EP1784507A2 - Technique d'analyse pour genes de levures industrielles - Google Patents

Technique d'analyse pour genes de levures industrielles

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Publication number
EP1784507A2
EP1784507A2 EP05783945A EP05783945A EP1784507A2 EP 1784507 A2 EP1784507 A2 EP 1784507A2 EP 05783945 A EP05783945 A EP 05783945A EP 05783945 A EP05783945 A EP 05783945A EP 1784507 A2 EP1784507 A2 EP 1784507A2
Authority
EP
European Patent Office
Prior art keywords
nucleotide sequence
seq
dna
gene
sequence represented
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP05783945A
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German (de)
English (en)
Inventor
Toshihiko Ashikari
Yoshihiro Nakao
Norihisa Nakamura
Yukiko Kodama
Tomoko Fujimura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suntory Holdings Ltd
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Suntory Ltd
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Publication date
Application filed by Suntory Ltd filed Critical Suntory Ltd
Publication of EP1784507A2 publication Critical patent/EP1784507A2/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method analyzing genes of an industrial yeast used for the production of an alcoholic beverage such as beer or sake, a fuel alcohol, etc. and particularly for genes of brewing yeast used for the production of an alcoholic beverage. More particularly, it relates to a method where, in the production of an alcoholic beverage, DNA sequence information of brewing yeast is compiled in a database so that the gene which involves in increase in productivity and/or improvement in flavor such as stabilization, reinforcement, etc. of the flavor is selected; a method for breeding yeast suitable for the brewing in which expression of a gene is controlled, such as yeast in which the selected gene is disrupted or yeast in which the gene is overexpressed; and a method for the production of an alcoholic beverage using the bred yeast.
  • the most consumed alcoholic beverage in the world is beer and the amount of beer produced in the world in 2001 was about 140,000,000 kL.
  • Type of beer is roughly classified into three depending upon types of yeast and fermentation methods. The three types are, naturally fermented beer where fermentation is carried out utilizing yeast and microorganisms inhabiting in breweries; ale-type beer where fermentation is carried out using a top fermenting yeast belonging to Saccharomyces cerevisiae (hereinafter, abbreviated as S.
  • lager-type beer where fermentation is carried out using a bottom fermenting yeast belonging to Saccharomyces pastorianus at the temperature of 6 to 15°C and then subjected to a low-temperature aging.
  • a bottom fermenting yeast belonging to Saccharomyces pastorianus at the temperature of 6 to 15°C and then subjected to a low-temperature aging.
  • the bottom fermenting yeast that is used for brewing of the lager-type beer has been most widely used in beer brewing.
  • a technique for selecting a desirable strain has widely been used rather than actual breeding.
  • Beer brewing per se has been performed since well before the discovery of microorganisms by Pasteur.
  • a method of selecting a more suitable strain of beer yeast from many strains of yeast used in the beer brewery has been traditionally carried out while there have been few cases where beer yeast with good traits is positively bred.
  • a positive breeding method there is a method where artificial mutagenesis by chemicals or radioactive rays is used.
  • brewing yeast particularly a bottom fermenting yeast which is widely used in beer brewing, is in many cases a polyploid.
  • Non-Patent Document 1 a breeding where mutation or cross ⁇ breeding is carried out by using spores isolated from bottom fermenting yeast (c.f.., for example, Non-Patent Document 1) has been tried.
  • the bottom fermenting yeast is a polyploid, and has complicated chromosome structure. Therefore, isolation of spores having a proliferation ability is difficult, and moreover, it is almost impossible to obtain a strain with good traits from them.
  • transcriptome analysis has been conducted using DNA array where DNA fragments or nucleotide oligomers, each of which has a partial sequence of structural genes or internal regions of the chromosomes are fixed on solid support.
  • Olesen, et al. conducted a comprehensive genetic expression analysis of bottom fermenting yeast during the brewing using GeneFilters (manufactured by Research Genetics Co.)(c.f., for example, Non-Patent Document 3).
  • GeneFilters manufactured by Research Genetics Co.
  • Non-Patent Document 3 Non-Patent Document 3
  • Non-Patent Document 6 the whole genome sequences of more than 100 species of microorganisms have been determined (c.f., for example, Non-Patent Document 6) including S. cerevisiae, Escherichia coli (c.f., for example, Non-Patent Document 4) and Mycobacterium tuberculosis (c.f., for example, Non-Patent Document 5).
  • genes of these microorganisms are identified and function of an enormous number of genes have been predicted without conducting genetic, biochemical or molecular biological experiments.
  • industrial yeast such as brewing yeast which has high ploidy and a complicated chromosome structure, and thus an assembly (an operation for connecting the DNA sequences) is presumed to be difficult. Therefore, the genome sequence of bottom fermenting yeast which contains two different types of genome (c.f., for example, Non-Patent Document 7) has not been reported yet.
  • Sulfite is known as a compound which has anti-oxidative activity, and has been widely used as an antioxidant in the fields of food, beverage and pharmaceuticals, and also in an alcoholic beverage.
  • Sulfite plays an important role for the preservation of its quality.
  • the quality preservation period becomes long in accordance with the increase in concentration of sulfite contained in the product.
  • the yeast used in brewing produces hydrogen sulfide by the reduction of sulfate in the medium in order to synthesize sulfur-containing metabolites such as sulfur- containing amino acids.
  • Sulfite is an intermediate metabolite of this pathway. If sulfite is efficiently excreted outside of the cells during fermentation period, it is possible to increase the amount of sulfite both in the wort and in the product.
  • Sulfite (SO 2 ) is an intermediate product of sulfur-containing amino acid and vitamin synthesis and is produced via a pathway of sulfate ion (SO 4 2" ) ⁇ APS (adenyl sulfate) ⁇ PAPS (phosphoadenylyl sulfate) ⁇ sulfite ion (SO 3 2" ) where the sulfate ion is incorporated from outside of the cells.
  • Non-Patent Document 1 C. Gjermansen: "Construction of a hybrid brewing strain of Saccharomyces carlsbergensis by mating of meiotic segregants", Carlsberg Res. Commun., volume 46, pages 1 to 11 (1981).
  • Non-Patent Document 2 A. Goffeau, et al.: "The
  • Non-Patent Document 3 K. Olesen, et al.: "The dynamics of the Saccharomyces carlsbergensis brewing yeast transcriptome during a production-scale lager beer fermentation", FEMS Yeast Research, volume 2, pages 563 to 573 (2000).
  • Non-Patent Document 4 F. R. Blattner, et al.: "The Complete Genome Sequence of Escherichia coli K-12", Science, volume 277, pages 1453-1462 (1997).
  • Non-Patent Document 5 S. T. Cole, et al.; "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence”. Nature, volume 393, pages 537-544 (1998).
  • Non-Patent Document 6 The National Center for Biotechnology Information, http;//www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html.
  • Non-Patent Document 7 Y. Tamai et al. : "Co-existence of two types of chromosome in the fermenting yeast, Sacchaomyces cerevisiae”. Yeast, volume 10, pages 923-933 (1998) .
  • Non-Patent Document 8 C. Korch, et al.: Proc. Eur. Brew. Conv. Congress, Lisbon, pages 201-208 (1991).
  • Non-Patent Document 9 J. Hansen, et al.: "Inactivation of MET 10 in brewer's yeast specifically increases SO 2 formation during beer production”. Nature Biotech., volume 14, pages 1587-1591 (1996).
  • Non-Patent Document 10 T. Sijen, et al.:
  • Non-Patent Document 11 N. Goto, et al.: "SSUl-R, a sulphite resistance gene of wine yeast, is an allele of SSU 1 with a different upstream sequence", J. Ferment. Bioeng., volume 86, pages 427-433 (1998).
  • Non-Patent Document 12 D. Avram, et al. : "SSU 1 encodes a plasma membrane protein with a central role in a network of proteins conferring sulfite tolerance in Saccharomyces cerevisiae” , J. Bacteriol., volume 179, pages 5971-5974 (1997).
  • Non-Patent Document 13 H. Park, et al.; "SSU 1 mediates sulphite efflux in Saccharomyces cerevisiae”. Yeast, volume 16, pages 881-888 (2000).
  • An object of the present invention is to provide a method of analyzing and selecting genes relating to the desired brewing characters, which is achieved in such a manner that a database compiling the genome sequence (hereinafter, may be abbreviated as genomic DB) of industrial yeast, particularly brewing yeast used for an alcoholic beverage such as beer, is prepared; gene that the brewing yeast possesses is selected from the database; functional analysis of the gene may be carried out by disruption or overexpression.
  • Another object of the present invention is to provide a DNA array which is useful for an analyzing method of genes of an industrial yeast. Further object is to provide a breeding method of the yeast showing the brewing character which the said gene is relating to and also a method of producing an alcohol or an alcoholic beverage where productivity and quality are improved using the said yeast. Still another object is to provide genes mentioned above and peptides encoded by the said genes.
  • bottom fermenting yeast is an allopolyploid which is composed of at least two kinds of genomes.
  • One of the genomes is thought to be a genome derived from S. cerevisiae of which the whole genome sequence has been clarified, while the source of another genome(s) has not been clarified yet.
  • the present inventors have analyzed the genome sequence of the bottom fermenting yeast in order to find unidentified genes displaying essential functions for excellent brewing. The amino acid sequences of the bottom fermenting yeast were then compared with those registered in the genomic DB for S. cerevisiae, and functions of proteins encoded by genes of the brewing yeast were estimated.
  • the genes of the bottom fermenting yeast are roughly classified into Sc type genes showing nearly 100% amino acid identity to those of S. cerevisiae and non-Sc type genes showing around 70 to 97% amino acid identify (this corresponds to around 60 to 94% identity in nucleotide level) .
  • the bottom fermenting yeast has a complicated chromosome structure consists of Sc-type chromosomes, non-Sc-type chromosomes and Sc/non-Sc-type chimera chromosomes. Structure of the whole chromosomes of the bottom fermenting yeast is shown in Fig. 1.
  • the present inventors have found such an unexpectedly complicated structure of chromosomes, and developed a screening method for the genes of bottom fermenting yeast. To be more specific, the inventors have achieved a method for analyzing gene of an industrial yeast comprising
  • (c-2) selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.
  • the method of the present invention comprises, (b) comparing the genome sequence with the genome sequence of Saccharomyces cerevisiae after the step (a) and before the step (c-1) or (c-2).
  • the analyzing method of the present invention comprises, (d) carrying out functional analysis of the selected gene, after the step (c-1) or (c-2), whereby the brewing character given to the yeast by the genes are identified.
  • the present inventors have repeatedly carried out intensive investigations on the basis of those findings and accomplished the present invention.
  • the present invention includes the following embodiments: 1. A method for analyzing gene of an industrial yeast comprising
  • ( ⁇ -2) selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.
  • Embodiment 3 which comprises screening genes involved in increase of productivity and/or improvement in flavor in the production of an alcohol or an alcoholic beverage by the functional analysis of the step (d) .
  • a gene of the industrial yeast encoding an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, which is obtained by the analyzing method according to any one of Embodiments 1-8.
  • a gene library comprising one or more of genes of the industrial yeast each of which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, which is obtained by the analyzing method according to any one of Embodiments 1-8. 11.
  • a DNA array comprising one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4) :
  • DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;
  • DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences; (3) DNA having a nucleotide sequence of any of SEQ ID NOs: 166490 to 167042, or DNA having a nucleotide sequence of any of SEQ ID NOs: 167043 to 173124;and (4) DNA having a nucleotide sequence of any of SEQ ID NOs: 173125 to 174603, or DNA having a nucleotide sequence of any of SEQ ID NOs:174604 to 190810.
  • the DNA array according to Embodiment 12 comprising DNAs wherein at least one DNA is selected from every group of (1) to (4) .
  • DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having identity of more than 97% to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;
  • DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having identity of more than 94% to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences, and optionally comprises DNAs of the following (5 1 ) and/or (6 1 ):
  • DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (1), or a nucleotide sequence complementary to the above nucleotide sequence; (8) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (2), or a nucleotide sequence complementary to the above nucleotide sequence; (9) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (3), or a nucleotide sequence complementary to the above nucleotide sequence; and (10) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence.
  • the DNA array according to Embodiment 17 which comprises, in addition to at least one group of DNAs selected from (1) to (4), DNAs selected from at least one of (7-1) to (10-1) :
  • a method for classifying an industrial yeast comprising
  • a method for screening a useful strain of industrial yeast comprising (a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of any one of Embodiments 12- 20; and
  • a method for screening a gene of an industrial yeast comprising (a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of any one of Embodiments 12-
  • a method for screening a gene of an industrial yeast comprising (a-1) hybridizing genomic DNA, cDNA or cRNA prepared from the industrial yeast to the DNA array of any one of Embodiments 12-20;
  • step (a-2) independently from the step (a-1), hybridizing genomic DNA, cDNA or cRNA prepared from another industrial yeast of any one of Embodiments 12-20;
  • step (b) selecting a gene wherein hybridization intensity thereof to the DNAs of any of (1) to (4) in the step (a-1) is significantly different from hybridization intensity thereof in the step (a-2).
  • a method for screening a gene of an industrial yeast comprising
  • step (a-2) independently from the step (a-1), hybridizing cDNA or cRNA prepared from the industrial yeast to the DNA array of any one of Embodiments 12-20, wherein the industrial yeast of (a-2) has been cultured in a different condition from the culture condition for the industrial yeast of (a- 1) ;
  • nucleic acid having a nucleotide sequence which hybridizes to the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:1 under a stringent condition, and encodes a polypeptide having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.
  • nucleic acid having the nucleotide sequence represented by SEQ ID N0:l (a) a nucleic acid having the nucleotide sequence represented by SEQ ID N0:l; and (b) a nucleic acid having a nucleotide sequence which hybridizes to the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID No.l under a stringent condition, and encodes a polypeptide having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.
  • a method for producing an alcohol or an alcoholic beverage using the transformant of Embodiment 34 or 35.
  • 39. A breeding method of yeast which is suitable for ⁇ the production of an alcohol or an alcoholic beverage, characterized in that, expression of the gene of Embodiment 9 or Embodiments 29-30, or the nucleic acid of Embodiments 31-32 is controlled.
  • a method for analyzing gene of an industrial yeast comprising
  • c-1 selecting a gene of the industrial yeast encoding an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae
  • c-2 selecting a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.
  • Fig. 1 shows total chromosome structures of bottom fermenting yeast.
  • a white bar represents an Sc type chromosome while a black bar represents a non-Sc type chromosome.
  • An ellipse represents a centromere.
  • Roman numerals show chromosome numbers for the corresponding S. cerevisiae.
  • a part marked out in black shows that ligation takes place at the region. For example, in nonScII-nonScIV, it is shown that nonScII and nonScIV are ligated at the part marked out in black (300 kb) .
  • Fig. 2 shows a distribution of identify of the DNA sequence at both ends of 3648 cosmids prepared from the genomic DNA of strain 34/70 with the genome sequence of S. cerevisiae.
  • the X-axis shows the identity to S. cerevisiae and, for example, 84% on the X-axis shows an identity of more than 82% and not more than 84%.
  • the Y-axis shows the numbers of cosmid end sequences showing the identity.
  • Fig. 3 shows a mapping example of cosmid and shotgun clones to genome sequence of S. cerevisiae. (1) and (2) show genes existing on Watson strand and Crick strand on the chromosome XVI of S. cerevisiae, respectively. (3)and (4) show Sc type and non-Sc type DNA fragments inserted in cosmid clones, respectively. (5) and (6) show Sc type and non-Sc type DNA fragments inserted in shotgun clones, respectively.
  • Fig. 4 shows a mapping example of contigs to the genome sequence of S. cerevisiae.
  • A is a schematic depiction of Chromosome XVI of S. cerevisiae.
  • B is a drawing where the parts of 857 to 886 kb of the Chromosome XVI of S. cerevisiae is enlarged.
  • Y-axis indicates % identity of contigs with S. cerevisiae genome sequence.
  • X- axis indicates position of contigs against S.cerevisiae genome sequence.
  • Contigs (solid lines) are connected with the forward-reverse links (dot lines) from the shotgun and cosmid reads, respectively.
  • FIG. 5 shows the result of DNA array-based comparative genomic hybridization.
  • the genomic DNA of strain 34/70 was hybridized to a DNA array (Affymetrix Gene Chip Yeast Genome S98 Array) and the signal of each ORF (open reading frame) was normalized to that of the haploid strain S288C and shown as Signal Log Ratio (2 n ).
  • Signal Log Ratios were lined following gene order in Chromosome XVI. The non-Sc type genes do not hybridize to this Sc type array, therefore, the points (indicated by arrows) where the Signal Log Ratios show vigorous changes were considered to be translocation sites.
