CTATCCAACTAGACCTCTAGCTA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

165
CNAG_06568
SKS1
L1
CNAG_06568 5′

AATAAGGTCTCCAGCCTCG

flanking region

primer 1

L2
CNAG_06568 5′

TCACTGGCCGTCGTTTTACCCAC

flanking region

CATCAATGAACTGC

primer 2

R1
CNAG_06568 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

CGACCTGTTGATGACG

primer 1

R2
CNAG_06568 3′

CAAGTTGAATGCTGGGAG

flanking region

primer 2

SO
CNAG_06568

AGCAAGTGGGCAAAGAAGC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06568

AACCGAAGTCACAGATGCG

Southern blot probe

primer

STM
NAT#211 STM

GCGGTCGCTTTATAGCGATT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

166
CNAG_06632
ABC1
L1
CNAG_06632 5′

ACGACCTGGTAAAGAGTGTG

flanking region

primer 1

L2
CNAG_06632 5′

TCACTGGCCGTCGTTTTACAGAT

flanking region

GGGCGAAATGTCTC

primer 2

R1
CNAG_06632 3′

CATGGTCATAGCTGTTTCCTGCA

flanking region

CCTCTTATCACCTCAATGAC

primer 1

R2
CNAG_06632 3′

ACCTTCACGACCAAGTGTC

flanking region

primer 2

SO
CNAG_06632

CTATCGCAGAAGAGGATGAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06632

AATACCCCTACAACCTCGTC

Southern blot probe

primer

STM
NAT#119 STM

CTCCCCACATAAAGAGAGCTAAA

primer

C

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

167
CNAG_06642

L1
CNAG_06642 5′
CCTTTTCCTTTTACCTGGC

flanking region

primer 1

L2
CNAG_06642 5′
TCACTGGCCGTCGTTTTACCGC

flanking region
TGAAAGATGTTGTCG

primer 2

R1
CNAG_06642 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
GGATTGACTGGACGAAAC

primer 1

R2
CNAG_06642 3′
CTGGTATGCGTAAAGACTTGA

flanking region
C

primer 2

SO
CNAG_06642
CCTGCTGAACGGATGATAG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06642
GAAGGTTAGTTCGCAAATGG

Southern blot probe

primer

STM
NAT#43 STM
CCAGCTACCAATCACGCTAC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

168
CNAG_06671
YKL1
L1
CNAG_06671 5′

CCGACCTACTGATTCGTCTAC

flanking region

primer 1

L2
CNAG_06671 5′

TCACTGGCCGTCGTTTTACCTCG

flanking region

CCCCTTTTCATAATG

primer 2

R1
CNAG_06671 3′

CATGGTCATAGCTGTTTCCTGGT

flanking region

CCAATCAACAACAGCG

primer 1

R2
CNAG_06671 3′

TGCGGAGGAGATTACCATAC

flanking region

primer 2

SO
CNAG_06671

TTCGCCTTTGAAGTTCCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06671

GGAAAGTGTAGATTGTCGGC

Southern blot probe

primer

STM
NAT#123 STM

CTATCGACCAACCAACACAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

169
CNAG_06697
MPS1
L1
CNAG_06697 5′

GCGATAACTTTTCATCCCC

flanking region

primer 1

L2
CNAG_06697 5′

TCACGGCCGTCGTTTTACGGTT

flanking region

TTTCCTTTCTCCAGTC

primer 2

R1
CNAG_06697 3′

CATGGTCATAGCTGTTTCCTGCG

flanking region

GAACTGTCAGATGGTAATC

primer 1

R2
CNAG_06697 3′

CCTTCTTCACCCTACTCTGG

flanking region

primer 2

SO
CNAG_06697

CCAATCTCGCATTTACACC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06697

TCCTTAGTTATCCTATCCCAGC

Southern blot probe

primer

STM
NAT#116 STM

GCACCCAAGAGCTCCATCTC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

170
CNAG_6730
GSK3
L1
CNAG_06730 5′

GTGAGTCTATCCTTCGTTTCTGT

flanking region

C

primer 1

L2
CNAG_06730 5′

TCACTGGCCGTCGTTTTACCGGC

flanking region

TTCCAAAAAAGTCAG

primer 2

R1
CNAG_06730 3′

CATGGTCATAGCTGTTTCCTGCT

flanking region

GAACAACTGCGTGTCAC

primer 1

R2
CNAG_06730 3′

CTTGAAAGATGACGCTCG

flanking region

primer 2

SO
CNAG_06730

ACATCCTTTGTCTCCCCCAC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_06730

CGGAAGACTTTGGTGAAGG

Southern blot probe

primer 1

STM
NAT#123 STM

CTATCGACCAACCAACACAG

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

171
CNAG_06809
IKS1
L1
CNAG_06809 5′

TGGAAGAGGATGAAAGACC

flanking region

primer 1

L2
CNAG_06809 5′

TCACTGGCCGTCGTTTTACACAA

flanking region

CTAAAGGCACAAGGG

primer 2

R1
CNAG_06809 3′

CATGGTCATAGCTGTTTCCTGAT

flanking region

GAGCGAGCAATGACCTGC

primer 1

R2
CNAG_06809 3′

CAGAACGGTCTTTTGCTTC

flanking region

primer 2

SO
CNAG_06809

TACAGTATCGCTGGTTGCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_06809

AGCGAGACTGGAATGTGGAG

Southern blot probe

primer

STM
NAT#116 STM

GCACCCAAGAGCTCCATCTC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

172
CNAG_06845

L1
CNAG_06845 5′
GTTATTTGGATGCCAGAGC

flanking region

primer 1

L2
CNAG_06845 5′
TCACTGGCCGTCGTTTTACATG

flanking region
CGGTTACCTCATTCG

primer 2

R1
CNAG_06845 3′
CATGGTCATAGCTGTTTCCTGA

flanking region
GGGAGAAGTAGTTTCGGG

primer 1

R2
CNAG_06845 3′
TGGAGGTTTCGGGTATCAC

flanking region

primer 2

SO
CNAG_06845
GCAAAAACCGAGACTGTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_06845
TTGAGGGGTTATGCCTTC

Southern blot probe

primer

STM
NAT#201 STM
CACCCTCTATCTCGAGAAAGC

primer
TCC

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

173
CNAG_06980
STE11
L1
CNAG_06980 5′

TCTCAGCCACATCAGTTAGC

flanking region

primer 1

L2
CNAG_06980 5′

CTGGCCGTCGTTTTACGGGTGC

flanking region

TCTAAATCTCCTTG

primer 2

R1
CNAG_06980 3′

GTCATAGCTGTTTCCTGCCATTT

flanking region

TCCGAGTCAGTAGG

primer 1

R2
CNAG_06980 3′

ATCCTGATGCCAGATTCG

flanking region

primer 2

SO
CNAG_06980

TCATCTGTCTCACCAACTGC

diagnostic screening

primer, pairing with

B79

PO1
CNAG_06980

GGACGCACAGTCTGGTTTAC

Southern blot probe

primer 1

PO2
CNAG_06980

TGGGTCAAGTTTAGGGATG

Southern blot probe

primer 2

STM
NAT#242 STM

GTAGCGATAGGGGTGTCGCTTT

primer

AG

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

174
CNAG_07359
IRK1
L1
CNAG_07359 5′

CGCATTTGGTGTATGATGAC

flanking region

primer 1

L2
CNAG_ 0359 5′

TCACTGGCCGTCGTTTTACGGAG

flanking region

GAAGAAGGAGATGAAG

primer 2

R1
CNAG_07359 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

CTTCGCCTTGATTGTC

primer 1

R2
CNAG_07359 3′

TGCTGAAGATTTCGGAGG

flanking region

primer 2

SO
CNAG_07359

TGATGGTAGAAATGGCGG

diagnostic screening

primer, pairing with

B79

PO1
CNAG_07359

GCATTCGGAGGTAGTTGAAG

Southern blot probe

primer 1

STM
NAT#5 STM primer

TGCTAGAGGGCGGGAGAGTT

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

175
CNAG_07372

L1
CNAG_07372 5′
CCAAACGGTGTGAAAAGG

flanking region

primer 1

L2
CNAG_07372 5′
TCACTGGCCGTCGTTTTACTGT

flanking region
AGTCGCCGATGGAGTAG

primer 2

R1
CNAG_07372 3′
CATGGTCATAGCTGTTTCCTGG

flanking region
GCAAGACGAGAAGTAGAGC

primer 1

R2
CNAG_07372 3′
GAACCTGAACCTGAACCAG

flanking region

primer 2

SO
CNAG_07372
TTTGTAGTTGGGTGTGGTG

diagnostic screening

primer, pairing with

B79

PO
CNAG_07372
CTTCGCCTTTTGCCTTTC

Southern blot probe

primer

STM
NAT#295 STM
ACACCTACATCAAACCCTCCC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