  • Fig. 6 shows the structure of the Chromosome XVI of strain 34/70 deduced from DNA array and PCR analysis.
  • Fig. 7 shows the fermentation profiles of SSUl disruptants and parental strain (BH96). a) shows yeast growth(OD 600), b) shows the change of apparent extract (w/w %) and c) shows sulfite concentration(ppm) .
  • Fig. 8 shows the fermentation profiles of SSUl overexpressed strains and parental strain (BH225) .
  • a) shows yeast growth(OD 600)
  • b) shows the change of apparent extract (w/w %)
  • c) shows sulfite concentration(ppm) .
  • Fig. 9 shows the change of sulfite concentration during fermentation using MET14 overexpressed strains and parental strains (KN009F and FOY227).
  • Fig. 10 shows DNA sequences of ScSSUl and non-ScSSUl.
  • Fig. 11 shows DNA sequences of ScMET14 and non- ScMETl4.
  • Fig. 12 shows the fermentation profiles of strain 34/70. a) shows yeast growth(OD 600) and b) shows the change of apparent extract (w/w %) .
  • a method for analyzing gene of an industrial yeast provides a method for analyzing gene of an industrial yeast.
  • the present method comprises (c-1) selecting a gene of the industrial yeast encoding an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or
  • the method of the present invention omprises, (a) analyzing nucleotide sequence of the genome sequence of the industrial yeast, before the step (c-1) or (c-2).
  • the method of the present invention comprises, (b) comparing the genome sequence with the genome sequence of Saccharomyces cerevisiae, after the step (a), if any, and before the step (c-1) or (c-2).
  • the analyzing method of the present invention further comprises the step of d) carrying out functional analysis of the selected gene, after the step ( ⁇ -1) or (c-2).
  • yeast of genus Saccharomyces, etc. may be listed, and in the present invention beer yeasts such as Saccharomyces pastorianus Weihenstephan 34/70, NCYC456, NBRC 1951, NBRC 1952, NBRC 1953, NBRC 1954, etc. may be used.
  • whisky yeasts such as S. cerevisiae NCYC 90, etc.
  • wine yeasts such as Kyokai wine yeast No. 1, No. 3, No. 4, etc.
  • sake yeasts such as Kyokai sake yeast No. 7, No. 9, etc. and the like.
  • the industrial yeast of the present invention is a brewing yeast, more preferably, a beer yeast.
  • the industrial yeast of the present invention is a bottom fermenting yeast.
  • the bottom fermenting yeast means a yeast used for the lager beer production.
  • Some strains of beer yeasts, such as Saccharomyces pastorianus Weihenstephan 34/70, NCYC456 or the like are classified as bottom fermenting yeasts.
  • the strain used in the following Examples is one of the bottom fermenting yeasts commonly used for the lager beer production. The most of the bottom fermenting yeasts seem to have almost the same genome with that of strain 34/70.
  • yeast having an excellent brewing character when the gene obtained by the method of the present invention is used for carrying out an expression control in such a way that the gene is overexpressed in the yeast, and/or the gene is disrupted. Accordingly, the gene which is obtained by the method of the present invention, peptide which is encoded by the gene, a breeding method of an industrial yeast using the gene, yeast which is obtained by the breeding method, and a method for the production of an alcohol or an alcoholic beverage using the yeast are also within a scope of the present invention.
  • step (A) Analysis of nucleotide sequence of the genome sequence of industrial yeast If the genome sequence of a target industrial yeast is not determined, "anayzing" at the step (a) can maen determining the genome sequence of industrial yeast.
  • the "genome sequence” means the whole genome sequence or a part of the genome sequence. If the genome sequence of a target industrial yeast is determined, “anayzing" in step (a) can mean obtaining information regarding the genome sequence of a target industrial yeast from an appropriate source, for example, from a publicly available database. Determination of the genome sequence of an industrial yeast can be achieved by any conventional methods.
  • genomic DNA is prepared from yeast
  • shotgun library and cosmid library are prepared from those genomic DNA
  • DNA fragments to be used for determination of DNA sequence are prepared from those library clones
  • DNA sequence of the library DNA fragments is determined by a sequence reaction
  • sequences of those DNA fragments are assembled to reconstruct the genome sequence.
  • Both or either one of (b) shotgun library and (c) cosmid library may be prepared for this purpose.
  • Yeast cells for the preparation of genomic DNA are cultured by a common method.
  • a medium any of natural and synthetic media may be used so far as the medium contains carbon source, nitrogen source, inorganic salt, etc. which are able to be metabolized by the yeast, whereby cultivation of the microorganism can be efficiently carried out.
  • YPD medium 2% (w/w) glucose, 1% (w/w) yeast extract and 2% (w/w) polypeptone
  • incubation incubation by shaking at about 25 to 35°C through the night is recommended. After the cultivation, cells are recovered from the culture medium by centrifugation. The resulting cell pellet is washed with a washing solution.
  • Example of the washing solution is buffer A (50 mM sodium phosphate, 25 mM EDTA and 1% (v/v) ⁇ -mercaptoethanol; pH 7.5), etc.
  • Preparation of the genomic DNA from the washed cells may be carried out according to a common preparation method of genomic DNA where cell walls are lysed using Zymolyase and SDS; protein, etc. are removed using a phenol and phenol/chloroform solution; and genomic DNA is precipitated using ethanol or the like. To be more specific, the following method may be exemplified.
  • Cultivated cells are washed and resuspended in buffer A, then about 5 to 10 mg of Zymolyase IOOT (Seikagaku Kogyo) are added and the mixture is gently shaken at about 25 to 40°C for about 30 minutes to 2 hours.
  • buffer containing SDS such as buffer B (0.2 M Tris-HCl, 80 mM EDTA and 1% SDS; pH 9.5) is added thereto and the mixture is allowed to stand at about 60 to 7O 0 C for about 30 minutes to lyse the cells.
  • the cell lysate is cooled on ice, mixed with 5 M potassium acetate and allowed to stand on ice for about 60 minutes further.
  • the resulting solution is centrifuged (for example, at 5,000 g for 10 minutes at 15°C) to take supernatant.
  • the same volume of ethanol is added to the supernatant to precipitate DNA and the mixture is immediately centrifuged
  • Bisbenzimide is removed by extracting the recovered DNA solution with isopropanol which is saturated with cesium chloride solution, then 4- fold by volume of 0.3 M sodium acetate are added to the recovered aqueous layer followed by mixing and the DNA is precipitated by ethanol and recovered by centrifugation.
  • the recovered DNA is treated with RNase and extracted with phenol/chloroform and DNA is purified from the recovered aqueous layer by precipitation with ethanol again.
  • the precipitate recovered by centrifugation is washed with 70% (v/v) ethanol, subjected to natural drying and dissolved in a TE buffer to prepare the genomic DNA solution.
  • a TE buffer is added to the genomic DNA prepared in (a) and the genomic DNA is fragmented using Hydroshear (manufactured by GeneMachines) or the like. Terminal of the genome fragment is blunted using a DNA Blunting Kit (manufactured by Takara Shuzo) or the like, and fractionated by means of an agarose gel electrophoresis.
  • genome fragments of about 1.5 to 2.5 kb are excised from the gel and a buffer for the elution of DNA such as an MG-elution buffer (0.5 mol/L ammonium acetate, 10 mmol/L magnesium acetate, 1 mmol/L EDTA and 0.1% SDS) or the like is added to the gel followed by shaking at about 25 to 40 0 C through the night to elute DNA.
  • MG-elution buffer 0.5 mol/L ammonium acetate, 10 mmol/L magnesium acetate, 1 mmol/L EDTA and 0.1% SDS
  • the DNA eluate is treated with phenol/chloroform and precipitated with ethanol to give a genomic library insert.
  • DH5 ⁇ strain manufactured by Takara Shuzo
  • the transformants into which recombinant vector containing the genomic DNA fragments is inserted are selected on an appropriate selective medium.
  • an LB plate medium an LB medium (10 g/L of bactotryptone, 5 g/L of yeast extract and 10 g/L of sodium chloride; pH 7.0) which contains 1.6% of agar
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • the transformants are cultured in LB medium containing about 0.1 mg/mL of ampicillin through the night at about 30 to 37 0 C using a 384-well titer plate, a 50% aqueous solution of glycerol in the same volume as the LB is added thereto and the mixture is stirred to give a glycerol stock.
  • the glycerol stock can be preserved at about -80 0 C.
  • (c) Preparation of a cosmid library
  • the genomic DNA prepared in (a) is subjected to a partial digestion using an appropriate restriction enzyme such as Sau3AI (manufactured by Takara Shuzo) . It is possible to insert the DNA fragment digested by Sau3AI into a BamHI site of a cosmid vector such as Super Cosl vector (manufactured by Stratagene) .
  • the treatment with the restriction enzyme and the ligation may be carried out according to the protocol attached thereto.
  • the ligated product obtained by such a method is subjected to a packaging using, for example, Gigapack III Gold (manufactured by Stratagene) , and according to the manual for the experimental procedure attached thereto, it is introduced into Escherichia coli such as an XLl-Blue MR strain (manufactured by Stratagene) . That is spread on an LB plate medium containing ampicillin and incubated through the night at about 30 to 37°C to get transformants.
  • Gigapack III Gold manufactured by Stratagene
  • the resultant transformants are cultured in LB medium containing about 0.1 mg/mL of ampicillin through the night at about 30 to 37 0 C using a 96-well titer plate, a 50% aqueous solution of glycerol in the same volume as the LB is added thereto and the mixture is stirred to give a glycerol stock.
  • the glycerol stock can be preserved at about -80 0 C.
  • the genome sequence of industrial yeast can be determined mainly using the genome shotgun method.
  • the DNA fragment of which DNA sequence is determined can be prepared by a PCR using the shotgun library prepared in the above (b) .
  • clone of the genome shotgun library is inoculated using a replicator (manufactured by Gene Solution) to a 384-well titer plate where about 50 ⁇ l each of an ampicillin-containing LB medium is placed to each well and cultured without shaking through the night at about 30 to 37°C.
  • the culture is transferred using a replicator (manufactured by Gene Solution) or the like to a 384-well reaction plate (manufactured by AB Gene) where about lO ⁇ l each of a reaction solution for PCR (TaKaRa Ex Taq manufactured by Takara Shuzo) is placed, and PCR is carried out according to a protocol by Makino, et al. (DNA Research, volume 5, pages 1 to 9 (1998)) or the like using a GeneAmp PCR System 9700 (manufactured by Applied
  • Excessive primer and nucleotide are removed using a kit for the purification of PCR products (manufactured by Amersham Bioscience) , etc. and a sequence reaction is carried out using the sample as a template.
  • Cosmid DNA from the cosmid library of (c) can be prepared by the following method. That is, clone derived from cosmid library is inoculated to each well of a 96-well plate where about 1.0 mL each of an ampicillin-containing appropriate medium such as a 2 x YT medium (1.6% bactotryptone, 1% yeast extract and 0.5% sodium chloride; pH 7.0) is placed and cultured with shaking through the night at about 30 to 37 0 C.
  • Cosmid DNA from the said culture can be prepared using KURABO PI-1100 AUTOMATIC DNA ISOLATION SYSTEM . (manufactured by KURABO) according to a manual of KURABO or the like, and they can be used as templates for sequencing reaction.
  • a Sequencing reaction can be carried out using a commercially available sequence kit, etc. Preferred examples of the present invention are shown below.
  • a sequence reaction mixture can be prepared as follows.
  • the PCR product or cosmid DNA prepared in the above (d) is mixed with about 2 ⁇ l of DYEnamic ET Terminator Sequencing Kit (manufactured by Amersham
  • An M13 forward (M13-21) primer and an M13 reverse (M13RV) primer are used for the sequence reaction of a PCR product derived from shotgun DNA, while a forward primer such as SS-cos F.I (SEQ ID NO: 7) and a reverse primer such as SS-cos R.I (SEQ ID NO: 8), etc. are used for cosmid DNA.
  • Amounts of the primer and the DNA fragment are about 1 to 4 pmole and about 50 to 200 ng, respectively.
  • a dye terminator sequence reaction of about 50 to 70 cycles can be carried out using the reaction solution and GeneAmp PCR System 9700 (manufactured by Applied Biosciences) .
  • GeneAmp PCR System 9700 manufactured by Applied Biosciences
  • a cycle parameter follows a manual attached thereto. Purification of the sample is carried out according to the manual of Millipore using Multiscreen HV plate (manufactured by Millipore), etc. The purified reaction product is precipitated with ethanol and the resulting precipitate is dried and stored in a dark place of about 4 0 C.
  • the dried product is analyzed using commercially available sequencer and analyzer such as MegaBACE 1000 Sequencing System (manufactured by Amersham Bioscience) and ABI PRISM 3700 DNA Analyzer (manufactured by Applied Biosystems), etc. according to the manuals attached thereto.
  • sequencer and analyzer such as MegaBACE 1000 Sequencing System (manufactured by Amersham Bioscience) and ABI PRISM 3700 DNA Analyzer (manufactured by Applied Biosystems), etc. according to the manuals attached thereto.
  • Reconstruction of genomic DNA may be carried out from sequence information of DNA fragments obtained in the above (e) . All operations of the reconstruction of genomic DNA sequence can be carried out on an UNIX ® platform. Base call can be conducted by a software such as phred (The
  • masking of vector sequence can be carried out by a software such as Cross Match (The University of Washington) or the like and assembly can be carried out by a software such as Phrap (The University of Washington) or the like.
  • Contig obtained as a result of assembly can be analyzed using a graphical editor such as consed, a graphical editor (The University of Washington) or the like.
  • a series of works from base call to assembly can be carried out en bloc utilizing phredPhrap, a script attached to the consed.
  • comparison of the genome sequence obtained in (A) with that of S. cerevisiae can be carried out by any known means for comparing two sequences. It may include (g) Preparation of a comparative database compiling the comparison data of each of DNA sequences of both ends of cosmid and shotgun clone and contig with S. cerevisiae genome sequence, and mapping of them on S. cerevisiae genome sequence.
  • cerevisiae genome sequences are accessible from public available data bank, such as, Saccharomyces Genome Database (SGD: http://genome- www.stanford.edu/Saccharomyces/) .
  • SGD Saccharomyces Genome Database
  • the percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program.
  • An exemplary, preferred computer program is the Genetics Computer Group (GCG; Madison, WI) Wisconsin package version 10.0 program, 1 GAP 1 (Devereux et al. , 1984, Nucl. Acids Res. 12: 387).
  • the preferred default parameters for the "GAP 1 program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds. , Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp.
  • Standard default parameter settings for WU- BLAST 2.0 are described at the following Internet site: sapiens.wustl.edu/blast/blast/#Features.
  • the BLAST algorithm uses the BLOSUM62 amino acid scoring matix, and optional parameters that can be used are as follows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993); also see Wootton and Federhen, 1996, Analysis of compositionally biased regions in sequence databases. Methods Enzymol.
  • E-score the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.
  • preferred E-score threshold values are 0.5, or in order of increasing preference, 0.25, 0.1, 0.05, 0.01,
  • le-100 0.001, 0.0001, le-5, le-10, le-15, le-20, le-25, le-30, Ie- 40, le-50, le-75, or le-100.
  • Fig. 2 An example of identity percentages distribution graph of cosmid DNA sequence corresponding to S. cerevisiae genomic DNA sequence is shown in Fig. 2.
  • the DNA sequence of cosmid is roughly classified into a DNA sequence group showing more than 94% identity to S. cerevisiae genome sequence and a DNA sequence group showing around 84% identity thereto. Accordingly, a DNA sequence having identity of more than 94% to the nucleotide sequence of the gene of Saccharomyces cerevisiae is named an Sc-type DNA sequence derived from S. cerevisiae.
  • a DNA sequence having 94% or less identity, more preferably,60 to 94% identity, most preferably around 84% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae is named a non-Sc-type DNA sequence derived from a closely- related species of S. cerevisiae.
  • a gene with the Sc type DNA sequence or the non-Sc type DNA sequence is named Sc type gene or non-Sc type gene, respectively.
  • a comparative database of the DNA sequence of both ends of shotgun clone prepared in (e) with genomic DNA sequence of S. cerevisiae is prepared.
  • a mapping of cosmid clone and shotgun clone on S. cerevisiae genome sequence is carried out (refer, for example, to Fig. 3) .
  • a comparative database of the DNA sequence of the contig prepared in (f) with S. cerevisiae genome sequence is also prepared and mapping is carried out.