176
CNAG_07377

L1
CNAG_07377 5′
CGATAACGCAACTTACGG

flanking region

primer 1

L2
CNAG_07377 5′
TCACTGGCCGTCGTTTTACTTT

flanking region
GGCTTGATTCTCCGC

primer 2

R1
CNAG_07377 3′
CATGGTCATAGCTGTTTCCTGC

flanking region
TCTCAATCTCGCTCAAATG

primer 1

R2
CNAG_07377 3′
CTGAGCCGATAGAGTTCAAC

flanking region

primer 2

SO
CNAG_07377
ACCAACGCACATCTACCTC

diagnostic screening

primer, pairing with

B79

PO
CNAG_07377
TTATCTACCGAAGTTGGCTG

Southern blot probe

primer

STM
NAT#296 STM
CGCCCGCCCTCACTATCCAC

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

177
CNAG_07408

L1
CNAG_07408 5′
GCTGGCATAAAACCGTTC

flanking region

primer 1

L2
CNAG_07408 5′
TCACTGGCCGTCGTTTTACCTC

flanking region
TTACTCCACATAAATGCCC

primer 2

R1
CNAG_07408 3′
CATGGTCATAGCTGTTTCCTGT

flanking region
TGAAGTCACCCGAGAAAC

primer 1

R2
CNAG_07408 3′
ACACTGCGGATTACGAAGC

flanking region

primer 2

SO
CNAG_07408
TGTGGCTGAGATGAGGTAGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_07408
TCTGGGCTGAAGTCTACTAAA

Southern blot probe
C

primer

STM
NAT#6 STM
ATAGCTACCACACGATAGCT

primer

STM
STM common
GCATGCCCTGCCCCTAAGAAT

common
primer
TCG

178
CNAG_07427
CCK2
L1
CNAG_07427 5′

AGATTCACTCGTCATCGCC

flanking region

primer 1

L2
CNAG_07427 5′

TCACTGGCCGTCGTTTTACTAAG

flanking region

ATGCGATAGGTGGGCG

primer 2

R1
CNAG_07427 3′

CATGGTCATAGCTGTTTCCTGCA

flanking region

GACTAAAGCCAGGGACAC

primer 1

R2
CNAG_07427 3′

GGAAGGTCAAGCCATTAGC

flanking region

primer 2

SO
CNAG_07427

TCAAGGCTTTCATCCCGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_07427

CGAGACCAGTTATGTTTGAGAG

Southern blot probe

primer

STM
NAT#230 STM

ATGTAGGTAGGGTGATAGGT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

179
CNAG_07580
TRM7
L1
CNAG_07580 5′

GGTGGAGAGATGTTATGGC

flanking region

primer 1

L2
CNAG_07580 5′

TCACTGGCCGTCGTTTTACATAG

flanking region

AGGACTTGGAGGTGGG

primer 2

R1
CNAG_07580 3′

CATGGTCATAGCTGTTTCCTGGC

flanking region

AATGCTGTGAATCTTGTG

primer 1

R2
CNAG_07580 3′

AGAGTAGGGCTGAGCAAGAC

flanking region

primer 2

SO
CNAG_07580

TGGAAAGACCTGTTGCGAC

diagnostic screening

primer, pairing with

B79

PO
CNAG_07580

TCTTCGGGAAATGGACTG

Southern blot probe

primer

STM
NAT#102 STM

CCATAGCGATATCTACCCCAATC

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

180
CNAG_07667
SAT4
L1
CNAG_07667 5′

GATTTTGTGGCTGTTGTGC

flanking region

primer 1

L2
CNAG_07667 5′

TCACTGGCCGTCGTTTTACTGCT

flanking region

TCAAAACCTGGGCTCC

primer 2

R1
CNAG_07667 3′

CATGGTCATAGCTGTTTCCTGGT

flanking region

GTAGATTGTTCAGGATGACG

primer 1

R2
CNAG_07667 3′

AGATAGGCGTGCTACCGATG

flanking region

primer 2

SO
CNAG_07667

ATCGGCTTACCATTCTGG

diagnostic screening

primer, pairing with

PO
CNAG_07667

TCGGTCCCATAATAGACGG

Southern blot probe

primer

STM
NAT#212 STM

AGAGCGATCGCGTTATAGAT

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

181
CNAG_07744
PIK1
L1
CNAG_07744 5′

TGGTAGTATGCCAAGAGGTG

flanking region

primer 1

L2
CNAG_07744 5′

TCACTGGCCGTCGTTTTACTGGG

flanking region

ATACTCTCTCTCTCTGAG

primer 2

R1
CNAG_07744 3′

CATGGTCATAGCTGTTTCCTGAA

flanking region

AGGGCAAAGGCAGAAG

primer 1

R2
CNAG_07744 3′

GGAGATGAAGTCAAGATGCG

flanking region

primer 2

SO
CNAG_07744

TCATCTTCATTGTCCTCCC

diagnostic screening

primer, pairing with

B79

PO
CNAG_07744

TAAAGAGCGGTAAGGCGAG

Southern blot probe

primer

STM
NAT#227 STM

TCGTGGTTTAGAGGGAGCGC

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

182
CNAG_07779
TDA10
L1
CNAG_07779 5′

TGGGAAGCGTTACTTATGC

flanking region

primer 1

L2
CNAG_07779 5′

TCACTGGCCGTCGTTTTACCTGT

flanking region

AGCAGTCATAATGGCTTG

primer 2

R1
CNAG_07779 3′

CATGGTCATAGCTGTTTCCTGTG

flanking region

AGCAGGTCCGACATTTC

primer 1

R2
CNAG_07779 3′

CATCGCTCTTTCCTACTCG

flanking region

primer 2

SO
CNAG_07779

TTTGGAGCCAGTTTAGGG

diagnostic screening

primer, pairing with

B79

PO
CNAG_07779

AAAACGAAGCCCTTTGCCCC

Southern blot probe

primer

STM
NAT#102 STM

CCATAGCGATATCTACCCCAATC

primer

T

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

183
CNAG_08022
PHO85
L1
CNAG_08022 5′

CCTTGCTTTTGAGCGAG

flanking region

primer 1

L2
CNAG_08022 5′

CTGGCCGTCGTTTTACCCTTCAC

flanking region

CAAGTTTCTCAAG

primer 2

R1
CNAG_08022 3′

GTCATAGCTGTTTCCTGCAAATG

flanking region

GCTCAACAAGGG

primer 1

R2
CNAG_08022 3′

CCACAGTGCGTCTTTTTATC

flanking region

primer 2

SO
CNAG_08022

ATAGGGGTGATTATCGGGC

diagnostic screening

primer, pairing with

B79

PO
CMG_08022

TCGGCATTATCTCTTCCTC

Southern blot probe

primer

STM
NAT#218 STM

CTCCACATCCATCGCTCCAA

primer

STM
STM common

GCATGCCCTGCCCCTAAGAATTC

common
primer

G

TABLE 3

Primers used in the construction and functional

characterization of kinase mutant library

Primer

Primer sequence

name
Primer description
(5′-3′)

B1026
M13 Forward
GTAAAACGACGGCCAGTGAGC

extended

B1027
M13 Reverse
CAGGAAACAGCTATGACCATG

extended

B1454
NAT split marker
AAGGTGTTCCCCGACGACGAA

primer (NSR)
TCG

B1455
NAT split marker
AACTCCGTCGCGAGCCCCATC

primer (NSL)
AAC

B1886
NEO split marker
TGGAAGAGATGGATGTGC

primer (GSR)

B1887
NEO split marker
ATTGTCTGTTGTGCCCAG

primer (GSL)