  • mapping technique is nearly the same as that mentioned above, when contigs linked by paired forward-reverse DNA sequence from the same cosmid and shotgun clone, those contigs are linked (refer, for example, to Fig. 4).
  • C-2 selection of a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae.
  • a stage for the selection of the gene of bottom fermenting yeast encoding an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of S. cerevisiae may include (h) a process of identification of ORF (open reading frame) and assignment of function.
  • Identification of ORF in the DNA sequence assembled in (f) is carried out. Preferred examples are specifically mentioned below. With regard to a certain length of DNA sequence (such as not less than 150 base) embraced by initiation codon and termination codon, there can be carried out identification of ORF existing in a DNA sequence assembled in (f) using a program, such as ORF finder (http://www.ncbi.nih.gov/gorf/gorf.html) or the like for the identification of ORF for six kinds of reading frames including complementary sequence.
  • ORF finder http://www.ncbi.nih.gov/gorf/gorf.html
  • Assignment of function of protein encoded by the identified ORF can be carried out using a homology searching such as BLAST
  • Example 8 the choromosome structure of the S. pastorianus strain 34/70 was analyzed by using a DNA array containing the DNA probes of known S. cerevisiae nucleotide sequences.
  • Yeast genomic DNA is prepared using a Quiagen Genomic Tip 100/G (#10243) and Qiagen Genomic DNA Buffer Set (#19060) according to the manual attached to the kit.
  • the DNA e.g., 10 ⁇ g
  • the DNA is digested with DNase I (manufactured by Invitrogen) according to a method of Winzeler, et al. (Science, volume 281, pages 1194-1197 (1998)), biotinylated using a terminal transferase (manufactured by Roche) and hybridized to a DNA array (Affymetrix Gene Chip Yeast
  • Genome S98 Array contains the DNA probes of known S. cerevisiae nucleotide sequences. Hybridization and detection of the signal intensity of the DNA array are carried out using a Gene Chip Analysis Basic System and analysis soft ware (Microarray Suite 5.0) manufactured by Affymetrix.
  • the signal of each probe hybridized with the DNA of brewing yeast is normalized to that of the haploid laboratory yeast strain S288C using an analysis soft ware (Microarray Suite 5.0) and shown as signal log ratio (2 n ).
  • Signal log ratios were lined following genes order in each chromosome using a spreadsheet program (Microsoft Excel 2000) and the signal log ratios are shown in bar graphs (refer, for example, to Fig. 5) .
  • the non-Sc type genes do not hybridize to the S. cerevisiae array, therefore, the Sc type gene dosage affects the signal log ratio and the points where the signal log ratios show vigorous changes are considered to be translocation sites between Sc type and non-Sc type chromosome.
  • the chimera chromosome structure can be confirmed by PCR, wherein a genomic DNA derived from brewing yeast is used as a template and Sc type and non-Sc type shotgun sequences are used as primers.
  • PCR may be carried out using a Takara PCR Thermal Cycler SP according to the attached manual using a Takara LA TaqTM and a buffer attached thereto.
  • a gene of the industrial yeast consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae can be selected. Identity of 60 to 94% in nucleotide sequence level corresponds approximately 70 to 97% in amino acid sequence level.
  • Some nucleotide sequences in non-Sc type chromosome other than open reading frames may have identity of less than 60% to the corresponding nucleotide sequences of Sc type chromosome.
  • functional analysis of the selected genes may optionally be subjected to functional analysis.
  • functional analysis of gene may include (i) selection of the gene, (i f ) cloning of the gene, (j) functional analysis of the gene by disruption or (k) functional analysis of the gene by overexpression.
  • One of the purposes of the present invention is to identify genes from industrial yeasts involved in increase of productivity and/or improvement in flavor in the production of an alcohol or an alcoholic beverage.
  • the whole genome sequence of Saccharomyces cerevisiae has been identified, and function of each gene of Saccharomyces cerevisiae has already been identified.
  • functions of proteins encoded by the non- Sc type genes can be assigned by a homology searching.
  • Non-Sc type genes which are expected from the assignment to have an useful activity, e.g., involved in increase of productivity and/or improvement in flavor in the production of an alcohol or an alcoholJ ⁇ fbeyerage, are selected as target genes for f unctional analysis. — ⁇ i
  • the SSUl gene and the MET14 gene are known to have the sulfite transporting activity, and the adenylsulfate kinase activity, respectively. Both genes are known to play an important role for stabilizing flavor of beer.
  • Other genes from the Sc gene analysis which are known to play some role in a step of producing beer are listed in Table 2 in the following Example 7. These genes, however, do not exert sufficient activity enough for producing tasty beer. Therefore, without any limitation, for the purpose of identifying genes from industrial yeasts involved in increse of productivity and/or improvement in flavor in the production of an alcohol or an alcoholic beverage, non-Sc genes corresponding to these Sc type genes can be selected.
  • the method using DNA array is, for example, gene expression analysis to identify genes, which show a characteristic expression profile under some conditions, or comparative genomic hybridization to identify genes, which have different copy numbers or different DNA sequences, by detecting deference of signal intensities of probes. (i f ) cloning of the gene
  • Genes selected in the above (i) can be obtained from the bottom fermenting yeast according to a common method mentioned, for example, in Molecular Cloning, Third Edition. That is, oligonucleotides having sequences adjacent to the gene are synthesized and a common PCR cloning method is carried out using a genomic DNA prepared from a bottom fermenting yeast as a template, whereupon the selected gene can be isolated and obtained. With regard to DNA sequences obtained as such, for example, SEQ ID NO: 1 may be listed.
  • the gene (1) or primer for amplifying the gene (1) by a PCR method may be also synthesized using a polynucleotide synthesizer on the basis of the above-mentioned sequence information.
  • gene (1) means not only a DNA fragment having the same DNA sequence as gene (1) but also a DNA fragment hybridizing to the above gene under stringent condition as is defined in the present specification.
  • the DNA fragment which hybridizes under stringent condition means a DNA fragment which is obtained by a colony hybridization method, a plaque hybridization method, a southern blot hybridization method or the like, using the DNA fragment containing the sequence of the gene (1) identified in the above as a probe.
  • under stringent condition means that two nucleic acid fragments can hybridize under a moderately stringent condition or a highly stringent condition. An artisan can easily choose an appropriate moderately or highly stringent condition.
  • moderately stringent conditions involves the use of a prewashing solution containing 5 x SSC, 0.5% SDS, 1.0 ⁇ iM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6 x SSC, and a hybridization temperature of about40-55°C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 0 C), and washing conditions of about 60 0 C, in 0.5 x SSC, 0.1% SDS.
  • a moderately stringent condition may include hybridization at about 50 0 C and 2 X SSC. Highly stringent conditions can also be appropriately selected based on factors like length of DNAs to be hybridized.
  • such conditions include hybridization and/or washing at higher temperature and/or lower salt concentration (such as hybridization at about 65°C, 6 X SCC-0.2 X SSC, preferably, 6 X SCC, more preferably 2 X SSC, most preferably, 0.2 X SSC), compared to the moderately stringent condition.
  • highly stringent conditions may include hybridization as defined above, and washing at approximately 68°C, 0.2 x SSC, 0.1% SDS.
  • IxSSPE 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15 mM sodium citrate
  • wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 2001).
  • the hybrid length is assumed to be that of the hybridizing nucleic acid.
  • the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity.
  • the hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 25°C, preferably 5 to 10 0 C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations.
  • Tm melting temperature
  • each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while
  • the hybridization may be carried out according to a method mentioned in "Molecular Cloning, Third Edition", “Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter, abbreviated as Current Protocols in Molecular Biology), "DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)", and the like.
  • shotgun clone containing full- length of the above-mentioned gene (1) can be retrieved using the comparative database obtained in (g) and, on the basis of homology and positional information, etc.
  • a DNA fragment encoding the full length of the gene is prepared by a PCR method.
  • a DNA fragment containing the above-mentioned gene is obtained using synthetic DNA primer pair represented by SEQ ID NO: 13 and SEQ ID NO: 14, etc.
  • PCR is carried out using a primer pair designed on the basis of the published information of SGD and using genomic DNA of S. cerevisiae or bottom fermenting yeast as a template, whereupon the full length of the Sc type gene corresponding to the non-Sc type gene is prepared.
  • synthetic oligonucleotides of SEQ ID NO: 15 and NO: 16 as the primer pair, the DNA fragment containing the Sc type gene can be obtained.
  • Sc or non-Sc type DNA fragment prepared as mentioned above is inserted into, for example, pCR 2.1-TOPO vector attached to a TA cloning kit (Invitrogen) using a TA cloning kit or the like, whereupon a recombinant vector TOPO/Sc gene and TOPO/non-Sc gene containing the DNA fragment having the Sc and the non-Sc type gene, respectively, are able to be prepared.
  • DNA sequences of the Sc and non-Sc type DNA fragments can be comfirmed by Sanger's method "F. Sanger, Science, volume 214, page 1215, 1981". (j) Functional analysis of the gene by disruption
  • Functional analysis of the selected non-Sc gene may be performed by disruption of the gene, for example.
  • Functional analysis by disruption of the gene can be done by any known conventional methods.
  • "method for disruption of a gene” includes any methods which stop, block, inhibit or suppress at least part of function of the gene, including deletion of the gene, blocking of the gene by antisense nucleic acid, RNAi method or the like.
  • disruption of the non-Sc SSUl gene which has been identified by the present invention.
  • a DNA fragment for gene disruption by PCR where a plasmid containing a drug-resistance gene (such as pFA 6a (G418 r ) , pAG 25 (natl)) is used as a template.
  • a primer pair for the PCR non-ScSSUl_for (SEQ ID NO: 17)/non- ScSSUl_rv (SEQ ID NO: 18) or the like may be used for the non-ScSSUl disruption.
  • ScSSUl_for SEQ ID NO: 19
  • ScSSUl_rv SEQ ID NO: 20
  • the like may be used.
  • a plasmid such as pPGAPAUR (AURl-C)
  • a primer pair such as non-ScSSUl_for + pGAPAUR (SEQ ID NO: 21)/non-ScSSUl_rv + AURI-C (SEQ ID NO: 22).
  • An industrial yeast is transformed with the DNA fragment for the gene disruption prepared by the above- mentioned method.
  • the transformation may follow a method mentioned in the Japanese Patent Laid-Open Gazette No. 07/303,475, for example. Further, the concentration of the drug for the selection of transformants may be appropriately determined by investigating the sensitivity of the yeast used as a host.
  • the genomic DNA extracted from the parental strain and the transformant are firstly digested by an appropriate restriction enzyme to distinguish Sc and non-Sc type gene (for example, at 37°C for 18 hours), then fractionated with 1.5% agarose gel electrophoresis and transferred to a membrane. After that, they are hybridized to a probe specific to an Sc-type or a non-Sc type gene for example at 55 0 C for 18 hours according to a protocol of Alkphos Direct Labelling Reagents (Amersham) and a signal is detected by CDP-Star.
  • the function of the gene obtained in (i 1 ) can be studied by fermentation test using a parental strain and non-Sc SSUl disruptants prepared in the above (j) and comparison of their fermentation character. Fermentation test can be carried out, for example, using wort under the following condition.
  • the sulfite concentration in the wort during the fermentation is analyzed.
  • Quantitative analysis of sulfite is carried out in such a manner that sulfite is captured in a hydrogen peroxide solution by means of distillation under an acidic condition and subjected to titration with an alkali (Revised Method for BCOJ Beer Analysis by the Brewing Society of Japan) .
  • the "substance relating with the function of the gene” includes, without any limitation, sulfite, ethanol, ester and fusel alcohol or the like. Substances, which are considered to have a negative effect on production of tasty beer, can also be a target of analysis, including diacetyl and phenolic compounds or the like.
  • a DNA fragment containing the full-length of the non- Sc type gene is excised by an appropriate restriction enzyme from the plasmid TOPO/non-Sc gene prepared in (i 1 )-
  • the DNA fragment is inserted into a cloning site of a vector for gene expression such as pNI-NUT to construct a vector (pYI-non-Sc type gene) for overexpression of the non-Sc type gene.
  • the vector pNI-NUT contains URA3 as a homologous recombination site and nourseothricin-resistance gene (natl) and ampicillin-resistance gene (Amp r ) as selective markers.
  • a vector for overexpression of the Sc type gene has a structure where the above-mentioned pYI-non-Sc type gene is substituted by the corresponding Sc type gene.
  • pNI-Sc type gene has a structure where the above-mentioned pYI-non-Sc type gene is substituted by the corresponding Sc type gene.
  • TDH3 glyceraldehyde-3-phosphate dehydrogenase gene
  • An industrial yeast is transformed using the overexpression vector, which is prepared by the above- mentioned method. The transformation is carried out by the method mentioned in the Japanese Patent Laid-Open Gazette No. 07/303,475 and transformants are selected on an appropriate selective medium.
  • Confirmation of the overexpression may be carried out by RT-PCR method, etc. Extraction of the total RNA may be carried out using an RNeasy Mini Kit (Qiagen) or the like, according to the manual of "for total RNA isolation from yeast" attached to the kit.
  • RNeasy Mini Kit Qiagen
  • the SSuI gene for example, it is possible to use ScSSUl_for331 (SEQ ID NO: 23)/ScSSUl_982rv (SEQ ID NO: 24) and nonSc-SSUl_for329 (SEQ ID NO: 25)/nonSc-SSUl_981rv (SEQ ID NO: 26) as specific primerpairs for the amplification of Sc and non-ScSSUl gene, respectively.
  • PDAl_forl SEQ ID NO: 27
  • PDAl_730rv SEQ ID NO: 28
  • the wort is periodically sampled and monitored the cell growth (OD600), apparent extract and the concentration of the substance relating with the function of the gene obtained in (i 1 ).
  • the present invention also provides genes obtained by the analyzing method of the present invention.
  • the genes of the present invention include both nucleotides of ORF encoding proteins, as well as nucleotides from non-coding regions. Nucleotides from non-coding regions may include regulatory regions, such as promoter, enhancer, silencer, terminator or the like, which are important for regulation of expression of a gene.
  • one embodiment of the present invention is a gene of the industrial yeast encoding an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or consisting of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, which is obtained by the present analyzing method.
  • the present invention also provides a gene library comprising one or more of genes of the industrial yeast as described above.
  • the gene of the industrial yeast is comprised in, consists of or has any nucleotide sequence selected from SEQ ID NOs:33 to 6236, SEQ ID Nos:166154 to 166181, SEQ ID Nos:166490 to 167042 and SEQ ID Nos:173125 to 174603.
  • SEQ ID NOs: 33-6236 represent 6204 DNA sequences of non-Sc type ORFs, which can be preferably used as probes of the microarry.
  • SEQ ID NOs: 166154-166181 represent 28 DNA sequences of mitochondrial ORFs from 34/70 strain, which can be preferably used as probes of the microarry.
  • SEQ ID Nos: 166490-167042 represent 553 DNA sequences which have not been identified as the above non-Sc ORFs but have significant similarity to the proteins of S. cerevisiae using NCBI-BlastX homology searching.
  • SEQ ID Nos: 173125-174603 represent 1479 DNA sequences ORFs which have not been identified as the above, but have significant similarity to the proteins of S. cerevisiae using NCBI-BlastP homology searching. 2908
  • genes of the present invention also include variants of the natural genes, which may include some modification in one or more nucleotide sequence(s) and/or amino acid sequence(s).
  • the genes of the present invention will be described in detail in the section IV of the present specification.
  • the "gene” and the “gene library” of the present invention can encompass not only genes in the form of chemical substan ⁇ e(s) , but also in a form of nucleic acid sequence information recorded in a computer-readable record medium or storage. Therefore, accessing and/or utilizing the nucleic acid seuqnece information of the "gene” and the “gene library” of the present invention are also included in the scope of the present invention.
  • the "computer-readable record medium or storage” includes any record medium or storage which is readable and accesible via a computer.
  • This may include, without any limitation, a magnetic record medium such as floppy diskette, hard diskette, and magnetic tape; an optical record medium such as CD-ROM, CD-R, CD-RW, DVD-ROM, DVD-RAM and DVD-RW; an electronic record medium such as RAM and ROM; as well as a hybrid of the above categories (e.g, a megnetic/optical record medium, such as MO) .
  • a magnetic record medium such as floppy diskette, hard diskette, and magnetic tape
  • an optical record medium such as CD-ROM, CD-R, CD-RW, DVD-ROM, DVD-RAM and DVD-RW
  • an electronic record medium such as RAM and ROM
  • a hybrid of the above categories e.g, a megnetic/optical record medium, such as MO
  • the present invention also provides a DNA array which is useful for an analyzing method of genes.
  • Genes which can be analyzed by using the DNA array of the present invention may include genomic DNA, cDNA or cRNA or the like of an industrial yeast.
  • the present DNA array can also be applied to the ChIP (Chromatin Immuno-Precipitation)-chip method for analyzing binding between DNA and a protein.
  • the DNA array of the present invention comprises probe(s) of DNA(s) based on the nucleotide sequences of non-Sc genes which have been identified by the present invention.
  • DNA oligonucleotide or cDNA which is fixed on a DNA array is sometimes called "probe".
  • the DNA array of the present invention comprises one or more of DNAs, wherein each DNA is selected from at least one group of (1) to (4): (1) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having 70 to 97% identity to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;
  • DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having 60 to 94% identity to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences;
  • the DNA array comprises DNAs wherein at least one DNA is selected from every group of (1) to (4) .
  • the DNAs of (1) is comprised in, consists of or has any nucleotide sequence selected from SEQ ID NOs:33 to 6236 and SEQ ID NOs: 166154 to 166181, and SEQ ID Nos:6237 to 75336 and SEQ ID Nos:166182 to 166489.
  • SEQ ID NOs:33 to 6236 correspond to ORF encoding non- SC proteins of the present invention. These sequences can be fixed to the DNA array as cDNA probes.
  • SEQ ID NOs:6237 to 75336 are short nucleotide sequences of about 25 continuous nucleotides selected from each of SEQ ID NOs:1 to 6236.
  • SEQ ID Nos: 166182-166489 represent sequences of oligonucleotides based on DNA sequences of SEQ ID NOs: 166154 to 166181.
  • the ORF sequences from yeast in the present specification may include intron. Such intron sequences can be easily recognized for an artisan.
  • nucleotide sequences of the DNA probes on the DNA array can include only exon sequences (without intron) which can be easily selected by referring to the ORF sequences disclosed in the present specification.
  • the term “have (has)” or “having” means that the nucleotide sequence of the present invention may include additional sequence which does not substantially affect the function of the DNA of the present invention.
  • DNAs encoding ORF may contain an additional signal or tag sequence which can facilitate secretion and/or purification of the protein encoded by the ORF.
  • oligonucleotides may contain additional one or several nucleotide sequences at its terminal which does not affect hybridization specificity of the oligonucleotides.
  • SEQ ID Nos. 167043-173124 represent sequences of oligonucleotides based on DNA sequences of SEQ ID Nos: 166490-167042.
  • SEQ ID Nos. 174604-190810 represent sequences of oligonucleotides based on DNA sequences of SEQ ID Nos: 173125-174603. These sequence can be fixed to the DNA array as oligonucleotide probes.
  • DNA array of the present invention is that of further comprising, in addition to at least one group of DNAs selected from (1) to (4), DNAs of the following (5) and/or (6): (5) DNA consisting of a nucleotide sequence of an open reading frame of the genome sequence of an industrial yeast which encodes an amino acid sequence having identity of more than 97% to an amino acid sequence encoded by the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a nucleotide sequence of continuous 10 or more nucleotides selected from the above nucleotide sequences; and (6) DNA consisting of a nucleotide sequence of the genome sequence of an industrial yeast other than from open reading frames which consists of a nucleotide sequence having identity of more than 94% to the nucleotide sequence of the gene of Saccharomyces cerevisiae, or a nucleotide sequence complementary to the above nucleotide sequence, or a
  • DNAs of (5) correspond to ORF of Sc genes
  • DNAs of (6) correspond to non-coding regions of Sc genes, respectively.
  • the DNA array comprising DNAs of (5) and/or (6) in addition to DNAs of any of (1) to (4), is useful for a method of classifying an industrial yeast, for example.
  • the DNAs of (5) has a nucleotide sequence of any one of SEQ ID NOs: 75337 to 82784, or SEQ ID NOs: 82785 to 166153.
  • SEQ ID NOs: 75337 to 82784 correspond to ORF encoding non-SC proteins of the present invention.
  • SEQ ID NOs: 82785 to 166153 are short nucleotide sequences of about 25 continuous nucleotides selected from each of SEQ ID NOs:33 to 6236.
  • the DNA array comprises DNAs selected from the group of (1) and/or (2), together with DNAs selected from the group of (5) and/or (6).
  • hybridization intensity of a sample to a DNA probe of (1) is compared to the corresponding DNA probe of (5) .
  • hybridization intensity of a sample to a DNA probe of (2) is compared to the corresponding DNA probe of (6).
  • the present DNA array of this embodiment may further comprise DNAs of the following (5 1 ) and/or
  • (6' ) DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (6), or a nucleotide sequence complementary to the above nucleotide sequence.
  • DNAs of (5) correspond to ORF of Sc genes
  • DNAs of (6) correspond to non-coding regions of Sc genes, respectively.
  • DNAs of (5 1 ) and (6 1 ) contain mismatch of one or more base(s), preferably one base to (5) and (6), respectively.
  • a sample of DNAs shows a different hybridization intensity to a probe having mismatch of one or more base(s) in the DNA array hybridization assay.
  • if a sample DNA hybridizes to a DNA probe of (5) (or (6)) and does not hybridize to the corresponding DNA probe of (5 1 ) (or (6 1 )) it can be concluded that the sample DNA is positive as regards the DNA probe of (5) (or (6)) of Sc type gene.
  • DNAs of (5 1 ) and/or (6 1 ) may be optionally used as DNA probes of the present DNA array for determining and confirming hybridization specificity of a sample to the DNAs of (5) and/or (6).
  • Still another aspect of the DNA array of the present invention is that of further comprising, in addition to at least one group of DNAs selected from (1) to (4), DNAs selected from at least one group of (7) to (10):
  • DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (1), or a nucleotide sequence complementary to the above nucleotide sequence;
  • DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (2), or a nucleotide sequence complementary to the above nucleotide sequence;
  • DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (3), or a nucleotide sequence complementary to the above nucleotide sequence;
  • DNA consisting of a nucleotide sequence which contains mismatch of 1 or more base(s) to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence.
  • a sample of DNAs shows a different hybridization intensity to a probe having mismatch of one or more base(s) in the DNA array hybridization assay.
  • This type of DNA array therefore, is useful for detecting a nucleotide polymorphism in genome of an industrial yeast. It can also be useful for the method to screen a useful strain of industrial yeast or useful genes themselves.
  • the DNAs of (7) to (10) includes mismatch of only one base. That is, the DNAs of (7) and (10) are the following DNAs of (7-1) to (10-1), respectively.
  • (10-1) DNA consisting of a nucleotide sequence which contains mismatch of 1 base to the DNA of (4), or a nucleotide sequence complementary to the above nucleotide sequence.
  • mismatch of one base is at the center or near the center of each DNA.
  • One of the most preferred embodiments of the present DNA array comprises at least one group of DNAs selected from (1) to (4) (preferably (1) and/or (2)), at least one group of DNAs selected from (7) to (10) (preferably (7) and/or (8)), DNAs of (5) and/or (6) and DNAs of (5 1 ) and/or (6 1 ).
  • the DNA array of this embodiment is especially useful for the present method to classify an industrial yeast, as well as a method to analyze the chromosomal structure of an industrial yeast by DNA array-based comparative genomic hybridization as discussed in the present specification below.
  • DNAs of (5 1 ) and/or (6 1 ) has a mismatch of only one base.
  • Probes which are attached to the DNA arrays of the present invention can be cDNA probes or oligonucleotids. There is no limitation to the length of cDNA probes, but preferably, no longer than 1,000 bases. Oligonucleotide probes range form about 5 to about 50 nucleotides, preferably, 10-30 nucleotides.
  • a DNA array of the present invention can be produced based on the DNA sequence information of the ORFs and/or non-coding regions (intergenic DNA sequence between two ORFs) obtained in the above (f) .
  • Examples include a DNA array comprising a solid support to which at least one of a polynucleotide comprising the DNA sequence obtained in the above items (f) .
  • DNA arrays of the present invention include substrates known in the art, such as a DNA microarray, a DNA chip, polynucleotide array and a DNA macroarray, or the like, and comprises a solid support and plural polynucleotides of fragments thereof which are adhered to the surface of the solid support.
  • substrates known in the art such as a DNA microarray, a DNA chip, polynucleotide array and a DNA macroarray, or the like
  • the polynucleotides or oligonucleotides adhered to the solid support the polynucleotides or oligonucleotides of the present invention obtained in the above (f) and (h) can be used.
  • the analysis described below can be efficiently performed by adhering the polynucleotides or oligonucleotides to the solid support at a high density, though a high fixation density is not always necessary.
  • Apparatus for achieving a high density such as an arrayer robot or the like, is commercially available from Takara Shuzo (GMS417 Arrayer) , and the commercially available product can be used.
  • the oligonucleotide of the present invention can be synthesized directly on the solid support by the photolithography method or the like (Nat. Genet. 21, 20-24 (1999)).
  • a linker having a protective group which can be removed by light irradiation is first adhered to a solid support, such as slide glass or the like. Then, it is irradiated with light through a mask (a photolithograph mask) permeating light exclusively at a definite part of the adhesion part. Next, an oligonucleotide having a protective group which can be removed by light irradiation is added to the part. Thus, a ligation reaction with the nucleotide arises exclusively at the irradiated part. By repeating this procedure, oligonucleotides each having a desired sequence and different from each other can be synthesized in respective parts.
  • the oligonucleotides to be synthesized have a length of 10 to 30 nucleotides.
  • cDNA probes which can be prepared by any known conventional methods based on the nucleotide sequences of mRNA obtained from the industrial yeast can be used.
  • a cDNA probe is fixed to the DNA array after it has been prepared. Examples of general methods for preparing and fixing the cDNA probes to the DNA array are explained in "Science volume 270, pages 467-470 (1995)", for example. There is no particular limitation for the methods used for the production of DNA array and the method may be conducted according to the known means, while preferred method for each of them is mentioned below.
  • any materials of which the polynucleotide or fragments can be adhere to the surface can be used as the solid supports using the invention DNA array.
  • the material and shape used for the solid support While preferred materials are some resinoids, such as polycarbonate, plasties or the like, as a material and a plate-like and film-like as a solid.
  • oligonucleotides to be fixed on the plate of a DNA array of this invention are as follows. Based on the DNA sequences of ORFs obtained in the above (h) and/or intergenic DNA sequences deduced from the above (h) , unique and perfect complementary probes (PM Probe; Perfect Match Probe) against genome sequence of brewing yeast can be designed using a certain method of probe production, such as GeneChip® (Affymetrix) technology or the like.
  • PM Probe Perfect Match Probe
  • the oligonucleotide probes range form about 5 to about 100 nucleotides, preferabaly about 5 to about 80 nucleotides, preferably about 5 to about 60 nucleotides, more preferably about 5 to about 50 nucleotides, more preferably from about 5 to about 45 nucleotides, still more preferably from about 10 to about 40 nucleotides and most preferably from about 10 to 30 nucletides in length.
  • Particularly preferred arrays contain probes ranging from about 20 to about 25 oligonucleotides in length.
  • the array may comprise more than 10, preferably more than 50, more preferably more than 100, and most preferably more than 1000 oligonucleotide probes specific to the each target gene (Where the array refers to the DNA array to be fixed the oligonucleitide probes using a certain method of probe production, such as GeneChip® (Affymetrix) technology or the like).
  • the array comprises at least 10 different oligonucleotide probes for each gene.
  • pastorianus has been regarded as a natural hybrid of S. cerevisiae and its closely related species (such as S. bayanus) as described above. So the bottom fermenting yeast has at least two different genes whose DNA sequences are similar to each other. Thereby, it is desirable to remove probes that hybridize to transcription products of more than one gene. One can then remove any probes that are similar to any other genes, and make a probe set for that genes using a certain method of probe production, such as GeneChip® (Affymetrix) technology or the like)
  • the array may further comprise mismatch control probes. Where such mismatch control probes are present, the quantifying step may comprise calculating the difference in hybridization signal intensity between each of the oligonucleotide probes and its corresponding mismatch control probe. The quantifying may further comprise calculating the average difference in hybridization signal intensity between each of the oligonucleotide probes and its corresponding mismatch control probe.
  • mismatch probe refer to the probe whose sequence is deliberately selected not to be perfectly complementary to a particular target sequences. For each mismatch (MM) control in a high-density array, there typically exists a corresponding perfect match (PM) probe that is perfectly complementary to the same particular target sequence.
  • the mismatch may comprise one or more bases, preferably one base.
  • the mismatch(es) may be located anywhere in the mismatch probe, terminal mismatche is less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequences.
  • the mismatch is located at or near the center of the probe so that the mismatch is most likely to destabilize the duplex with the target sequence under the optimal hybridization conditions.
  • the mismatch is adenine (A) to thymine (T) and vice versa, or guanine (G) to cytosine (c) and vice versa.
  • each PM probe can be designed with a closely related mismatch probe (MM probe) that is identical to PM probe with the exception of a mismatched base, i.e. base 13.
  • MM probe closely related mismatch probe
  • the preferable length of oligonucleotide which is used in this invention is 25 base, but no particular limitation for the length of oligonucleotide.
  • the mismatch is adenine (A) to thymine (T) and vice versa, or guanine (G) to cytosine (c) and vice versa.
  • (l)-3 Adhering oligonucleotides to solid support
  • the methods used for adhering oligonucleotides to solid support may be conducted according to the known means, while preferred method is mentioned below.
  • all of designed PM and MM probes as above ((l)-2) can be adhered to the surface of solid support to produce a DNA array using a certain method, such as GeneChip® technology or the like.
  • the arrays of the invention can be used for various methods to analyze genomic DNA, cDNA or cRNA of an industrial yeast.
  • the analyzing methods include, for example, 1) a method for determining expression of each gene of non-Sc type, 2) a method for classifying an industrial yeast, 3) a method for detecting nucleotide polymorphism in genome of an industrial yeast, 4) a method for screening a useful strain of industrial yeasts, and 5) a method for screening a gene of an industrial yeast. (m) Gene expression analysis
  • Gene expression analysis of an industrial yeast can be carried out using the DNA array of the present invention produced according to the method described in (1). It is possible to identify the highly inducible or reducible gene(s) according to changes of not only medium but also environment using the DNA array. It is also possible to identify the specific gene(s) for lager brewing yeast in brewing using the DNA array. But it is not limited for these examples.
  • Gene expression analysis includes culturing of a industrial yeast, preparation of mRNA, synthesis of labeled cRNA(or cDNA) , hybridization, and data analysis. There is no particular limitation for the methods of Gene expression, while preferred methods for each of them is mentioned below.
  • Industrial yeast can be cultivated under various conditions for any purpose. For example, the cultivation for identification of genes which respond to the change of composition of culture medium can be carried out as mentioned below.
  • Industrial yeast can be grown overnight in a Zinc replete medium, such as LZMM medium + 40 ⁇ M zinc sulfate at 30°C with shaking.
  • LZMM medium contains 0.17 % yeast nitrogen base w/o amino acids (manufactured by DIFCO), 0.5 % ammonium sulfate, 20 mM sodium citrate (pH 4.2), 125 ⁇ M MnC12, 10 ⁇ M FeC12, 2 % maltose, 10 mM EDTA
  • Preparations of total RNA from each cell can be carried out using an RNeasy® Mini Kit (manufactured by
  • GCOS GeneChip Operating Software manufactured by Affymetrix; GeneSpring manufactured by Silicon Genetics; ImaGene manufactured by Takara Shuzo; Array Gauge manufactured by Fuji Photo Film; ImageQuant manufactured by Amersham Pharmacia Biotech, or the like
  • GCOS GeneChip Operating Software manufactured by Affymetrix; GeneSpring manufactured by Silicon Genetics; ImaGene manufactured by Takara Shuzo; Array Gauge manufactured by Fuji Photo Film; ImageQuant manufactured by Amersham Pharmacia Biotech, or the like
  • Genes which show characteristic expression profiles can be identified and selected for functional analysis. Furthermore, the identified gene can be used as a gene marker to figure out a condition of the yeast cells. A fluctuation in the expression level of a specific gene can be monitored using a nucleic acid molecule obtained in the time course of culture as the nucleic acid molecule derived from brewing yeast. The culture conditions can be optimized by analyzing the fluctuation. The expression profile of the brewing yeast at the total gene level
  • the expression level of the genes determined by the genome sequence can be analyzed and, in its turn, the biological conditions of the yeast can be recognized as the expression pattern at the full gene level.
  • a certain condition may include, without any limitation, changes in environment, such as, temperature, pH, or osmotic pressure, as well as changes in components of medium.