B4017
Primer 1 for
GCATGCAGGATTCGAGTG

overexpression

promoter with NEO

marker

B4018
Primer 2 for
GTGATAGATGTGTTGTGGTG

overexpression

promoter with NEO

marker

B678
Northern probe
TTCAGGGAACTTGGGAACAGC

primer1 for ERG11

B1598
Northern probe
CAGGAGCAGAAACAAAGC

primer2 for ERG11

B3294
Northern probe
GCACCATACCTTCTACAATGA

primer1 for ACT1
G

B3295
Northern probe
ACTTTCGGTGGACGATTG

primer2 for ACT1

B5251
RT-PCR primer for
CACTCCATTCCTTTCTGC

HXL1 of H99

B5252
RT-PCR primer for
CGTAACTCCACTGTGTCC

HXL1 of H99

B7030
qRT-PCR primer for
AGACTGTTTACAATGCCTGC

CNA1 of H99

B7031
qRT-PCR primer for
TCTGGCGACAAGCCACCATG

CNA1 of H99

B7032
qRT-PCR primer for
AAGATGGAAGTGGAACGG

CNB1 of H99

B7033
qRT-PCR primer for
TTGAAAGCGAATCTCAGCTT

CNB1 of H99

B7034
qRT-PCR primer for
ACCACGGACATTATCTTCAG

CRZ1 of H99

B7035
qRT-PCR primer for
AGCCCAGCCTTGCTGTTCGT

CRZ1 of H99

B7036
qRT-PCR primer for
TTTCTATGCCCATCTACAGC

UTR2 of H99

B7037
qRT-PCR primer for
CTTCGTGGGAGTACAGTGGC

UTR2 of H99

B679
qRT-PCR primer for
CGCCCTTGCTCCTTCTTCTAT

ACT1 of H99
G

B680
qRT-PCR primer for
GACTCGTCGTATTCGCTCTTC

ACT1 of H99
G

Example 3
Systematic Phenotypic Profiling and Clustering of Cryptococcus neoformans Kinom Network

With the kinase mutant library constructed in the above Example, the present inventors performed a series of in vitro phenotypic analyses (a total of 30 phenotypic traits) under distinct growth conditions covering six major phenotypic classes (growth, differentiation, stress responses and adaptations, antifungal drug resistance and production of virulence factors), thereby making more than 6,600 phenotype data. Such comprehensive kinase phenome data are freely accessible to the public through the Cryptococcus neoformans kinome database (http://kinase.cryptococcus.org). To gain insights into the functional and regulatory connectivity among kinases, the present inventors attempted to group kinases by phenotypic clustering through Pearson correlation analysis (see FIG. 3). The rationale behind this analysis was that a group of kinases in a given signaling pathway tended to cluster together in teams of shared phenotypic traits. For example, mutants in three-tier mitogen-activated protein kinase (MAPK) cascades should cluster together because they exhibit almost identical phenotypic traits. In fact, the present inventors found that the three-tier kinase mutants in the cell wall integrity MAPK (bck1Δ, mkk1Δ, mpk1Δ), the high osmolarity glycerol response (HOG) MAPK (ssk2Δ, pbs2Δ, hog1Δ), and the pheromone-responsive MAPK (ste11Δ, ste7Δ, cpk1Δ) pathways were clustered together based on their shared functions (FIG. 4). Therefore, groups of kinases clustered together by this analysis are highly likely to function in the same or related signaling cascades. The present inventors identified several hitherto uncharacterized kinases that are functionally correlated with these known signaling pathways. First, the present inventors identified CNAG_06553, encoding a protein orthologous to yeast Ga183 that is one of three possible β-subunits of the Snf1 kinase complex in S. cerevisiae. The yeast Snf1 kinase complex consists of Snf1, catalytic α-subunit, Snf4, regulatory γ subunit, and one of three possible β-subunits (Ga183, Sip1 and Sip2), and controls the transcriptional changes under glucose derepression (Jiang, R. & Carlson, M. The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Ga183 component in the kinase complex. Mol Cell Biol 17, 2099-2106, 1997; Schuller, H. J. Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr Genet 43, 139-160, doi:10.1007/s00294-003-0381-8, 2003). In C. neoformans, Snf1 functions have been previously characterized (Hu, G., Cheng, P. Y., Sham, A., Perfect, J. R. & Kronstad, J. W. Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Molecular microbiology 69, 1456-1475, doi:10.1111/j.1365-2958.2008.06374.x, 2008). Several lines of experimental evidence showed that Ga183 is likely to function in association with Snf1 in C. neoformans. First, the in vitro phenotypic traits of the ga183Δ mutant were almost equivalent to those of the snf1Δ mutant (FIG. 3). Both snf1Δ and ga183Δ mutants exhibited increased susceptibility to fludioxonil and increased resistance to organic peroxide (tert-butyl hydroperoxide). Second, growth defects in the snf1Δ mutant in alternative carbon sources (for example, potassium acetate, sodium acetate and ethanol) were also observed in ga183Δ mutants (FIG. 4). Therefore, Ga183 is likely to be one of the possible β-subunits of the Snf1 kinase complex in C. neoformans.

The present inventors also identified several kinases that potentially work upstream or downstream of the TOR kinase complex. Although the present inventors were not able to disrupt Tor1 kinase, which has been suggested to be essential in C. neoformans, the present inventors found three kinases (Ipk1, Ypk1 and Gsk3 found to be clustered in most eukaryotes) that are potentially related to Tor1-dependent signaling cascades clustered in C. neoformans. Recently, Lev et al. proposed that Ipk1 could be involved in the production of inositol hexaphosphate (IP₆) based on its limited sequence homology to S. cerevisiae Ipk1 (Lev, S. et al. Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity. MBio 6, e00531-00515, doi:10.1128/mBio.00531-15 (2015)). In mammals, inositol polyphosphate multikinase (IPMK), identified as Arg82 in yeast, produces IP6, a precursor of 5-IP₇that inhibits Akt activity and thereby decreases mTORC1-mediated protein translation and increases GSK3-mediated glucose homeostasis, adipogenesis, and activity (Chakraborty, A., Kim, S. & Snyder, S. H. Inositol pyrophosphates as mammalian cell signals. Sci Signal 4, rel, doi:10.1126/scisignal.2001958 (2011)). It was reported that in S. cerevisiae, Ypk1 is the direct target of TORC2 by promoting autophagy during amino acid starvation (Vlahakis, A. & Powers, T. A role for TOR complex 2 signaling in promoting autophagy. Autophagy 10, 2085-2086, doi:10.4161/auto.36262 (2014)). In C. neoformans, Ypk1, which is a potential downstream target of Tor1, is involved in sphingolipid synthesis and deletion of YPK1 resulted in a significant reduction in virulence (Lee, H., Khanal Lamichhane, A., Garraffo, H. M., Kwon-Chung, K. J. & Chang, Y. C. Involvement of PDK1, PKC and TOR signalling pathways in basal fluconazole tolerance in Cryptococcus neoformans. Mol. Microbiol. 84, 130-146, doi:10.1111/j.1365-2958.2012.08016.x (2012)). Reflecting the essential role of Tor1, all of the mutants (ipk1Δ, ypk1Δ, and gsk3Δ) exhibited growth defects, particularly at high temperature.

However, there are two major limitations in this phenotypic clustering analysis. First, kinases that are oppositely regulated in the same pathway cannot be clustered. Second, a kinase that regulates a subset of phenotypes governed by a signaling pathway may not be clustered with its upstream kinases; this is the case of the Hog1-regulated kinase 1 (CNAG_00130; Hrk1). Although the present inventors previously demonstrated that Hrk1 is regulated by Hog1, Hrk1 and Hog1 are not clustered together as Hrk1 regulates only subsets of Hog1-dependent phenotypes. Phospholipid flippase kinase 1 (Fpk1) is another example. In S. cerevisiae, the activity of Fpk1 is inhibited by direct phosphorylation by Ypk1. As expected, Fpk1 and Ypk1 were clustered together. To examine whether Fpk1 regulates Ypk1-dependent phenotypic traits in C. neoformans, the present inventors performed epistatic analyses by constructing and analyzing FPK1 overexpression strains constructed in the ypk1Δ and wild-type strain backgrounds. As expected, overexpression of FPK1 partly restored normal growth, resistance to some stresses (osmotic, oxidative, genotoxic, and cell wall/membrane stresses) and antifungal drug (amphotericin B) in ypk1Δ mutants (FIG. 5). However, azole susceptibility of ypk1Δ mutants could not be restored by FPK1 overexpression (see FIG. 5). These results suggest that Fpk1 could be one of the downstream targets of Ypk1 and may be positively regulated by Ypk1.