  • a screening method for identifying genes showing distinctive expression under the certain condition for example, a gene wherein significant changes in expression of the gene are recognized according to changes in environment, can be selected.
  • Signal changes mean preferably, 1.5 times higher, or 2/3 or less, more preferably, 2 times higher, or 1/2 or less, even more preferably 3 times higher, or 1/3 or less, but not limited thereto.
  • the DNA array of the present invention can also be used for comparative hybridization analysis using genomic DNA, cDNA or cRNA as a sample for hybridization, (n) Classification of industrial yeast
  • An embodiments of the present invention includes a method for classifying an industrial yeast comprising T/IB2005/002908
  • the DNA array comprises, in addition to the DNAs of any of (1) to (4), DNAs of the (5) and/or (6) is preferably used (Embodiment 15 or 16) .
  • hybridization intensity to the DNAs of (1) and to the DNAs of (5) is compared.
  • hybridization intensity to the DNAs of (2) and to the DNAs of (6) is compared.
  • the present DNA array may optionally comprise DNAs of the (5 1 ) and/or (6 1 ) and/or (7) and/or (8)having one or more mismatch(es) to the DNA of (5) and/or (6) and/or (l) and/or (2) respectively.
  • DNAs of (5 1 ) and/or (6 1 ) and/or (7) and/or (8) may be optionally used as DNA probes of the present DNA array for determining and confirming hybridization specificity of a sample to the DNAs of (5) and/or (6) and/or (l) and/or (2) .respectively.
  • the DNA array comprises at least one group of DNAs selected from (1) to (4) (preferably (1) and/or (2)), at least one group of DNAs selected from (7) to (10) (preferably (7) and/or (8)), DNAs of (5) and/or (6) and DNAs of (5') and/or (6 1 ).
  • percentage of genes which hybridize to DNAs of (1) and/or (2), but not to DNAs of (7) and/or (8) is determined as hybridization ratio to any of (1) to (4)
  • percentage of genes which hybridize to DNAs of (5) and/or (6), but not to DNAs of (5 1 ) and/or (6 1 ) is determined as hybridization ratio to any of (5) and/or (6).
  • GCOS GeneChip Operating
  • An embodiment of the present invention includes a method for detecting nucleotide polymorphism in genome of an industrial yeast comprising
  • the DNA array comprises, in addition to the DNAs of any of (1) to (4), DNAs of any of (7) to (10) is preferably used (Embodiments 17-19) .
  • the probes on the array are preferably oligonuclotide probes.
  • the sets of oligonucleotides for each probe consist of Perfect Match oligonucleotide (PM) which is identical to the sequence of strain 34/70 and MisMatch oligonucleotide (MM) which contains a single base mismatch, e.g., in the central position of the oligonucleotide. It is possible to detect nucleotide polymorphism from the gene whose signal intensity in MM is higher (for example, more than 5-fold) than that in PM.
  • PM Perfect Match oligonucleotide
  • MM MisMatch oligonucleotide
  • Genes comprising one or more mismat ⁇ he(es) can be preferable candidates of target genes for further functional analysis.
  • An embodiment of the present invention includes a method for screening a useful strain of industrial yeast, comprising (a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of the present invention;
  • a gene of genomic DNA which shows hybridization intensity significantly different from other genes may be selected as a candidate of useful gene for further functional analysis. Therefore, strains of industrial yeasts comprising 1 or more such genes, preferably 2 or more such genes can be selected as candidate of useful strains.
  • hybridization intensity of such gene is significantly different from that of other genes, more preferably, thereof is 1.5 times higher or less than 2/3 compared to an average hybridization intensity to the DNA of (1) and/or (2). More preferably, the difference in hibridization intensity is 2 times higher, or 1/2 or less, even more preferably 3 times higher, or 1/3 or less, but not limited thereto.
  • Further embodiment of the present invention is a method for screening a gene of an industrial yeast, comprising (a) hybridizing genomic DNA prepared from the industrial yeast strain to the DNA array of the present invention;
  • Another embodiment of the present invention is a method for screening a gene of an industrial yeast, comprising
  • step (a-2) independently from the step (a-1), hybridizing genomic DNA, cDNA or cRNA prepared from the industrial yeast of the strain used for preparing the DNA array to the DNA array of the present invention
  • step (b) selecting a gene wherein hybridization intensity thereof to the DNA any of (1) to (4) in the step (a-1) is significantly different from hybridization intensity thereof in the step (a-2).
  • the above screening method enable to select genes of which hybridization intensity to the DNA array are different between strains. From the results of comparative genomic hybridization analysis, a gene which has probe sets showing low signal intensities may be lost or have different sequence from that of the strain used for probes of the array (e.g., 34/70). In contrast, a gene which has probe sets showing high signal intensities may be high in copy number. For example, as for a gene, which show hybridization intensity of 2 times higher in the step (a-1) than that of the strain used for preparing the DNA array in T/IB2005/002908
  • the strain of (a-1) is likely to have two or more copies of the gene.
  • Another embodiment of the present invention is a method for screening a gene of an industrial yeast, comprising
  • step (b) selecting a gene wherein hybridization intensity thereof to the DNA of any of (1) to (4) in the step (a-1) is significantly different from hybridization intensity thereof in the step (a-2).
  • the above screening method enable to select genes wherein expression of the genes may be changed due to difference in culture condition.
  • the "industrial yeast cultured in a different condition” includes yeasts cultured in different medium or temperature, or sequential samples during the process of producing an alcohol or an alcoholic beverage, for example.
  • Genes obtained by the various screening methods of the present invention may be subjected for further functional analysis, because differences in hybridization intensity may contribute to the difference of fermentation character between strains.
  • the genes which have nucleotide polymorphism detected by the method mentioned above can be also selected for functional analysis.
  • the DNA array of the present invention may be used to analyze the chromosomal structure of an industrial yeast by DNA array-based comparative genomic hybridization.
  • Yeast genomic DNA is prepared using a Quiagen Genomic Tip 100/G (#10243) and Qiagen Genomic DNA Buffer Set
  • the DNA (10 ⁇ g) is digested with DNase I (manufactured by Invitrogen) according to a method of Winzeler, et al. (Science, volume 281, pages 1194-1197 (1998)), biotinylated using a terminal transferase (manufactured by Roche) and hybridized to a DNA array (Affymetrix Gene Chip Yeast Genome S98 Array) .
  • the DNA array of the present invention may include DNAs of any one of (1) to (4) and (7) to (10) from Non-Sc type genes as well as (5) and/or (6) and/or (5 r ) and/or (6')of the Sc type.
  • Hybridization and detection of the signal intensity of DNA array are carried out using a Gene Chip Analysis Basic System and analysis soft ware (GCOS; GeneChip Operating Software 1.0) manufactured by Affymetrix.
  • GCOS Gene Chip Analysis Basic System and analysis soft ware
  • the signals of probes hybridized with the DNA of brewing yeast were lined following genes order in each chromosome using a spreadsheet program (Microsoft Excel 2000) and the signals are shown in bar graphs.
  • the Sc type genes do not hybridize to the non-Sc type DNA probes (i.e., DNAs of (1) to (4)). Contrary, the non-Sc type genes do not hybridize to the Sc type DNA probes (i.e., DNAs of (5) and/or (6)).
  • hybridization to DNAs of any of (1) to (4) can be determined by hybridization to DNAs of (1) and/or (2), but not to DNAs of (7) and/or (8), and hybridization to DNAs of any of (5) and/or (6) can be determined by hybridization to DNAs of (5) and/or (6), but not to DNAs of (5 1 ) and/or
  • the present invention further provides a non-Sc gene obtained by the analyzing method and the screening method of the present invention.
  • genes of the present invention involved in increase in productivity and/or improvement in flavor in the production of an alcohol or an alcoholic beverage.
  • genes of the present invention is, preferably, characterized in that concentration of sulfite in a culture medium of an industrial yeast increases when the gene is expressed in the yeast.
  • the present invention also provides polypeptides or proteins encoded by the genes.
  • the polypeptides of the present invention may be obtained by any known conventional methods including expression of the gene by genetic engineering, or via polypeptide synthesizing technique.
  • genes of the present invention may include both ORF and non-coding regions (non-ORF regions).
  • "Gene” includes DNA, cDNA, mRNA, cRNA, without any limitation.
  • the gene of the present invention can be prepared by any known conventional methods, for example, by screening a cDNA library, or via polynucleotide synthesizing techniques. In the present specification, the term
  • nucleic acid is used interchangeably with “gene”.
  • DNA will be explained as an example of gene.
  • a DNA containing the DNA sequence of the non-Sc type gene obtained in the above and a DNA which hybridizes to the said DNA under stringent condition and a protein having the substantially the same biological activity may be listed.
  • the DNA obtained by the method of the present invention includes single-stranded and double-stranded DNAs although they are non-limitative.
  • a DNA which hybridizes to the DNA containing a DNA sequence of the non-Sc type gene obtained in the above under stringent condition includes a degenerated mutant of codon for the protein encoded by the said gene, for example.
  • a degenerated mutant means a polynucleotide fragment encoding the same amino acid sequence by degeneration of codon, although in terms of a DNA sequence, it is different from a DNA sequence of the non-Sc type selected by the present invention.
  • the gene of the present invention includes a nucleic acid encoding a polypeptide of any one of the following i) and ii) : i) a polypeptide having the amino acid sequence represented by SEQ ID NO:3; and ii) a polypeptide having an amino acid sequence wherein one or more amino acid residue(s) is deleted from, substituted for and/or added to the amino acid sequence represented by SEQ ID NO:3, and having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.
  • the nucleic acid according to the present invention is selected from the following a) and b):
  • nucleic acid having the nucleotide sequence represented by SEQ ID N0:l (a) a nucleic acid having the nucleotide sequence represented by SEQ ID N0:l; and (b) a nucleic acid having a nucleotide sequence which hybridizes to the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:1 under a stringent condition, and encodes a polypeptide having an activity to increase concentration of sulfite in a culture medium of an industrial yeast when the gene is expressed in the yeast.
  • the DNA which hybridizes under stringent condition means a DNA which is prepared by a colony hybridization method, a plaque hybridization method, a southern blot hybridization method or the like using a DNA fragment with the sequence of the non-Sc type identified hereinabove as a probe.
  • An appropriate stringent condition can be selected as described above.
  • Hybridization may be carried out according to a method mentioned in "Molecular Cloning, Third Edition", “Current Protocols in Molecular Biology”, “DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)", etc.
  • hybridizable DNA is a DNA which shows at least not less than 60% identity, preferably a DNA which shows not less than 80% identity and, more preferably, a DNA which shows not less than 95% identity to a DNA sequence as shown in SEQ ID NO:1 when calculation is conducted using a parameter of the default setting (initial setting) by a software for homology searching such as
  • An example of the DNA obtained by the screening method of the present invention is a DNA encoding a polypeptide comprising an amino acid sequence shown by SEQ ID NO:3 , or a DNA which hybridizes to the said DNA under stringent condition.
  • An example of the polypeptide which is encoded by the DNA obtained by the screening method of the present invention is a polypeptide encoded by the DNA containing the DNA sequence of ORF obtained in the above and a polypeptide encoded by the DNA which is hybridized to the said DNA under stringent condition or a polypeptide comprising an amino acid sequence shown by SEQ ID NO:3 .
  • polypeptide having an amino acid sequence wherein one or more amino acid residue(s) is deleted from, substituted for and/or added to the amino acid sequence of the said polypeptide, and has substantially same biological activity as the activity of the said polypeptide is also included in the present invention.
  • substantially same activity as the activity of the said polypeptide means the same activity as the activity which is represented by enzymatic activity or the function inherent to the polypeptide before the deletion, substitution or addition.
  • the said polypeptide can be prepared by a site-specific mutation introduction which is mentioned in "Molecular Cloning, Third Edition", “Current Protocols in Molecular Biology", “Nuc. Acids. Res., volume 10, page 6487 (1982)", “Proc. Natl. Acad.
  • the number of the amino acid residue(s) which is/are deleted and/or substituted and/or added is within such an extent that is able to be deleted and/or substituted and/or added by known methods such as the above-mentioned site-specific mutation method and is one to several tens, preferably 1 to 20, more preferably 1 to 10 and, still more preferably, 1 to 5.
  • a polypeptide having an amino acid sequence wherein one or more amino acid residue(s) is deleted from, substituted for and/or added to the amino acid of the polypeptide of the present invention means that there is/are one or more deletion(s) and/or substitution(s) and/or addition(s) of one or more amino acid residue(s) at any one or more site(s) of the amino acid sequence in the same sequence. Those deletion(s) and/or substitution(s) and/or addition(s) may take place at the same time and the substituted or added amino acid residue may be either naturally occurring type or a non-naturally occurring type.
  • amino acid residue of a natural type examples include L- alanine, L-asparagine, L-aspartic acid, L-glutamine, L- glutamic acid, glycine, L-histidine, L-isoleu ⁇ ine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-cysteine, etc.
  • amino acid residues in the same group may be substituted each other (conservative substitution) .
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, 0- methylserine, tert-butylglycine, tert-butylalanine and cyclohexylalanine.
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid and 2- aminosuberic acid.
  • Group C asparagine and glutamine.
  • Group D lysine, arginine, ornithine, 2,4- diaminobutanoic acid and 2,3-diaminopropionic acid.
  • Group E proline, 3-hydroxyproline and 4- hydroxyproline.
  • Group F serine, threonine and homoserine.
  • Group G phenyl alanine and tyrosine.
  • the amino acid sequence of mutated one is at least 60% or more, usually 80% or more or, particularly, 95% or more of identity to the amino acid sequence of the polypeptide before the mutation when calculation is carried out using a parameter of the default setting (initial setting) by a software for the analysis such as BLAST and FASTA.
  • the method of the present invention it is possible to determine the genome sequence of industrial yeast, to identify the useful genes of industrial yeast and to assign the functions of the said genes. There are many cases where the genes in industrial yeast are industrially useful and, when the genes are classified on the basis of the assigned functions, character of the yeast is clarified and precious information for breeding of industrial yeast is able to be obtained.
  • the industrial yeast is a brewing yeast
  • a gene involved in increase in productivity and improvement in flavor in the production of alcoholic beverage is identified and, in case the gene is disadvantageous for the increase of productivity or for the improvement of flavor, the gene expression is suppressed by a gene disruption or an antisense method or an RNAi method (c.f., for example, Non-Patent Document 10), whereupon yeast which shows an excellent brewing character can be bred.
  • the gene is advantageous for the increase of productivity, improvement of flavor, etc., then for example the gene is overexpressed in the yeast, whereupon brewing yeast which shows an excellent brewing character, which is industrially useful, can be bred.
  • SSUl is a gene, which has been isolated and shown complement the sulfite-sensitive mutation (c.f., for example, Non-Patent Document 11).
  • SSUl gene product comprises 485 amino acid residues, and the structural analysis suggests that it is a transporter with 9 to 10 membrane-spanning domains (c.f., for example, Non-Patent Document 12). Further, as a result of experiment using a SSUl overexpressed strain, it has been already proved that the SSUl gene product effects the discharge of sulfite (c.f., for example, Non-Patent Document 13).
  • Bottom fermenting yeast usually has a high production ability of sulfite, while top fermenting yeast rarely produces it.
  • a screening method of the present invention it is possible to select non-ScSSUl gene which is specific to bottom fermenting yeast in addition to ScSSUl gene which exists in both top and bottom fermenting yeast.
  • a recombinant vector containing the gene of the present invention is provided.
  • a transformant comprising the recombinant vector.
  • the transformant is preferably, a yeast of genus Saccharomyces.
  • a method for producing an alcohol or an alcoholic beverage, using the transformant of the present invention may comprise culturing the transformant of the present invention in a sugar-containing medium.
  • the sugar containing medium is selected, from a group consisting of wort, grape juice, rice juice and. glucose and/or maltose syrup.
  • yeast which is suitable for the production of an alcohol or an alcoholic beverage, characterized in that, expression of the gene or the nucleic acid of the present invention is controlled.
  • the yeast belongs to the genus Saccharomyces.
  • Yeast obtained by the breeding method of the present invention A method for producing an alcohol or an alcoholic beverage using the yeast of the present invention.
  • An alcohol or an alcoholic beverage which is produced by the producing method of the present invention.
  • yeast used as a host in the introduction of the gene selected by the screening method of the present invention
  • yeast which is usable for brewing
  • any yeast which is widely used as a brewing yeast at present such as beer yeast including NCYC456, NBRC 1951, NBRC 1952, NBRC 1953 and. NBRC 1954.
  • whisky yeasts such as S. cerevisiae NCYC 90
  • wine yeasts such as wine yeast Kyokai No. 1, No. 3, No. 4, etc.