Example 4
Pathogenic Kinome Networks in C. neoformans

To identify pathogenicity-regulating kinases which are controlled by both infectivity and virulence, the present inventors two large-scale in vivo animal studies: a wax moth-killing virulence assay and a signature-tagged mutagenesis (STM)-based murine infectivity assay. In the two assays, two independent mutants for each of kinases, excluding kinases with single mutants, were monitored. As a result, 31 virulence-regulating kinases in the insect killing assay (FIGS. 6 and 7) and 54 infectivity-regulating kinases in the STM-based murine infectivity assay were found (FIGS. 9 and 10). Among these kinases, 25 kinases were co-identified by both assays (FIG. 11a), indicating that virulence in the insect host and infectivity in the murine host are closely related to each other as reported previously (Jung, K. W. et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757, 2015). Only 6 kinase mutants were identified by the insect killing assay (FIG. 11b). The present inventors discovered a total of 60 kinase mutants involved in the pathogenicity of C. neoformans.

Additionally, a large number of known virulence-regulating kinases (a total of 15 kinases) were rediscovered in the present invention (kinases indicated in black in FIG. 11a). These kinases include Mpk1 MAPK (Gerik, K. J., Bhimireddy, S. R., Ryerse, J. S., Specht, C. A. & Lodge, J. K. PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans. Eukaryot. Cell 7, 1685-1698, 2008; Kraus, P. R., Fox, D. S., Cox, G. M. & Heitman, J. The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function. Mol. Microbiol. 48, 1377-1387, 2003); Ssk2 in the high osmolarity glycerol response (HOG) pathway (Bahn, Y. S., Geunes-Boyer, S. & Heitman, J. Ssk2 mitogen-activated protein kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans. Eukaryot. Cell 6, 2278-2289, 2007), an essential catalytic subunit (Pka1) of protein kinase A in the cAMP pathway (D'Souza, C. A. et al. Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol. Cell. Biol. 21, 3179-3191, 2001); Ire1 kinase/endoribonuclease in the unfolded protein response (UPR) pathway (Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hx11, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177, 2011); Ypk1 (Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767, 2015; Lee, H., Khanal Lamichhane, A., Garraffo, H. M., Kwon-Chung, K. J. & Chang, Y. C. Involvement of PDK1, PKC and TOR signalling pathways in basal fluconazole tolerance in Cryptococcus neoformans. Mol. Microbiol. 84, 130-146, doi:10.1111/j.1365-2958.2012.08016.x, 2012); and Snf1 (Hu, G., Cheng, P. Y., Sham, A., Perfect, J. R. & Kronstad, J. W. Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Molecular microbiology 69, 1456-1475, doi:10.1111/j.1365-2958.2008.06374.x, 2008. The function of (B3501A) Gsk3 in serotype D was examined, and it was demonstrated that Gsk3 survives at low oxygen partial pressure (1%) in C. neoformans and is required for the virulence of serotype D in a murine model system (Chang, Y. C., Ingavale, S. S., Bien, C., Espenshade, P. & Kwon-Chung, K. J. Conservation of the sterol regulatory element-binding protein pathway and its pathobiological importance in Cryptococcus neoformans. Eukaryot Cell 8, 1770-1779, doi:10.1128/EC.00207-09, 2009). The present inventors found that Gsk3 is also required for the virulence of serotype A C. neoformans (H99S). Although not previously reported, deletion mutants of kinases functionally connected to these known virulence-regulating kinases were also found to be attenuated in virulence or infectivity. These include bck1Δ and mkk1/2Δ mutants (related to Mpk1) and the ga183Δ mutant (related to Snf1). Notably, among them, 44 kinases have been for the first time identified to be involved in the fungal pathogenicity of C. neoformans.

For the 60 pathogenicity-related kinases in C. neoformans, the present inventors analyzed phylogenetic relationships among orthologs, if any, in fungal species and other eukaryotic kingdoms. To inhibit a broad spectrum of fungal pathogens, it is ideal to target kinases which are not present in humans and are required in a number of fungal pathogens (broad-spectrum antifungal targets). The present inventors compared these large-scale virulence data of C. neoformans with those of other fungal pathogens. A large-scale kinome analysis was performed for the pathogenic fungus Fusarium graminearum, which causes scab in wheat plants, and 42 virulence-related protein kinases were identified (Wang, C. et al. Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathog 7, e1002460, doi:10.1371/journal.ppat.1002460, 2011). Among them, a total of 21 were involved in the pathogenicity of both types of fungi, and thus were regarded as broad-spectrum antifungal targets: BUD32 (Fg10037), ATG1 (Fg05547), CDC28 (Fg08468), KIC1 (Fg05734), MEC1 (Fg13318), KIN4 (Fg11812), MKK1/2 (Fg07295), BCK1 (Fb06326), SNF1 (Fg09897), SSK2 (Fg00408), PKA1 (Fg07251), GSK3 (Fg07329), CBK1 (Fg01188), KIN1 (Fg09274), SCH9 (Fg00472), RIM15 (Fg01312), HOG1 (Fg09612), and YAK1 (Fg05418). In another human fungal pathogen C. albicans, genome-wide pathogenic kinome analysis has not been performed. Based on information from the Candida genome database (http://www.candidagenome.org/), 33 kinases are known to be involved in the pathogenicity of C. albicans. Among them, 13 were involved in the pathogenicity of both C. neoformans and C. albicans. Notably, five kinases (Sch9, Snf1, Pka1, Hog1, and Swe1) appear to be core-pathogenicity kinases as they are involved in the pathogenicity of all three fungal pathogens.

On the contrary, to selectively inhibit C. neoformans, it is ideal to target pathogenicity-related kinases which are present in C. neoformans but are not present in other fungi or humans (narrow-spectrum anti-cryptococcosis targets). Among them, CNAG_01294 (named IPK1), encoding a protein similar to inositol 1,3,4,5,6-pentakisphosphate 2-kinase from plants, is either not present or distantly related to those in ascomycete fungi and humans, and is considered a potential anti-cryptococcal target. In addition to lacking virulence, the ipk1Δ mutants exhibited pleiotropic phenotypes (FIG. 12). Deletion of IPK1 increased slightly capsule production, but inhibited melanin and urease production. Its deletion also rendered cells to be defective in sexual differentiation and hypersensitive to high temperature and multiple stresses, and enhances susceptibility to multiple antifungal drugs. In particular, Ipk1 can be an useful target in combination therapy, because its deletion significantly increases susceptibility to various kinds of antifungal drugs. Therefore, the present inventors revealed narrow- and broad-spectrum anticryptococcal and antifungal drug targets by kinome analysis of C. neoformans pathogenicity.

Example 5
Biological Functions of Kinases Regulating Pathogenicity of C. neoformans

To further clarify a functional network of pathogenicity-related kinases, the present inventors employed a genome-scale co-functional network CryptoNet (www.inetbio.org/cryptonet) for C. neoformans, recently constructed by the present inventors (Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767 (2015)). To search for any proteins functionally linked to the pathogenicity-related kinases, previously reported information on C. neoformans and the Gene Ontology (GO) teams of corresponding kinase orthologs and its interacting proteins in S. cerevisiae and other fungi were used. This analysis revealed that the biological functions of pathogenicity-related kinases include cell cycle regulation, metabolic process, cell wall biogenesis and organization, DNA damage repair, histone modification, transmembrane transport and vacuole trafficking, tRNA processing, cytoskeleton organization, stress response and signal transduction, protein folding, mRNA processing, and transcriptional regulation, suggesting that various biological and physiological functions affect virulence of C. neoformans. Among pathogenicity-related kinases, kinases involved in the cell cycle and growth control were identified most frequently. These include CDC7, SSN3, CKA1, and MEC1. In particular, Cdc7 is an essential catalytic subunit of the Dbf4-dependent protein kinase in S. cerevisiae, and Cdc7-Dbf4 is required for firing of the replication of origin throughout the S phase in S. cerevisiae (Diffley, J. F., Cocker, J. H., Dowell, S. J., Harwood, J. & Rowley, A. Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle. J Cell Sci Suppl 19, 67-72, 1995). Although not essential at ambient temperature, cdc7Δ mutants exhibit serious growth effects at high temperature (FIG. 13a), indicating that they are likely to affect virulence of C. neoformans. The cdc7Δ mutants in C. neoformans are very susceptible to genotoxic agents such as methyl methanesulfonate (MMS) and hydroxyurea (HU), suggesting that Cdc7 can cause DNA replication and repair (FIG. 13a). Mec1 is required for cell cycle checkpoint, telomere maintenance and silencing and DNA damage repair in S. cerevisiae (Mills, K. D., Sinclair, D. A. & Guarente, L. MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97, 609-620, 1999). Reflecting these roles, deletion of MEC1 increased cellular sensitivity to genotoxic agents in C. neoformans (FIG. 13b), indicating that the role of Mec1 in chromosome integrity can be retained. Deletion of MEC1 did not cause any lethality or growth defects in C. neoformans, as was the case in C. albicans (Legrand, M., Chan, C. L., Jauert, P. A. & Kirkpatrick, D. T. The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans. Fungal Genet Biol 48, 823-830, doi:10.1016/j.fgb.2011.04.005, 2011). Cka1 and Cka2 are catalytic α-subunits of protein kinase CK2, which have essential roles in growth and proliferation of S. cerevisiae; deletion of both kinases causes lethality (Padmanabha, R., Chen-Wu, J. L., Hanna, D. E. & Glover, C. V. Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae. Mol Cell Biol 10, 4089-4099, 1990). Interestingly, C. neoformans appears to have a single protein (CKA1) that is orthologous to both Cka1 and Cka2. Although deletion of CKA1 is not essential, it severely affected the growth of C. neoformans (FIG. 13c). Notably, the cka1Δ mutant showed elongated, abnormal cell morphology (FIG. 13d), which is comparable to that of two kinase mutants in the RAM pathway (cbk1Δ and kic1Δ). Cbk1 and Kic1 are known to control the cellular polarity and morphology of C. neoforman, but their correlation with virulence is not yet known (Walton, F. J., Heitman, J. & Idnurm, A. Conserved Elements of the RAM Signaling Pathway Establish Cell Polarity in the Basidiomycete Cryptococcus neoformans in a Divergent Fashion from Other Fungi. Mol. Biol. Cell, 2006). The present inventors revealed that the cellular polarity and morphology of C. neoforman is related to virulence.