  • sake yeasts such as sake yeast Kyokai No. 7, No. 9, etc.
  • a vector used for the introduction of gene into the above-mentioned, host there is no particular limitation so far as it is a vector which can express gene in the yeast, and a multicopy plasmid (YEp type), a single- copy plasmid (YCp type) and a chromosomal DNA-integrating plasmid (YIp type) may be utilized.
  • YEp type multicopy plasmid
  • YCp type single- copy plasmid
  • YIp type chromosomal DNA-integrating plasmid
  • An example of a YEp vector is YEp 51 (J. R. Broach, et al. , Experimental Manipulation of Gene Expression, Academic Press, New York, 83, 1983), etc.
  • an example of a YCp vector is YCp 50 (M. D. Rose, et al.
  • YIp vector YIp 5 (K. Struhl, et al., Proc. Natl. Acad. Sci. USA, volume 76, page 1035, 1979), etc.
  • Those plasmids are put into the market and are easily available.
  • the above-mentioned vector may have other sequence for controlling expression of a gene in yeast such as, promoter, operator, enhancer, silencer, ribosome binding sequence, terminator, etc.
  • a promoter and a terminator for a constitutive expression of a gene but any combination may be used as long as it functions in a brewing yeast and is independent from sulfite concentration in the product.
  • a promoter for example, it is possible to use a promoter for glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene, a promoter for phosphoglycerate kinase (PGKl) gene, etc.
  • TDH3 glyceraldehyde-3-phosphate dehydrogenase
  • PGKl phosphoglycerate kinase
  • Those promoters have been known, and PGKl gene, for example, is mentioned in detail in publicly known documents such as M. F. Tuite, et al., EMBO J., volume 1, page 603 (1982) and easily available.
  • a method for the transformation of the above vector to a host may follow known procedures. For example, the following methods may be used; an electroporation method "Meth. Enzym. , volume 194, page 182 (1991)", a spheroplast method “Proc. Natl. Acad. Sci. USA, volume 75, page 1929 (1978)", a lithium acetate method "J. Bacteriology, volume 153, page 163 (1983)", a method mentioned in “Proc. Natl. Acad. Sci. USA, volume 75, page 1929 (1978)", etc.
  • a host is cultivated in a standard yeast nutrient medium (such as YEPD medium "Genetic Engineering, vol.
  • the cells are incubated at about 30 0 C for about 60 minutes, they are incubated together with DNA to be introduced (about 1 to 20 ⁇ g) at about 30 0 C for about 60 minutes.
  • DNA to be introduced about 1 to 20 ⁇ g
  • Polyethyleneglycol or, preferably, polyethyleneglycol of about 4,000 daltons is added as the final concentration will be about 20% to 50%.
  • the incubation is carried out at about 30 0 C for about 30 minutes, the cells are subjected to a heating treatment at about 42°C for about 5 minutes.
  • the cell suspension is washed with a standard yeast nutrient medium and placed in a predetermined amount of a fresh standard yeast nutrient medium, then incubated at about 30 0 C for about 1 hour. After the incubation, it is spread on an appropriate selective medium plate.
  • G 418-resistance gene G 418 r
  • CUP 1 copper- resistance gene
  • serulenin- resistance gene fas2m, PDR 4
  • M. Hussain, et al., Gene, volume 101, page 149, (1991)", etc. are applicable.
  • the brewing yeast bred according to the present invention is not different from a parental strain in terms of growth and fermentation ability. Accordingly, materials, facilities for the production, production control, etc. may be entirely the same as those in the conventional methods, which is an important aspect of the present invention. However, it goes without saying that, conditions such as fermentation period may be changed on a case-by-case, if desired. For example, when a brewing yeast in which discharging ability of sulfite is intensified and an alcoholic beverage is produced using such yeast, only the content of sulfite in the product changes, and there is no difference from the case where a parental strain is used, in terms of growth and fermentation ability of the yeast. Accordingly, materials, facilities for the production, production control, etc. may be entirely the same as those in the conventional methods, and there is no increase in the cost of production of an alcoholic beverage in which sulfite content increases and of which flavor is improved.
  • strain 34/70 Saccharomyces pastorianus Weihenstephan 34/70 (hereinafter, abbreviated as strain 34/70)
  • chromosomal DNA was carried out by a method mentioned in "Yeast, a practical approach (IRL Press) 6.2.1 (pages 228-229)", which was partially modified.
  • Cells were inoculated and grown in 200 mL of YPD medium (2% glucose, 1% yeast extract and 2% polypeptone) at 30 0 C until absorbance of the culture at 660 nm became 4. Cells were collected by centrifligation, washed with buffer
  • the resulting solution was centrifuged at 5,000 g for 10 minutes at 15°C.
  • To the recovered supernatant was added the same volume of ethanol to precipitate DNA, and the mixture was immediately centrifuged at 5,000 g for 10 minutes at 15°C to collect the precipitate.
  • the resulting precipitate was washed with 70% (v/v) ethanol, subjected to natural drying and dissolved in 5 mL of TE buffer (10 mM Tris-HCl and 1 mM of EDTA; pH 8.0) to give a crude DNA solution.
  • Cesium chloride (4.06 g) and 840 ⁇ g of bisbenzimide (Hoechst 33258) were added and dissolved in 3.5 mL of the crude DNA solution, the mixture was subjected to centrifugal separation at 100,000 g for 17 hours at 25°C and exposed to UV light to make DNA bands visible, whereupon the band of the lower layer was recovered.
  • the recovered DNA solution was extracted with isopropanol which was saturated with a cesium chloride solution to remove bisbenzimide (Hoechst 33258).
  • To the recovered aqueous layer was added 4-fold by volume of 0.3 M sodium acetate followed by mixing. Then, 3-fold by volume of ethanol was added thereto to precipitate the DNA, which was recovered by centrifligation.
  • the recovered DNA was dissolved in TE buffer containing 75 ⁇ g/mL of RNase, kept at 37°C for 5 minutes, and extracted with phenol/chloroform for three times and the aqueous layer was further subjected to precipitation with ethanol.
  • the precipitate recovered by centrifugation was washed with 70% (v/v) ethanol, subjected to natural drying and dissolved in TE buffer to prepare a chromosomal DNA solution.
  • Example 2 Preparation of a shotgun library
  • concentration of a genome solution of strain 34/70 prepared in Example 1 was adjusted to 1 mg/mL using a TE buffer. 0.1 mL thereof was treated with a Hydroshear (manufactured by GeneMachines; speed: 6; cycle: 20) to fragmentize the genomic DNA. End of the genomic fragment was blunted using a DNA Blunting Kit (manufactured by Takara Shuzo), fractionated by 0.8% agarose gel electrophoresis. Genomic fragments of 1.5 to 2.5 kb were excised from the gel, and DNA was eluted. The DNA eluate was treated with phenol/chloroform and precipitated with ethanol to give a genome library insert.
  • the resulting product is spread on an LB plate medium containing 1.6% of agar (the LB medium (1% bactotryptone, 0.5% yeast extract and 1% sodium chloride; pH 7.0)) containing 0.1 mg/mL of ampicillin, 0.1 mg/mL of X-gal and 1 mmol/L of isopropyl- ⁇ - D-thiogalactopyranoside (IPTG), and incubated through the night at 37°C.
  • the LB medium 1% bactotryptone, 0.5% yeast extract and 1% sodium chloride; pH 7.0
  • the transformant obtained from colonies formed on the said plate medium was subjected to cultivation without shaking through the night at 37°C in a 384-well titer plate to which 50 ⁇ L of an LB medium containing 0.1 mg/mL of ampicillin was added. Then 50 ⁇ L of a 50% aqueous solution of glycerol was added thereto followed by stirring, and the mixture was used as a glycerol stock.
  • Example 3 Preparation of a cosmid library About 0.1 mg of the genomic DNA obtained in Example 1 was partially digested with Sau3AI (manufactured by Takara Shuzo) . Insertion of the fragment into a BamHI site of Super Cos I vector (manufactured by Stratagene) was carried out according to a manual. A ligated product prepared by this method was subjected to packaging using Gigapack III Gold (manufactured by Stratagene) and introduced into Escherichia coli XLl-Blue MR strain (manufactured by Stratagene) according to a manual. It was spread on an LB plate medium containing 0.1 mg/mL of ampicillin and incubated through the night at 37°C.
  • the resulting transformants were cultured through the night at 37°C in an LB medium (each well: 50 ⁇ L) containing 0.1 mg/mL of ampicillin using a 96-well titer plate, and then 50 ⁇ L of 50% glycerol solution was added thereto followed by stirring and the mixture was used as a glycerol stock.
  • the genome sequence of strain 34/70 was determined mainly using the genome shotgun method.
  • a DNA fragment of which DNA sequence is to be determined by that method was prepared by a PCR method from the shotgun library prepared in the above Example 2.
  • clone derived from the genome shotgun library was inoculated using a replicator (manufactured by Gene Solution) to a 384-well titer plate where 50 ⁇ L of an LB medium containing 0.1 mg/mL of ampicillin was placed to each well and cultivated without shaking through the night at 37°C. The culture liquid was transferred to a 384-well reaction plate
  • a DNA fragment from the cosmid library of the above Example 3 was prepared according to the following method. That is, clone derived from the cosmid library was inoculated to each well of a 96-well plate to which 1.0 mL each of a 2 x YT medium (1.6% bactotryptone, 0.1% yeast extract and 0.5% sodium chloride; pH 7.0) containing
  • a cosmid DNA was prepared from the said culture using KURABO PI-1100
  • a sequence reaction mixture was prepared as follows.
  • the PCR product or cosmid DNA prepared in the above (4-1) is mixed with about 2 ⁇ l of DYEnami ⁇ ET Terminator Sequencing Kit (manufactured by Amersham Bioscience) and appropriate primers to give about 8 ⁇ l of reaction mixture.
  • An M13 forward (M13-21) primer and an M13 reverse (M13RV) primer (manufactured by Takara Bio) , are used for the sequence reaction of a PCR product derived from shotgun DNA, while a forward primer SS-cos F.I (SEQ ID NO: 7) and a reverse primer SS-cos R.I (SEQ ID NO: 8) are used for cosmid DNA.
  • Amounts of the primers and the DNA fragment were 3.2 pmol and 50 to 200 ng, respectively.
  • the said reaction solution was subjected to dye terminator sequence reaction of 60 cycles using a GeneAmp PCR System 9700.
  • S. pastorianus is believed to be a natural hybrid of S. cerevisiae with its closely-related species "Int. J. Syst. Bacteriol., volume 35, pages 508 to 511 (1985)". Therefore, a DNA sequence (comprising 10,044 bases) of both ends of the cosmid DNA clone obtained in (4-2) was subjected to a homology searching by a homology searching algorithm to S. cerevisiae genome sequence, whereupon for each DNA sequence alignment of homologous region on S. cerevisiae genome sequence and the identity thereof were determined to prepare a database. An identity distribution chart for cosmid DNA sequence with the corresponding genomic DNA sequence of S. cerevisiae is shown in Fig. 2.
  • the DNA sequence of cosmid was roughly classified into a DNA sequence group showing not less than 94% identity to the genomic DNA sequence of S. cerevisiae and a DNA sequence group showing around 84% identity thereto.
  • the DNA sequence group showing not less than 94% identity is named a DNA sequence of an Sc type derived from S. cerevisiae and the DNA sequence group showing around 84% identity is named a DNA sequence of a non-Sc type derived from genome of closely related species.
  • a comparative database (Table 1) was prepared for the DNA sequence of both ends of shotgun clone obtained in (4-1) with the genomic DNA sequence of S. cerevisiae.
  • Table 1 shows an example of the comparative database of DNA sequence of both ends of 3,648-cosmid clone with the genomic DNA sequence of S. cerevisiae. There are shown the homologous region and the identity of forward sequence and reverse sequence of cosmid subjected to the DNA sequence determination on each genomic DNA sequence of S. cerevisiae.
  • mapping of cosmid clone and shotgun clone on S. cerevisiae genome sequence was carried out (Fig. 3).
  • a comparative database (Table 1) of contig DNA sequence obtained in Example 5 with S. cerevisiae genome sequence was prepared, then mapping was carried out.
  • the means for the mapping was almost the same as the above-mentioned method, if forward and reverse sequences of cosmid and shotgun clones were present in different contigs, these contigs were connected by forward-reverse link (Fig. 4).
  • Example 7 Identification and assignment of function of ORF Identification of ORF (open reading frame) in the DNA sequence assembled in Example 5 was carried out. The examples are specifically shown below. Identification of ORF existing in the DNA sequence assembled in Example 5 was carried out using a available program using ORF finder (http;//www.ncbi.nih.gov/gorf/gorf.html) . The identification of ORF was done for six kinds of reading frames in the sequence with the length of not less than 150 bases from initiation codon to termination codon including its complementary sequence. Assignment of function of the extracted ORF was carried out by homology searching of amino acid sequence of ORF of S. cerevisiae that has been registered at the SGD and published.
  • Table 2 is a table showing examples of the name of ORF in S. cerevisiae corresponding to the result to assignment of function of ORF existing in the non-Sc genome. From the left side of the table, there are shown name of the ORF existing in the brewing yeast, ORF length in polynucleotide, ORF length in polypeptide, name of the ORF in S. cerevisiae determined by homology searching, identity, coincided length and functions of the gene.
  • the signal of each probe hybridized with the DNA of strain 34/70 is normalized to that of the haploid laboratory yeast strain S288C using an analysis soft ware (Microarray Suite 5.0: manufactured by Affymetrix) and shown as signal log ratio (2 n ).
  • Signal log ratios were lined following gene order in each chromosome using a spreadsheet program (Microsoft Excel 2000) and the signal log ratios are shown in bar graphs as shown in Fig. 5.
  • the non-Sc type genes do not hybridize to the S. cerevisiae array, therefore, the Sc type gene dosage affects the signal log ratio and the points where the signal log ratios show vigorous changes and which are considered to be translocation sites between Sc type and non-Sc type chromosome.
  • the chimera chromosome structure was confirmed by PCR where two pairs of primers having DNA sequences in which one side is Sc type while the other side is a non-Sc type (XVI-l(L)cer-95894 (SEQ ID NO: 9)/XVI-l(R)nonSc-106302rv (SEQ ID NO: 10) and XVI-2(L)cer- 859737 (SEQ ID NO: 11)/XVI-2(R)nonSc-864595rv (SEQ ID NO: 12)) were designed and the genomic DNA derived from strain 34/70 was used as a template. Two examples of translocation of chromosome XVI are shown as follows.
  • the PCR was carried out using Takara LA TaqTM and a buffer attached thereto in accordance with the attached manual by a Takara PCR Thermal Cycler SP.
  • Example 9 Cloning of SSUl genes of strain 34/70 The shotgun clone containing non-ScSSUl gene was retrieved using a comparative database obtained in Example 6. There was SSS103_G08 which comprises about 2.4 kb of fragment containing full-length of non-ScSSUl ORF, where identity of forward and reverse sequence of shotgun clone to those of S. cerevisiae were 62.9% and 82.9%, respectively.
  • SSS103-G08 was selected from a library of genomic DNA, then full length of non-ScSSUl was prepared by PCR.
  • Synthetic DNAs of SacI-non-Sc-SSUl_forl (SEQ ID NO: 13) and BgIII-non-Sc-SSUl_rvl460 (SEQ ID NO: 14) were used as primers.
  • base numbers 1 to 1460 of nonScSSUl was amplified to give a Sad-BgIII fragment of about 1.5 kb.
  • an ScSSUl gene full length of gene was obtained by PCR using a primer pair designed on the basis of the information of SGD using a genomic DNA of strain 34/70 as a template.
  • Synthetic DNAs of SacI-ScSSUl_forl (SEQ ID NO: 15) and BglII-ScSSUl_rvl406 (SEQ ID NO: 16) were used as primers.
  • base numbers 1 to 1406 of ScSSUl gene was amplified to give a Sad-BgIII fragment of about 1.4 kb.
  • ScSSUl and non-ScSSUl genes obtained as above were inserted using TA cloning kit (Invitrogen) into pCR 2.1- TOPO vector attached to the kit, and they were named TOPO/ScSSUl and TOPO/non-ScSSUl, respectively. Sequences of the resulting ScSSUl and non-ScSSUl genes were confirmed by a method of Sanger "F. Sanger, Science, volume 214, page 1215, 1981" (Fig. 10).
  • ScSSUl_rv SEQ ID NO: 20
  • a plasmid pPGAPAUR AURl-C
  • primers non-Sc-SSUl_for + pGAPAUR SEQ ID NO: 21
  • non-Sc-SSUl_rv + AURI-C SEQ ID NO: 22
  • the bottom fermenting yeast BH 96 was transformed using the DNA fragments for gene disruption prepared with above method.