Bud32 is also required for growth, potentially through involvement of tRNA modification. Bud32 belongs to the piD261 family of atypical protein kinases, which are conversed in bacteria, Archaea and eukaryotes, and it recognizes acidic agents, unlike other eukaryotic protein kinases that recognize basic agents (Stocchetto, S., Marin, O., Carignani, G. & Pinna, L. A. Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase. FEBS Lett 414, 171-175, 1997). In S. cerevisiae, Bud32 is a component of the highly conserved EKC (Endopetidase-like and Kinase-associated to transcribed Chromatin)/KEOPS (Kinase, putative endopetidase and other proteins of small size) complex. This complex is required for N⁶-threonylcarbamoyladenosine (t⁶A) tRNA modification, which is important in maintaining codon-anticodon interactions for all tRNAs. Therefore, damaged cells in the EKC/KEOPS complex are likely to have increased frameshift mutation rate and low growth rate (Srinivasan, M. et al. The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A. EMBO J 30, 873-881, doi:10.1038/emboj.2010.343, 2011). As expected, these defects in tRNA modification had dramatic effects on various biological aspects of C. neoformans, and thus affected virulence. The bud32Δ mutants exhibited very defective growth under basal and most of the stress conditions (FIG. 12a), and also produced smaller amounts of capsule, melanin and urease (FIG. 12b). In addition, the bud32 mutant was significantly defective in mating (FIG. 14c). One exception was fluconazole resistance (FIG. 14a). Interestingly, the present inventors found that deletion of BUD32 abolished the induction of ERG11 upon sterol depletion by fluconazole treatment (FIG. 14d), suggesting a potential role of Bud32 in ergosterol gene expression and sterol biosynthesis in C. neoformans.

Kinases involved in nutrient metabolism are also involved in the pathogenicity of C. neoformans. In S. cerevisiae, Arg5, 6p is synthesized as a single protein and is subsequently processed into two separate enzymes (acetylglutamate kinase and N-acetyl-γ-glutamyl-phosphate reductase) (Boonchird, C., Messenguy, F. & Dubois, E. Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae. Eur J Biochem 199, 325-335, 1991). These enzymes catalyze biosynthesis of ornithine, an arginine intermediate. Consistent with this, the present inventors found that the arg5, 6pΔ mutant was auxotrophic for arginine (FIG. 15a). In S. cerevisiae, MET3, encoding ATP sulfurylase, catalyzes the initial state of the sulfur assimilation pathway that produces hydrogen sulfide, a precursor for biosynthesis of homocysteine, cysteine and methionine (Cherest, H., Nguyen, N. T. & Surdin-Kerjan, Y. Transcriptional regulation of the MET3 gene of Saccharomyces cerevisiae. Gene 34, 269-281, 1985; Ullrich, T. C., Blaesse, M. & Huber, R. Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation. EMBO J 20, 316-329, doi:10.1093/emboj/20.3.316, 2001). In fact, the met3Δ mutant was found to be auxotrophic for both methionine and cysteine (FIG. 15b). Notably, both arg5, 6pΔ and met3Δ mutants did not exhibit growth defects in nutrient-rich media (YPD), but exhibited severe growth defects under various stress conditions (FIG. 15c), which may contribute to virulence defects observed in the arg5,6pΔ and met3Δ mutants.

Example 6
Retrograde Vacuole Trafficking Affecting Pathogenicity of C. neoformans

A notable biological function unknown as a cause of the pathogenicity of C. neoformans is retrograde vacuole trafficking. It was already reported that, in C. neoformans, the ESCRT complex-mediated vacuolar sorting process is involved in virulence, because some virulence factors such as capsule and melanin need to be secreted extracellularly (Godinho, R. M. et al. The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex. Scientific reports 4, 6198, doi:10.1038/srep06198, 2014; Hu, G. et al. Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence. Infect Immun 81, 292-302, doi:10.1128/IAI.01037-12, 2013). However, the role of endosome-to-Golgi retrograde transport in the virulence of C. neoformans has not previously been characterized. Here the present inventors discovered that deletion of CNAG_02680, encoding a VPS15 orthologue involved in the vacuolar sorting process, significantly reduced virulence (FIG. 16a). This result is consistent with the finding that mutation of VPS15 also attenuates virulence of C. albicans (Liu, Y. et al. Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans. Eukaryot Cell 13, 279-287, doi:10.1128/EC.00295-13, 2014), strongly suggesting that the role of Vps15 in fungal virulence is evolutionarily conserved. In S. cerevisiae, Vps15 constitutes the vacuolar protein sorting complex (Vps15/30/34/38) that mediates endosome-to-Golgi retrograde protein trafficking (Stack, J. H., Horazdovsky, B. & Emr, S. D. Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Annu Rev Cell Dev Biol 11, 1-33, doi:10.1146/annurev.cb.11.110195.000245, 1995).

To examine the role of Vps15 in vacuolar sorting and retrograde protein trafficking, the vacuolar morphology of the vps15Δ mutant was examined comparatively with that of the wild-type strain. Similar to the vps15Δ null mutant in C. albicans, the C. neoformans vps15Δ mutant also exhibited highly enlarged vacuole morphology (FIG. 16b). It is known that defects in retrograde vacuole trafficking can cause extracellular secretion of an endoplasmic reticulum (ER)-resident chaperon protein, Kar2 (Liu, Y. et al. Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans. Eukaryot Cell 13, 279-287, doi:10.1128/EC.00295-13 (2014)). Supporting this, the present inventors found that vps15Δ mutants were highly susceptible to ER stress agents, such as dithiothreitol (DTT) and tunicamycin (TM) (FIG. 16c). Growth defects at 37° C. strongly attenuated the virulence and infectivity of the vps15Δ mutant (FIG. 16d). This may result from increased cell wall and membrane instability by the vps15Δ mutant. In C. albicans, impaired retrograde trafficking in the vps15Δ mutant also causes cell wall stress, activating the calcineurin signaling pathway by transcriptionally up-regulating CRZ1, CHR1 and UTR2 (Liu, Y. et al. Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans. Eukaryot Cell 13, 279-287, doi:10.1128/EC.00295-13, 2014). In C. neoformans, however, the present inventors did not observe such activation of signaling components in the calcineurin pathway of the vps15Δ mutant (FIG. 16e). Expression levels of CHR1, CRZ1 and UTR2 in the vps15Δ mutant were equivalent to those in the wild-type strain. In C. neoformans, cell wall integrity is also governed by the unfolded protein response (UPR) pathway (Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hx11, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177 (2011)). Previously, the present inventors demonstrated that activation of the UPR pathway through Ire1 kinase results in an unconventional splicing event in HXL1 mRNA, which subsequently controls an ER stress response (Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hx11, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177 (2011)). Indeed, the present inventors found that cells with the VPS15 deletion were more enriched with spliced HXL1 mRNA (HXL1s) under basal conditions than the wild-type strain, indicating that the UPR pathway may be activated instead of the calcineurin pathway in C. neoformans when retrograde vacuole trafficking is perturbed.

Example 7
Novel Virulence- and Infectivity-Regulating Kinases in C. neoformans

Eight of the 60 pathogenicity-related kinases did not appear to have apparent orthologs in model yeasts, and thus were named virulence-regulating kinase (Vrk1) or infectivity-regulating kinase 1-7 (Irk1-7). Particularly, the present inventors paid attention to Vrk1 (CNAG_06161) (FIG. 17) because its deletion reduced the virulence of C. neoformans in the insect host model (FIGS. 6 to 8) and diminished infectivity in the murine host model (FIGS. 9 and 10). A yeast ortholog closest thereto is Fab1 (score: 140.9, e-value: 3.2E-34), but the closest Fab1 ortholog in C. neoformans is CNAG_01209 (score: 349.7, e-value: 0.0). Surprisingly, deletion of VRK1 increased cellular resistance to hydrogen peroxide and capsule production (FIGS. 17a and 17b). In addition, it increased cellular resistance to 5-flucytosine and increased fludioxonil susceptibility (FIG. 17a). Based on the kinase mutant phenome clustering data of the present inventors, Vrk1 was not clearly grouped with other kinases.

To gain further insight into the regulatory mechanism of Vrk1, the present inventors performed comparative phosphoproteomic analysis of the wild-type and vrk1A strains to identify Vrk1-specific phospho-target proteins. TiO₂enrichment-based phosphoproteomic analysis showed eight potential Vrk1 substrates: CNAG_04190 (TOP1, Topoisomerase I), CNAG_01744 (GPP2, a DL-glycerol-3-phosphate phosphatase), CNAG_05661 (POB3, heterodimeric FACT complex subunit), CNAG_01972, CNAG_07381, CNAG_00055, CNAG_02943 (SLRU, a phosphatidylinositol-4,5-bisphosphate binding protein), and CNAG_07878 (NOC2, a nucleolar complex associated protein). CNAG_01972, 07381 and 00055 did not have clear fungal orthologues. Although it is not clear whether candidate proteins are phosphorylated by Vrk1 directly or indirectly, it was found that five candidate proteins (TOP1, GPP2, POB3, CNAG_01972 and CNAG_07381) in the vrk1Δ mutant were damaged (FIG. 17c), suggesting that these proteins can be phosphorylated directly by Vrk1. To gain further insight into Vrk1-dependent functional networks, the present inventors used CryptoNet to search for any proteins that were functionally linked to the Vrk1-regulated target proteins and Vrk1 itself, and constructed the functional networks for those proteins. CNAG_01972 and 00055 did not have meaningful connections with any known proteins. Among a variety of potential biological functions connected to Vrk1 and its substrates, rRNA processing were mostly over-represented, suggesting that Vrk1 could be involved in the ribosome biosynthesis and trafficking, either directly or indirectly (FIG. 17d).

Example 8
Analysis of Antifungal Drug Resistance-Related Kinases in C. neoformans

Based on antifungal drug analysis using the kinas mutant library, 43, 38 and 42 kinases showed increased or reduced susceptibility to amphotericin B (a polyene), fluconazole (an azole) and flucytosine (a nucleotide analog), respectively, which are antifungal drugs used in clinical applications (Table 4). For kinases with deletions that increase susceptibility to these drugs, the present inventors discovered 39 kinases (to amphotericin B), 24 kinases (to fluconazole) and 28 kinases (to flucytosine), which can be developed as targets of drugs in combination therapy.

TABLE 4

Analysis of Antifungal Drug Resistance-Related Kinases in C. neoformans

Antifungal agents
Kinase mutant showingincreased resistance
Kinase mutants showingincreased susceptibility

Polyene(Amphotericin B)
HRK1/NPH1, SPS1,

YPK1, VPS15, CBK1, HOG1, SSK2, PBS2,

SWE102, TCO4

ARG5.6, GAL83, SNF1, MKK2, MPK1,

BUD32, CKA1, IPK1, IRE1,

CDC7, KIC1, PKA1, CRK1,

BCK1, TCO2, IRK5, IGI1,

GSK3, UTR1, MEC1, MET3, PAN3, MPS1,

PKH201, PIK1, HRK1, KIC102,

ALK1, TLK1, ARK1, IRK3, KIN1, POS5

Azole(Fluconazole)

GAL83, PAN3, ALK1, TCO1,

YPK1, VPS15, CBK1, MKK2, MPK1, IPK1,

STE11, TCO2, SCH9, SSK2,

IRE1, BCK1, IGI1, GSK3, UTR1, PIK1,

PBS2, HOG1, BUD32, PKA1,
HRK1/NPH1, CDC7, HRK1, PSK201, MPK2,

CHK1, YAK1
RAD53, ARG5.6, KIC1, KIC102, SPS1,

IRK6, MAK322

5-flucyotosine

BCK1, PSK201, ARG5.6, GAL83,

YPK1, VPS15, GSK3, UTR1, HRK1/NPH1,

TCO2, SNF1, IRK5, PKH201,

SCH9, BUD32, CKA1, MEC1, FBP26, CBK1,

VRK1, CKI1, TCO5, STE7, IGI1,

HOG1, IPK1, IRE1, SSK2, PBS2,

URK1

MET3, CDC7, KIC1, PAN3, TCO1, PKA1,

CHK1, CRK1, MPS1, CDC2801, TCO6, BUB1

* Underlined kinases are those identified for the first time in the present invention.

Example 9
Growth and Chemical Susceptibility Test

To analyze the growth and chemical susceptibility of the kinase mutant library, C. neoformans cells grown overnight at 30° C. were serially diluted tenfold (1 to 10⁴) and spotted on YPD media containing the indicated concentrations of chemical agents as follows: 2M sorbitol for osmotic stress and 1-1.5M NaCl and KCl for cation/salt stresses under either glucose-rich (YPD) or glucose-starved (YPD without dextrose; YP) conditions; hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (an organic peroxide), menadione (a superoxide anion generator), diamide (a thiol-specific oxidant) for oxidative stress; cadmium sulphate (CdSO₄) for toxic heavy metal stress; methyl methanesulphonate and hydroxyurea for genotoxic stress; sodium dodecyl sulphate (SDS) for membrane destabilizing stress; calcofluor white and Congo red for cell wall destabilizing stress; tunicamycin (TM) and dithiothreitol (DTT) for ER stress and reducing stress; fludioxonil, fluconazole, amphotericin B, flucytosine for antifungal drug susceptibility. Cells were incubated at 30° C. and photographed post-treatment from day 2 to day 5. To test the growth rate of each mutant at distinct temperatures, YPD plates spotted with serially diluted cells were incubated at 25° C., 30° C., 37° C., and 39° C., and photographed after 2 to 4 days.

Example 10
Mating Assay

To examine the mating efficiency of each kinase mutant, the MATα kinase mutant in Table 1 above was co-cultured with serotype A MATα wild-type strain KN99a as a unilateral mating partner. Each kinase mutant MATα strain and MATα WT KN99a strain (obtained from the Joeseph Heitman Laboratory at Duke University in USA) was cultured in YPD medium at 30° C. for 16 hours, pelleted, washed and resuspended in distilled water. The resuspended a and a cells were mixed at equal concentrations (10⁷cells per ml) and 5 μl of the mixture was spotted on V8 mating media (pH 5). The mating plate was incubated at room temperature in the dark for 7 to 14 days and was observed weekly.

Example 11
In Vitro Virulence-Factor Production Assay

For virulence-factor production assay, capsule production, melanin production and urease production were examined for each kinase mutant. Capsule production was examined qualitatively by India ink staining (Bahn, Y. S., Hicks, J. K., Giles, S. S., Cox, G. M. & Heitman, J. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade. Eukaryot. Cell 3, 1476-1491 (2004). To measure the capsule production levels quantitatively by Cryptocrit, each kinase mutant was grown overnight in YPD medium at 30° C., spotted onto Dulbecco's Modified Eagle's (DME) solid medium, and then incubated at 37° C. for 2 days for capsule induction. The cells were scraped, washed with phosphate buffered saline (PBS), fixed with 10% of formalin solution, and washed again with PBS. The cell concentration was adjusted to 3×10⁸cells per ml for each mutant and 50 μl of the cell suspension was injected into microhaematocrit capillary tubes (Kimble Chase) in triplicates. All capillary tubes were placed in an upright vertical position for 3 days. The packed cell volume ratio was measured by calculating the ratio of the lengths of the packed cell phase to the total phase (cells plus liquid phases). The relative packed cell volume ratio was calculated by normalizing the packed cell volume ratio of each mutant with that of the wild-type strain. Statistical differences in relative packed cell volume ratios were determined by one-way analysis of variance tests employing the Bonferroni correction method by using the Prism 6 (GraphPad) software.