  • the transformation was carried out by a method mentioned in the Japanese Patent Laid-Open Gazette No. H07/303.475 and concentrations of the drugs were 300 mg/L for geneticin, 50 mg/L for nourseothricin and 1 mg/L for aureobasidin A.
  • ScSSUl gene disruption and HindiII for the confirmation of non-ScSSUl gene disruption, and then fractionated by 1.5% agarose gel electrophoresis and transferred to a membrane. After that, hybridization was carried out (at 55 0 C for 18 hours) with a probe specific to the ScSSUl or non-ScSSUl following a protocol of the Alkphos Direct Labelling Reagents (Amersham) and signals were detected by CDP-Star.
  • Example 12 Overexpression of each SSUl gene From the plasmid TOPO/non-ScSSUl mentioned in Example 9, a fragment of about 1.5 kb including the full length of non-ScSSUl ORF was excised by a treatment with a restriction enzyme (Sacl-Bglll) . Then this fragment was inserted into a plasmid pNI-NUT which was similarly treated with a restriction enzyme (Sad-BgIII) to construct a non- ScSSUl overexpression vector pYI-non-ScSSUl.
  • a restriction enzyme Sad-Bglll
  • the vector pNI-NUT contains URA3 as a homologous recombination site and nourseothricin-resistance gene (natl) and ampicillin- resistance gene (Amp r ) as selective markers.
  • the ScSSUl overexpression vector pNI-ScSSUl has a structure wherein the non-ScSSUl gene of the above- mentioned pYI-non-ScSSUl has been substituted with the SSUl-R of about 2 kb derived from S. cerevisiae "J. Ferment. Bioeng., volume 86, page . 427 (1998)".
  • promoter and terminator of glyceraldehyde-3-phosphate dehydrogenase gene were used for overexpression of each SSUl gene.
  • Bottom fermenting yeast BH225 was transformed with a overexpression vector prepared in the above-mentioned method. Transformation was carried out by a method mentioned in the Japanese Patent Laid-Open Gazette No. H07/303,475 and selected on YPD plate medium containing 50 mg/L of nourseothricin.
  • PCR product was fractionated by 1.2% agarose gel electrophoresis, stained with an ethidium bromide solution and signal value of each SSUl gene of transformant was normalized with a signal value of PDA 1 and compared with that of the parental strain.
  • the overexpressed strains confirmed as such were named as ScSSUl overexpressed strain and non-ScSSUl overexpressed strain.
  • DNA sequence of non-Sc MET14 gene was retrieved from the comparative database obtained in Example 6.
  • a shotgun clone SSS 134_021 containing about 1.9 kb (full-length) of non-Sc MET14 gene was obtained; its forward and reverse DNA sequence identity to S. cerevisiae were 79.0% and 56.0%, respectively.
  • the shotgun clone 134_021 was selected from shotgun library and a full length of non-Sc MET14 gene was obtained by PCR.
  • synthetic DNAs of SacI-nonSc- MET14_for-21 SEQ ID NO: 29
  • BamHI-nonSc-MET14_rv618 SEQ ID NO: 30
  • a non-Sc MET14 gene about 0.6 kb embraced by Sad and BamHI restriction sites was obtained.
  • Sc MET14 gene a full length of the structural gene was obtained by PCR using a primer pair designed on the basis of the information of SGD and using genomic DNA of strain 34/70 as a template. Synthetic DNAs of SacI-ScMET14_for (SEQ ID NO: 31) and BamHI-ScMET14_rv (SEQ ID NO: 32) were used as primers. As a result of using such a pair of primers, Sc METl4 gene (about 0.6 kb) embraced by Sacl and BamHI restriction sites was obtained.
  • Example 15 Overexpression of each MET14 gene in Sc SSUl overexpressed strain A fragment of about 0.6 kb containing Sc MET14 or non- Sc MET14 mentioned in Example 14 was inserted into a multi- cloning site of a expression vector pUP3GLP (Japanese Patent Laid-Open Gazette No. 2000/316,559) to construct overexpression vectors pUP3Sc MET14 and pUP3nonSc-MET14 in which each MET14 gene was expressed under control of glyceraldehyde-3-phosphate dehydrogenase promoter and terminator.
  • pUP3GLP Japanese Patent Laid-Open Gazette No. 2000/316,559
  • top fermenting yeast strain KN009F
  • Sc SSUl overexpression vector pNI-SSUl mentioned in Example 12
  • strain FOY227 which is an Sc SSUl overexpressed strain.
  • the strain FOY227 was transformed with the above pUP3ScMET14 and pUP3nonSc-MET14 to prepare strain FOY306 and strain FOY 307 in which Sc MET14 and non-Sc MET14, together with Sc SSUl, are overexpressed, respectively.
  • Fermentation tests were carried out under the following condition using strains prepared in Example 15; strain FOY227 which is Sc SSUl overexpressed strain, strain FOY306 which is Sc MET14 overexpressed strain in strain FOY227, strain FOY307 which is a non-Sc MET14 overexpressed strain in strain FOY227and the parental strain KN009 F.
  • DNA array of bottom fermenting yeast was produced based on the DNA sequence information of the ORFs obtained in the above (h) and intergenic DNA sequences located between ORFs deduced from the whole genome sequence of strain 34/70.
  • PM probe Perfect Match Probe; 25 base long
  • GeneChip® GeneChip® (Affymetrix) technology
  • mismatch probe that has sequences identical to PM probe with the exception of one mismatched base, which substitutes the complementary base (adenine (A) to thymine (T) and vice versa, or guanine (G) to cytosine (C) and vice versa) at the central position (i.e. base 13), was also designed.
  • Group (1) consists of:
  • C) 28 DNA sequences of mitochondrial ORFs from 34/70 strain represented from SEQ ID NOS: 166154 to 166181; D) 553 DNA sequences represented from SEQ ID NOS: 166490 to 167042, which have not been identified as the above ORFs but have significant similarity (E value ⁇ 10e- 7) to the proteins of S. cerevisiae using NCBI-BlastX homology searching,
  • E-value is an expected value for a possibility that a query sequence is found in a data base by chance.
  • E-value 10 means that 10 sequences can be found accidentally. The smaller the E-value is, the lesser is the chance of finding the sequence by chance, and thus meaning that similarity between the found sequence in data base and the query sequence is significant.
  • the E-value as the result of homology searching such as NCBI-BLAST can be significant when it is less than 10 "7 , preferably less than 10 "10 , more preferably less than 10 "20 .
  • the function can be assigned as having the least value with the DNA sequences of the ORFs as the result of the homology searching.
  • Group (2) consists of:
  • YLR154W-F YLR155C, YLR156W, YLR157C-B, YLR157W-C, YLR162W, YLR205C, YLR207W, YLR209C, YLR227W-B, YLR236C, YLR237W, YLR238W, YLR245C, YLR251W, YLR271W, YLR278C, YLR287C-A, YLR305C, YLR306W, YLR311C, YLR317W, YLR338W, YLR344W, YLR345W, YLR354C, YLR364W, YLR380W, YLR401C, YLR402W,
  • YLR410W-B YLR411W, YLR412C-A, YLR412W, YLR413W, YLR448W, YLR460C, YLR461W, YLR463C, YLR465C, YML003W, YML039W, YMLO73C, YMRO87W, YMRl43W, YMRl75W-A, YMR247W-A, YMR268W-A, YMR324C, YMR325W, YNL020C, YNL035C, YNL054W-B, YNL243W, YNR034W-A, YNR075C-A, YNR077C, YOL038C-A, YOL053W, YOLlOlC, YOL103W-B, YOL162W, YOL163W, YOL164W,
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6237, 6238, 6239, 6240, 6241, 6242, 6243, 6244, 6245, 6246, 6247 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 33.
  • SEQ ID NOS: 6248, 6249, 6250, 6251, 6252, 6253, 6254, 6255, 6256, 6257, 6258 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 34.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6259, 6260, 6261, 6262, 6263, 6264, 6265, 6266, 6267, 6268, 6269 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 35.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6270, 6271, 6272, 6273, 6274, 6275, 6276, 6277, 6278, 6279, 6280 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 36.
  • SEQ ID NOS: 6281, 6282, 6283, 6284, 6285, 6286, 6287, 6288, 6289, 6290, 6291 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 37.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6292, 6293, 6294, 6295, 6296, 6297, 6298, 6299, 6300, 6301, 6302 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 38.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6303, 6304, 6305, 6306, 6307, 6308, 6309, 6310, 6311, 6312, 6313 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 39.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6314, 6315, 6316, 6317, 6318, 6319, 6320, 6321, 6322, 6323, 6324 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 40.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6325, 6326, 6327, 6328, 6329, 6330, 6331, 6332, 6333, 6334, 6335 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 41.
  • SEQ ID NOS: 6336, 6337, 6338, 6339, 6340, 6341, 6342, 6343, 6344, 6345, 6346 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 42.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6347, 6348, 6349, 6350, 6351, 6352, 6353, 6354, 6355, 6356, 6357 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 43.
  • SEQ ID NOS: 6358, 6359, 6360, 6361, 6362, 6363, 6364, 6365, 6366, 6367, 6368 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 44.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6369, 6370, 6371, 6372, 6373, 6374, 6375, 6376, 6377, 6378, 6379 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 45.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6380, 6381, 6382, 6383, 6384, 6385» 6386, 6387, 6388, 6389, 6390 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 46.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6391, 6392, 6393, 6394, 6395, 6396, 6397, 6398, 6399, 6400, 6401 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 47.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6402, 6403, 6404, 6405, 6406, 6407, 6408, 6409, 6410, 6411, 6412 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 48.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6402, 6403, 6404, 6405, 6406, 6407, 6408, 6409, 6410, 6411, 6412 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 48.
  • SEQ ID NOS: 6413, 6414, 6415, 6416, 6417, 6418, 6419, 6420, 6421, 6422, 6423 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 49.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6424, 6425, 6426, 6427, 6428, 6429, 6430, 6431, 6432, 6433, 6434 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 50.
  • SEQ ID NOS: 6435, 6436, 6437, 6438, 6439, 6440, 6441, 6442, 6443, 6444, 6445 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 51.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6446, 6447, 6448, 6449, 6450, 6451, 6452, 6453, 6454, 6455, 6456 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 52.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6457, 6458, 6459, 6460, 6461, 6462, 6463, 6464, 6465, 6466, 6467 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 53.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6468, 6469, 6470, 6471, 6472, 6473, 6474, 6475, 6476, 6477, 6478 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 54.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6479, 6480, 6481, 6482, 6483, 6484, 6485, 6486, 6487, 6488, 6489 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 55.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6479, 6480, 6481, 6482, 6483, 6484, 6485, 6486, 6487, 6488, 6489 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 55.
  • SEQ ID NOS: 6490, 6491, 6492, 6493, 6494, 6495, 6496, 6497, 6498, 6499, 6500 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 56.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6501, 6502, 6503, 6504, 6505, 6506, 6507, 6508, 6509, 6510, 6511 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 57.
  • SEQ ID NOS: 6512, 6513, 6514, 6515, 6516, 6517, 6518, 6519, 6520, 6521, 6522 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 58.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6523, 6524, 6525, 6526, 6527, 6528, 6529, 6530, 6531, 6532, 6533 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 59.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6534, 6535, 6536, 6537, 6538, 6539, 6540, 6541, 6542, 6543, 6544 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 60.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6545, 6546, 6547, 6548, 6549, 6550, 6551, 6552, 6553, 6554, 6555 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 61.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6556, 6557, 6558, 6559, 6560, 6561, 6562, 6563, 6564, 6565, 6566 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 62.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6556, 6557, 6558, 6559, 6560, 6561, 6562, 6563, 6564, 6565, 6566 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 62.
  • SEQ ID NOS: 6567, 6568, 6569, 6570, 6571, 6572, 6573, 6574, 6575, 6576, 6577 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 63.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6578, 6579, 6580, 6581, 6582, 6583, 6584, 6585, 6586, 6587, 6588 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 64.
  • SEQ ID NOS: 6589, 6590, 6591, 6592, 6593, 6594, 6595, 6596, 6597, 6598, 6599 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 65.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6600, 6601, 6602, 6603, 6604, 6605, 6606, 6607, 6608, 6609, 6610 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 66.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6611, 6612, 6613, 6614, 6615, 6616, 6617, 6618, 6619, 6620, 6621 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 67.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6622, 6623, 6624, 6625, 6626, 6627, 6628, 6629, 6630, 6631, 6632 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 68.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6633, 6634, 6635, 6636, 6637, 6638, 6639, 6640, 6641, 6642, 6643 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 69.
  • SEQ ID NOS: 6644, 6645, 6646, 6647, 6648, 6649, 6650, 6651, 6652, 6653, 6654 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 70.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6655, 6656, 6657, 6658, 6659, 6660, 6661, 6662, 6663, 6664, 6665 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 71.
  • SEQ ID NOS: 6666, 6667, 6668, 6669, 6670, 6671, 6672, 6673, 6674, 6675, 6676 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 72.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6677, 6678, 6679, 6680, 6681, 6682, 6683, 6684, 6685, 6686, 6687 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 73.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6688, 6689, 6690, 6691, 6692, 6693, 6694, 6695, 6696, 6697, 6698 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 74.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6699, 6700, 6701, 6702, 6703, 6704, 6705, 6706, 6707, 6708, 6709 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 75.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6710, 6711, 6712, 6713, 6714, 6715, 6716, 6717, 6718, 6719, 6720 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 76.
  • SEQ ID NOS: 6721, 6722, 6723, 6724, 6725, 6726, 6727, 6728, 6729, 6730, 6731 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 77.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6732, 6733, 6734, 6735, 6736, 6737, 6738, 6739, 6740, 6741, 6742 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 78.
  • SEQ ID NOS: 6743, 6744, 6745, 6746, 6747, 6748, 6749, 6750, 6751, 6752, 6753 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 79.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6754, 6755, 6756, 6757, 6758, 6759, 6760, 6761, 6762, 6763, 6764 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 80.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6765, 6766, 6767, 6768, 6769, 6770, 6771, 6772, 6773, 6774, 6775 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 81.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6776, 6777, 6778, 6779, 6780, 6781, 6782, 6783, 6784, 6785, 6786 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 82.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6787, 6788, 6789, 6790, 6791, 6792, 6793, 6794, 6795, 6796, 6797 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 83.
  • SEQ ID NOS: 6798, 6799, 6800, 6801, 6802, 6803, 6804, 6805, 6806, 6807, 6808 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 84.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6809, 6810, 6811, 6812, 6813, 6814, 6815, 6816, 6817, 6818, 6819 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 85.
  • SEQ ID NOS: 6820, 6821, 6822, 6823, 6824, 6825, 6826, 6827, 6828, 6829, 6830 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 86.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6831, 6832, 6833, 6834, 6835, 6836, 6837, 6838, 6839, 6840, 6841 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 87.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6842, 6843, 6844, 6845, 6846, 6847, 6848, 6849. 6850, 6851, 6852 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 88.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6853, 6854, 6855, 6856, 6857, 6858, 6859, 6860, 6861, 6862, 6863 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 89.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6864, 6865, 6866, 6867, 6868, 6869, 6870, 6871, 6872, 6873, 6874 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 90.
  • SEQ ID NOS: 6875, 6876, 6877, 6878, 6879, 6880, 6881, 6882, 6883, 6884, 6885 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 91.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6886, 6887, 6888, 6889, 6890, 6891, 6892, 6893, 6894, 6895, 6896 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 92.
  • SEQ ID NOS: 6897, 6898, 6899, 6900, 6901, 6902, 6903, 6904, 6905, 6906, 6907 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 93.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6908, 6909, 6910, 6911, 6912, 6913, 6914, 6915, 6916, 6917, 6918 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 94.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6919, 6920, 6921, 6922, 6923, 6924, 6925, 6926, 6927, 6928, 6929 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 95.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6930, 6931, 6932, 6933, 6934, 6935, 6936, 6937, 6938, 6939, 6940 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 96.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6941, 6942, 6943, 6944, 6945, 6946, 6947, 6948, 6949, 6950, 6951 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 97.
  • SEQ ID NOS: 6952, 6953, 6954, 6955, 6956, 6957, 6958, 6959, 6960, 6961, 6962 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 98.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6963, 6964, 6965, 6966, 6967, 6968, 6969, 6970, 6971, 6972, 6973 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 99.