To examine melanin production, each kinase mutant was grown overnight in YPD medium at 30° C.; 5 μl of each culture was spotted on Niger seed media containing 0.1% or 0.2% glucose. The Niger seed plates were incubated at 37° C. and photographed after 3-4 days. For kinase mutants showing growth defects at 37° C., the melanin and capsule production were assessed at 30° C. To examine urease production, each kinase mutant was grown in YPD medium at 30° C. overnight, washed with distilled water, and then an equal number of cells (5×10⁴) was spotted onto Christensen's agar media. The plates were incubated for 2-3 days at 30° C. and photographed.

Example 12
Insect-Based In Vivo Virulence Assay

For each tested C. neoformans strain, the present inventors randomly selected a group of 15 Galleria mellonella caterpillars in the final instar larval stage with a body weight of 200-300 mg, which arrived within 7 days from the day of shipment (Vanderhorst Inc. St Marys, Ohio, USA). Each C. neoformans strain was grown overnight at 30° C. in YPD liquid medium, washed three times with PBS, pelleted and resuspended in PBS at equal concentrations (10⁶cells per ml). A total of 4,000 C. neoformans cells in a 4-μl volume per larva was inoculated through the second to last prolegs by using a 100-μl Hamilton syringe equipped with a 10 μl-size needle and a repeating dispenser (PB600-1, Hamilton). The same volume (4 μl) of PBS was injected as a non-infectious control. Infected larvae were placed in petri dishes in a humidified chamber, incubated at 37° C., and monitored daily. Larvae were considered dead when they showed a lack of movement upon touching. Larvae that pupated during experiments were censored for statistical analysis. Survival curves were illustrated using the Prism 6 software (GraphPad). The Log-rank (Mantel-Cox) test was used for statistical analysis. The present inventors examined two independent mutant strains for each kinase mutant. For kinase mutants with single strains, the experiment was performed in duplicate.

Example 13
Signature-Tagged Mutagenesis (STM)-Based Murine Infectivity Assay

For the high-throughput murine infectivity test, a group of kinase mutant strains with the NAT selection marker containing 45 unique signature-tags (a total of four groups) was pooled. The ste50Δ and hx11Δ mutants were used as virulent and avirulent control strains, respectively (Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hx11, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177 (2011), Jung, K. W., Kim, S. Y., Okagaki, L. H., Nielsen, K. & Bahn, Y. S. Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet. Biol. 48, 154-165, doi:S1087-1845(10)00191-X [pii] 10.1016/j.fgb.2010.10.006 (2011)). Each group of the kinase mutant library was grown at 30° C. in YPD medium for 16 hours separately and washed three times with PBS. The concentration of each mutant was adjusted to 10⁷cells per ml and 50 μl of each sample was pooled into a tube. For preparation of the input genomic DNA of each kinase mutant pool, 200 μl of the mutant pool was spread on YPD plate, incubated at 30° C. for 2 days, and then scraped. For preparation of the output genomic DNA samples, 50 μl of the mutant pool (5×10⁵cells per mouse) was infected into seven-week-old female A/J mice (Jackson Laboratory) through intranasal inhalation. The infected mice were sacrificed with an overdose of Avertin 15 days post-infection, their infected lungs were recovered and homogenized in 4 ml PBS, spread onto the YPD plates containing 100 μg/ml of chloramphenicol, incubated at 30° C. for 2 days, and then scraped. Total genomic DNA was extracted from scraped input and output cells by the CTAB method (Jung, K. W., Kim, S. Y., Okagaki, L. H., Nielsen, K. & Bahn, Y. S. Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet. Biol. 48, 154-165, doi:S1087-1845(10)00191-X [pii]10.1016/j.fgb.2010.10.006 (2011)). Quantitative PCR was performed with the tag-specific primers listed in Tables 2 and 3 above by using MyiQ2 Real-Time PCR detection system (Bio-Rad). The STM score was calculated (Jung, K. W. et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757 (2015)). To determine the STM score, relative changes in genomic DNA amounts were calculated by the 2^−ΔΔCTmethod (Choi, J. et al. CFGP 2.0: a versatile web-based platform for supporting comparative and evolutionary genomics of fungi and Oomycetes. Nucleic Acids Res 41, D714-719, doi:10.1093/nar/gks1163 (2013)). The mean fold changes in input verses output samples were calculated in Log score (Log₂2^{(Ct, Target-Ct, Actin) output-(Ct, Target-Ct, Actin) input}).

Example 14
Vacuole Staining

To visualize vacuole morphology, the wild-type H99S strain and vsp15Δ strains (YSB1500 and YSB1501) (obtained from the Joeseph Heitman Laboratory at Duke University in USA) were cultured in liquid YPD medium at 30° C. for 16hours. FM4-64 dye (Life Technologies) was added to each culture at a final concentration of 10 μM and further incubated at 30° C. for 30 minutes. The cells were pelleted by centrifugation, resuspended with fresh liquid YPD medium, and further incubated at 30° C. for 30 minutes. The cells were pelleted again, washed three times with PBS, and then resuspended in 1 ml of PBS. On the glass slide, 10 ml of the cells and 10 ml of mounting solution (Biomeda) were mixed and spotted. The glass slides were observed by confocal microscope (Olympus BX51 microscope).

Example 15
TiO₂Enrichment-Based Phosphoproteomics

To identify the phosphorylated targets of Vrk1 on a genome-wide scale, the H99S and vrk1Δ mutant strains were incubated in YPD liquid medium at 30° C. for 16 hours, sub-cultured into 1 liter of fresh YPD liquid medium, and further incubated at 30° C. until it approximately reached an optical density at 600 nm (OD₆₀₀) of 0.9. Each whole-cell lysate was prepared with lysis buffer (Calbiochem) containing 50 mM Tris-Cl (pH 7.5), 1% sodium deoxycholate, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 50 mM sodium fluoride (NaF), 0.1% sodium dodecyl sulphate, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 2.5× protease inhibitor cocktail solution (Merck Millipore). The protein concentration of each cell lysate was measured using a Pierce BCA protein kit (Life Technologies). Sulfhydryl bonds between cysteine residues in protein lysates were reduced by incubating 10 mg of total protein lysate with 10 mM DTT at room temperature for 1 hour and then alkylated with 50 mM iodoacetamide in the dark at room temperature for 1 hour. These samples were treated again with 40 mM DTT at room temperature for 30 min and then digested using trypsin (Sequencing grade trypsin, Promega) at an enzyme: substrate ratio of 1:50 (w/w) with overnight incubation at 37° C. The trypsin-digested protein lysates were then purified with Sep-Pak C18 columns (Waters Corporation, Milford, Mass.), lyophilized and stored at −80° C. Phosphopeptides were enriched using TiO₂Mag Sepharose beads (GE Healthcare) and then lyophilized for LC-MS/MS. Mass spectrometric analyses were performed using a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, MA, USA) equipped with Dionex U 3000 RSLC nano high-performance liquid chromatography system, a nano-electrospray ionization source and fitted with a fused silica emitter tip (New Objective, Wobum, Mass.). All phosphopeptide samples were reconstituted in solution A (water/acetonitrile (98:2, v/v), 0.1% formic acid), and then injected into an LC-nano ESI-MS/MS system. Samples were first trapped on a Acclaim PepMap 100 trap column (100 μm i.d.×2 cm, nanoViper C₁₈, 5 μm particle size, 100 Å pore size, Thermo Scientific) and washed for 6 min with 98% solution A at a flow rate of 4 μl/min, and then separated on an Acclaim PepMap 100 capillary column (75 μm i.d.×15 cm, nanoViper C₁₈, 3 μm particle size, 100 Å pore size, Thermo Scientific) at a flow rate of 400 nl/min. Peptides were analyzed with a gradient of 2 to 35% solution B (water/acetonitrile (2:98, v/v), 0.1% formic acid) over 90 min, 35 to 90% over 10 min, followed by 90% for 5 min, and finally 5% for 15 min. The resulting peptides were electrosprayed through a coated silica tip (PicoTip emitter, New Objective, MA, USA) at an ion spray voltage of 2,000 eV. To assign peptides, MS/MS spectra were searched against the C. neoformans var. grubii H99S protein database (http://www.uniprot.org) using the SEQUEST search algorithms through the Proteome Discoverer platform (version 1.4, Thermo Scientific). The following search parameters were applied: cysteine carbamidomethylation as fixed modifications, methionine oxidation and serine/threonine/tyrosine phosphorylation as variable modifications. Two missed trypsin cleavages were allowed to identify the peptide. Peptide identification was filtered by a 1% false discovery rate cut-off. Spectral counts were used to estimate relative phosphopeptide abundance between the wild-type and mutant strains. The Student's t-test was used to assess the statistically significant difference between the samples.

Example 16
ER Stress Assay

To monitor the ER stress-mediated UPR induction, the H99S and vps15Δ mutant strains were incubated in YPD at 30° C. for 16 hours, sub-cultured with fresh YPD liquid medium, and then further incubated at 30° C. until they reached the early-logarithmic phase (OD₆₀₀=0.6). The cells were treated with 0.3 μg/ml tunicamycin (TM) for 1 hour. The cell pellets were immediately frozen with liquid nitrogen and then lyophilized. Total RNAs were extracted using easy-BLUE (Total RNA Extraction Kit, Intron Biotechnology) and subsequently cDNA was synthesized using an MMLV reverse transcriptase (Invitrogen). HXL1 splicing patterns (UPR-induced spliced foam of HXL1 (HXL1S) and unspliced foam of HXL1 (HXL1U)) were analyzed by PCR using cDNA samples of each strain and primers (B5251 and B5252) (Table 3).

Example 17
Expression Analysis

To measure the expression level of ERG11, the H99S strain and bud32Δ mutants were incubated in liquid YPD medium at 30° C. for 16 hours and sub-cultured with fresh liquid YPD medium. When the cells reach the early-logarithmic phase (OD600=0.6), the culture was divided into two samples: one was treated with fluconazole (FCZ) for 90 minutes and the other was not treated. The cell pellets were immediately frozen with liquid nitrogen and then lyophilized. Total RNA was extracted and northern blot analysis was performed with the total RNA samples for each strain as previously reported (Jung, K. W., Kim, S. Y., Okagaki, L. H., Nielsen, K. & Bahn, Y. S. Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet. Biol. 48, 154-165, doi:S1087-1845(10)00191-X [pii]10.1016/j.fgb.2010.10.006 (2011)). For quantitative reverse transcription-PCR (qRT-PCR) analysis of genes involved in the calcineurin pathway, the H99S strain and vps15Δ mutants were incubated in liquid YPD medium at 30° C. for 16hours and were sub-cultured in fresh liquid YPD medium until they reached to the early-logarithmic phase (OD₆₀₀=0.8). The cells were then pelleted by centrifugation, immediately frozen with liquid nitrogen, and lyophilized. After total RNA was extracted, cDNA was synthesized using RTase (Thermo Scientific). CNA1, CNB1, CRZ1, UTR2 and ACT1-specific primer pairs (B7030 and B7031, B7032 and B7033, B7034 and B7035, B7036 and B7037, B679 and B680, respectively) (Table 3) were used for qRT-PCR.

Example 18
Construction of FPK1 Overexpression Strains

To construct the FPK1 overexpression strain, the native promoter of FPK1 was replaced with histone H3 promoter using an amplified homologous recombination cassette (FIG. 5a). In the first round of PCR, primer pairs L1/OEL2 and OER1/PO were used for amplification of the 5′-flaking region and 5′-coding region of FPK1, respectively. The NEO-H3 promoter region was amplified with the primer pair B4017/B4018. For second-round PCR for the 5′ or 3′ region of the PH3:FPK1 cassette, the first-round PCR product was overlap-amplified by DJ-PCR with the primer pair L1/GSL or GSR/PO (primers in Tables 2 and 3 above). Then, the PH3:FPK1 cassettes were introduced into the wild-type strain H99S (obtained from the Joeseph Heitman Laboratory at Duke University in USA) and the ypk1A mutant (YSB1736) by biolistic transformation. Stable transformants selected on YPD medium containing G418 were screened by diagnostic PCR with a primer pair (SO/B79). The correct genotype was verified by Southern blotting using a specific probe amplified by PCR with primers L1/PO. Overexpression of FPK1 was verified using a specific Northern blot probe amplified by PCR with primers NP1 and PO (FIGS. 5b and 5c).

Example 19
Kinase Phenome Clustering

In vitro phenotypic traits of each kinase mutant were scored with the following qualitative scale: −3 (strongly sensitive or defective), −2 (moderately sensitive or defective), −1 (weakly sensitive or defective), 0 (wild-type-like), +1 (weakly resistant or enhanced), +2 (moderately resistant or enhanced), and +3 (strongly resistant or enhanced). The excel file containing the phenotype scores of each kinase mutant was uploaded by Gene-E software (http://www.broadinstitute.org/cancer/software/GENE-E/) and then kinase phenome clustering was drawn using one minus Pearson correlation.

Example 20

Cryptococcus Kinome Web-Database

For public access to the phenome and genome data for the C. neoformans kinase mutant library constructed by the present inventors, the Cryptococcus Kinase Phenome Database was developed (http://kinase.cryptococcus.org/). Genome sequences of C. neoformans var. grubii H99 were downloaded from the Broad Institute (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html), and incorporated into the standardized genome data warehouse in the Comparative Fungal Genomics Platform database (CFGP 2.0; http://cfgp.snu.ac.kr/) (Choi, J. et al. CFGP 2.0: a versatile web-based platform for supporting comparative and evolutionary genomics of fungi and Oomycetes. Nucleic Acids Res 41, D714-719, doi:10.1093/nar/gks1163 (2013)). Classification of protein kinases was performed by using the hidden Markov model-based sequence profiles of SUPERFAMILY (version 1.73) (Wilson, D. et al. SUPERFAMILY—sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res 37, D380-386, doi:10.1093/nar/gkn762 (2009)). A total of 64 family identifiers belonging to 38 superfamilies were used to predict putative kinases. In addition, the sequence profiles of Kinomer (version 1.0) (Martin, D. M., Miranda-Saavedra, D. & Barton, G. J. Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar/gkn834 (2009); Miranda-Saavedra, D. & Barton, G. J. Classification and functional annotation of eukaryotic protein kinases. Proteins 68, 893-914, doi:10.1002/prot.21444 (2007)) and the Microbial Kinome (Kannan, N., Taylor, S. S., Zhai, Y., Venter, J. C. & Manning, G. Structural and functional diversity of the microbial kinome. PLoS Biol 5, e17, doi:10.1371/journal.pbio.0050017 (2007)) were used to supplement the kinase prediction. Information from genome annotation of C. neoformans var. grubii H99 and protein domain predictions of InterProScan 62 was also adopted to capture the maximal extent of possible kinase-encoding genes. For each gene, results from the eight bioinformatics programs were also provided to suggest clues for gene annotations. In addition, results from SUPERFAMILY, Kinomer and Microbial Kinome were displayed for supporting robustness of the prediction. If a gene has an orthologue in C. neoformans var. neoformans JEC21, a link to the KEGG database was also provided. To browse genomic data in context to important biological features, the Seoul National University genome browser (SNUGB; http://genomebrowser.snu.ac.kr/) (Jung, K. et al. SNUGB: a versatile genome browser supporting comparative and functional fungal genomics. BMC Genomics 9, 586, doi:10.1186/1471-2164-9-586 (2008)) was integrated into the Cryptococcus kinase phenome database. In kinase browser, a direct link to the SNUGB module was provided for each gene. The Cryptococcus kinase phenome database was developed by using MySQL 5.0.81 (source code distribution) for database management and PHP 5.2.6 for web interfaces. The web-based user interface is served through the Apache 2.2.9 web server.

INDUSTRIAL APPLICABILITY

The present invention relates to kinases making it possible to effectively screen novel antifungal agent candidates. The use of the kinases according to the present invention makes it possible to effectively screen novel antifungal agent candidates. In addition, the use of an antifungal pharmaceutical composition comprising an agent (antagonist or inhibitor) for the kinase according to the present invention can effectively prevent, treatment and/or diagnose fungal infection.

NOVEL KINASE FOR TREATING AND PREVENTING FUNGAL INFECTIONS, AND USE THEREOF

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