  • SEQ ID NOS: 6974, 6975, 6976, 6977, 6978, 6979, 6980, 6981, 6982, 6983, 6984 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 100.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6985, 6986, 6987, 6988, 6989, 6990, 6991, 6992, 6993, 6994, 6995 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 101.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 6996, 6997, 6998, 6999, 7000, 7001, 7002, 7003, 7004, 7005, 7006 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 102.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7007, 7008, 7009, 7010, 7011, 7012, 7013, 7014, 7015, 7016, 7017 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 103.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7018, 7019, 7020, 7021, 7022, 7023, 7024, 7025, 7026, 7027, 7028 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 104.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7018, 7019, 7020, 7021, 7022, 7023, 7024, 7025, 7026, 7027, 7028 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 104.
  • SEQ ID NOS: 7029, 7030, 7031, 7032, 7033, 7034, 7035, 7036, 7037, 7038, 7039 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 105.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7040, 7041, 7042, 7043, 7044, 7045, 7046, 7047, 7048, 7049, 7050 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 106.
  • SEQ ID NOS: 7051, 7052, 7053, 7054, 7055, 7056, 7057, 7058, 7059, 7060, 7061 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 107.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7062, 7063, 7064, 7065, 7066, 7067, 7068, 7069, 7070, 7071, 7072 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 108.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7073, 7074, 7075, 7076, 7077, 7078, 7079, 7080, 7081, 7082, 7083 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 109.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7084, 7085, 7086, 7087, 7088, 7089, 7090, 7091, 7092, 7093, 7094 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 110.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7095, 7096, 7097, 7098, 7099, 7100, 7101, 7102, 7103, 7104, 7105 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 111.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7095, 7096, 7097, 7098, 7099, 7100, 7101, 7102, 7103, 7104, 7105 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 111.
  • SEQ ID NOS: 7106, 7107, 7108, 7109, 7110, 7111, 7112, 7113, 7114, 7115, 7116 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 112.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7117, 7118, 7119, 7120, 7121, 7122, 7123, 7124, 7125 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 113.
  • SEQ ID NOS: 7126, 7127, 7128, 7129, 7130, 7131, 7132, 7133, 7134, 7135, 7136 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 114.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7137, 7138, 7139, 7140, 7141, 7142, 7143, 7144, 7145, 7146, 7147 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 115.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7148, 7149, 7150, 7151, 7152, 7153, 7154, 7155, 7156, 7157, 7158 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 116.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7159, 7160, 7161, 7162, 7163, 7164, 7165, 7166, 7167, 7168, 7169 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 117.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7170, 7171, 7172, 7173, 7174, 7175, 7176, 7177, 7178, 7179, 7180 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 118.
  • SEQ ID NOS: 7181, 7182, 7183, 7184, 7185, 7186, 7187, 7188, 7189, 7190, 7191 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 119.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7192, 7193, 7194, 7195, 7196, 7197, 7198, 7199, 7200, 7201, 7202 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 120.
  • SEQ ID NOS: 7203, 7204, 7205, 7206, 7207, 7208, 7209, 7210, 7211, 7212, 7213 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 121.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7214, 7215, 7216, 7217, 7218, 7219, 7220, 7221, 7222, 7223, 7224 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 122.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7225, 7226, 7227, 7228, 7229, 7230, 7231, 7232, 7233, 7234, 7235 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 123.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7236, 7237, 7238, 7239, 7240, 7241, 7242, 7243, 7244, 7245, 7246 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 124.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 125.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7247, 7248, 7249, 7250, 7251, 7252, 7253, 7254, 7255, 7256, 7257 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 125.
  • SEQ ID NOS: 7258, 7259, 7260, 7261, 7262, 7263, 7264, 7265, 7266, 7267, 7268 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 126.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7269, 7270, 7271, 7272, 7273, 7274, 7275, 7276, 7277, 7278, 7279 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 127.
  • SEQ ID NOS: 7280, 7281, 7282, 7283, 7284, 7285, 7286, 7287, 7288, 7289, 7290 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 128.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7291, 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7299, 7300, 7301 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 129.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7302, 7303, 7304, 7305, 7306, 7307, 7308, 7309, 7310, 7311, 7312 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 130.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7313, 7314, 7315, 7316, 7317, 7318, 7319, 7320, 7321, 7322, 7323 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 131.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7324, 7325, 7326, 7327, 7328, 7329, 7330, 7331, 7332, 7333, 7334 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 132.
  • SEQ ID NOS: 7335, 7336, 7337, 7338, 7339, 7340, 7341, 7342, 7343, 7344, 7345 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 133.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7346, 7347, 7348, 7349, 7350, 7351, 7352, 7353, 7354, 7355, 7356 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 134.
  • SEQ ID NOS: 7357, 7358, 7359, 7360, 7361, 7362, 7363, 7364, 7365, 7366, 7367 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 135.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7368, 7369, 7370, 7371, 7372, 7373, 7374, 7375, 7376, 7377, 7378 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 136.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7379, 7380, 7381, 7382, 7383, 7384, 7385, 7386, 7387, 7388, 7389 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 137.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7390, 7391, 7392, 7393, 7394, 7395, 7396, 7397, 7398, 7399, 7400 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 138.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7401, 7402, 7403, 7404, 7405, 7406, 7407, 7408, 7409, 7410, 7411 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 139.
  • SEQ ID NOS: 7412, 7413, 7414, 7415, 7416, 7417, 7418, 7419, 7420, 7421, 7422 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 140.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7423, 7424, 7425, 7426, 7427, 7428, 7429, 7430, 7431, 7432, 7433 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 141.
  • SEQ ID NOS: 7434, 7435, 7436, 7437, 7438, 7439, 7440, 7441, 7442, 7443, 7444 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 142.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7445, 7446, 7447, 7448, 7449, 7450, 7451, 7452, 7453, 7454, 7455 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 143.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7456, 7457, 7458, 7459, 7460, 7461, 7462, 7463, 7464, 7465, 7466 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 144.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7467, 7468, 7469, 7470, 7471, 7472, 7473, 7474, 7475, 7476, 7477 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 145.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7478, 7479, 7480, 7481, 7482, 7483, 7484, 7485, 7486, 7487, 7488 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 146.
  • SEQ ID NOS: 7489, 7490, 7491, 7492, 7493, 7494, 7495, 7496, 7497, 7498, 7499 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 147.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7500, 7501, 7502, 7503, 7504, 7505, 7506, 7507, 7508, 7509, 7510 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 148.
  • SEQ ID NOS: 7511, 7512, 7513, 7514, 7515, 7516, 7517, 7518, 7519, 7520, 7521 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 149.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7522, 7523, 7524, 7525, 7526, 7527, 7528, 7529, 7530, 7531, 7532 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 150.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7533, 7534, 7535, 7536, 7537, 7538, 7539, 7540, 7541, 7542, 7543 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 151.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7544, 7545, 7546, 7547, 7548, 7549, 7550, 7551, 7552, 7553, 7554 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 152.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7555, 7556, 7557, 7558, 7559, 7560, 7561, 7562, 7563, 7564, 7565 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 153.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7555, 7556, 7557, 7558, 7559, 7560, 7561, 7562, 7563, 7564, 7565 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 153.
  • SEQ ID NOS: 7566, 7567, 7568, 7569, 7570, 7571, 7572, 7573, 7574, 7575, 7576 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 154.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7577, 7578, 7579, 7580, 7581, 7582, 7583, 7584, 7585, 7586, 7587 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 155.
  • SEQ ID NOS: 7588, 7589, 7590, 7591, 7592, 7593, 7594, 7595, 7596, 7597, 7598 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 156.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7599, 7600, 7601, 7602, 7603, 7604, 7605, 7606, 7607, 7608, 7609 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 157.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7610, 7611, 7612, 7613, 7614, 7615, 7616, 7617, 7618, 7619, 7620 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 158.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7621, 7622, 7623, 7624, 7625, 7626, 7627, 7628, 7629, 7630, 7631 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 159.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7632, 7633, 7634, 7635, 7636, 7637, 7638, 7639, 7640, 7641, 7642 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 160.
  • SEQ ID NOS: 7643, 7644, 7645, 7646, 7647, 7648, 7649, 7650, 7651, 7652, 7653 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 161.
  • the probes having the nucleotide sequence represented, by SEQ ID NOS: 7654, 7655, 7656, 7657, 7658, 7659, 7660, 7661, 7662, 7663, 7664 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 162.
  • SEQ ID NOS: 7665, 7666, 7667, 7668, 7669, 7670, 7671, 7672, 7673, 7674, 7675 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 163.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7676, 7677, 7678, 7679, 7680, 7681, 7682, 7683, 7684, 7685, 7686 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 164.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7687, 7688, 7689, 7690, 7691, 7692, 7693, 7694, 7695, 7696, 7697 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 165.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7698, 7699, 7700, 7701, 7702, 7703, 7704, 7705, 7706, 7707, 7708 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 166.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7709, 7710, 7711, 7712, 7713, 7714, 7715, 7716, 7717, 7718, 7719 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 167.
  • SEQ ID NOS: 7720, 7721, 7722, 7723, 7724, 7725, 7726, 7727, 7728, 7729, 7730 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 168.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7731, 7732, 7733, 7734, 7735, 7736, 7737, 7738, 7739, 7740, 7741 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 169.
  • SEQ ID NOS: 7742, 7743, 7744, 7745, 7746, 7747, 7748, 7749, 7750, 7751, 7752 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 170.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7753, 7754, 7755, 7756, 7757, 7758, 7759, 7760, 7761, 7762, 7763 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 171.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7764, 7765, 7766, 7767, 7768, 7769, 7770, 7771, 7772, 7773, 7774 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 172.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7775, 7776, 7777, 7778, 7779, 7780, 7781, 7782, 7783, 7784, 7785 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 173.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7786, 7787, 7788, 7789, 7790, 7791, 7792, 7793, 7794, 7795, 7796 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 174.
  • SEQ ID NOS: 7797, 7798, 7799, 7800, 7801, 7802, 7803, 7804, 7805, 7806, 7807 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 175.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7808, 7809, 7810, 7811, 7812, 7813, 7814, 7815, 7816, 7817, 7818 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 176.
  • SEQ ID NOS: 7819, 7820, 7821, 7822, 7823, 7824, 7825, 7826, 7827, 7828, 7829 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 177.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7830, 7831, 7832, 7833, 7834, 7835, 7836, 7837, 7838, 7839, 7840 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 178.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7841, 7842, 7843, 7844, 7845, 7846, 7847, 7848, 7849, 7850, 7851 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 179.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7852, 7853, 7854, 7855, 7856, 7857, 7858, 7859, 7860, 7861, 7862 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 180.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7863, 7864, 7865, 7866, 7867, 7868, 7869, 7870, 7871, 7872, 7873 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 181.
  • SEQ ID NOS: 7874, 7875, 7876, 7877, 7878, 7879, 7880, 7881, 7882, 7883, 7884 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 182.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7885, 7886, 7887, 7888, 7889, 7890, 7891, 7892, 7893, 7894, 7895 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 183.
  • SEQ ID NOS: 7896, 7897, 7898, 7899, 7900, 7901, 7902, 7903, 7904, 7905, 7906 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 184.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7907, 7908, 7909, 7910, 7911, 7912, 7913, 7914, 7915, 7916, 7917 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 185.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7918, 7919, 7920, 7921, 7922, 7923, 7924, 7925, 7926, 7927, 7928 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 186.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7929, 7930, 7931, 7932, 7933, 7934, 7935, 7936, 7937, 7938, 7939 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 187.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7940, 7941, 7942, 7943, 7944, 7945, 7946, 7947, 7948, 7949, 7950 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 188.
  • SEQ ID NOS: 7951, 7952, 7953, 7954, 7955, 7956, 7957, 7958, 7959, 7960, 7961 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 189.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7962, 7963, 7964, 7965, 7966, 7967, 7968, 7969, 7970, 7971, 7972 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 190.
  • SEQ ID NOS: 7973, 7974, 7975, 7976, 7977, 7978, 7979, 7980, 7981, 7982, 7983 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 191.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7984, 7985, 7986, 7987, 7988, 7989, 7990, 7991, 7992, 7993, 7994 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 192.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 7995, 7996, 7997, 7998, 7999, 8000, 8001, 8002, 8003, 8004, 8005 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 193.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8006, 8007, 8008, 8009, 8010, 8011, 8012, 8013, 8014, 8015, 8016 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 194.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 195.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 195.
  • SEQ ID NOS: 8028, 8029, 8030, 8031, 8032, 8033, 8034, 8035, 8036, 8037, 8038 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 196.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8039, 8040, 8041, 8042, 8043, 8044, 8045, 8046, 8047, 8048, 8049 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 197.
  • SEQ ID NOS: 8050, 8051, 8052, 8053, 8054, 8055, 8056, 8057, 8058, 8059, 8060 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 198.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8061, 8062, 8063, 8064, 8065, 8066, 8067, 8068, 8069, 8070, 8071 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 199.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8072, 8073, 8074, 8075, 8076, 8077, 8078, 8079, 8080, 8081, 8082 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 200.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8083, 8084, 8085, 8086, 8087, 8088, 8089, 8090, 8091, 8092, 8093 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 201.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8094, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 202.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8094, 8095, 8096, 8097, 8098, 8099, 8100, 8101, 8102, 8103, 8104 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 202.
  • SEQ ID NOS: 8105, 8106, 8107, 8108, 8109, 8110, 8111, 8112, 8113, 8114, 8115 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 203.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8116, 8117, 8118, 8119, 8120, 8121, 8122, 8123, 8124, 8125, 8126 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 204.
  • SEQ ID NOS: 8127, 8128, 8129, 8130, 8131, 8132, 8133, 8134, 8135, 8136, 8137 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 205.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8138, 8139, 8140, 8141, 8142, 8143, 8144, 8145, 8146, 8147, 8148 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 206.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8149, 8150, 8151, 8152, 8153, 8154, 8155, 8156, 8157, 8158, 8159 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 207.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8160, 8161, 8162, 8163, 8164, 8165, 8166, 8167, 8168, 8169, 8170 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 208.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8171, 8172, 8173, 8174, 8175, 8176, 8177, 8178, 8179, 8180, 8181 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 209.
  • SEQ ID NOS: 8182, 8183, 8184, 8185, 8186, 8187, 8188, 8189, 8190, 8191, 8192 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 210.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8193, 8194, 8195, 8196, 8197, 8198, 8199, 8200, 8201, 8202, 8203 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 211.
  • SEQ ID NOS: 8204, 8205, 8206, 8207, 8208, 8209, 8210, 8211, 8212; 8213, 8214 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 212.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8215, 8216, 8217, 8218, 8219, 8220, 8221, 8222, 8223, 8224, 8225 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 213.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8226, 8227, 8228, 8229, 8230, 8231, 8232, 8233, 8234, 8235, 8236 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 214.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8237, 8238, 8239, 8240, 8241, 8242, 8243, 8244, 8245, 8246, 8247 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 215.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8248, 8249, 8250, 8251, 8252, 8253, 8254, 8255, 8256, 8257, 8258 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 216.
  • SEQ ID NOS: 8259, 8260, 8261, 8262, 8263, 8264, 8265, 8266, 8267, 8268, 8269 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 217.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8270, 8271, 8272, 8273, 8274, 8275, 8276, 8277, 8278, 8279, 8280 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 218.
  • SEQ ID NOS: 8281, 8282, 8283, 8284, 8285, 8286, 8287, 8288, 8289, 8290, 8291 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 219.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8292, 8293, 8294, 8295, 8296, 8297, 8298, 8299, 8300, 8301, 8302 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 220.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8303, 8304, 8305, 8306, 8307, 8308, 8309, 8310, 8311, 8312, 8313 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 221.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8314, 8315, 8316, 8317, 8318, 8319, 8320, 8321, 8322, 8323, 8324 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 222.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8325, 8326, 8327, 8328, 8329, 8330, 8331, 8332, 8333, 8334, 8335 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 223.
  • SEQ ID NOS: 8336, 8337, 8338, 8339, 8340, 8341, 8342, 8343, 8344, 8345, 8346 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 224.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8347, 8348, 8349, 8350, 8351, 8352, 8353, 8354, 8355, 8356, 8357 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 225.
  • SEQ ID NOS: 8358, 8359, 8360, 8361, 8362, 8363, 8364, 8365, 8366, 8367, 8368 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 226.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8369, 8370, 8371, 8372, 8373, 8374, 8375, 8376, 8377, 8378, 8379 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 227.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8380, 8381, 8382, 8383, 8384, 8385, 8386, 8387, 8388, 8389, 8390 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 228.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8391, 8392, 8393, 8394, 8395, 8396, 8397, 8398, 8399, 8400, 8401 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 229.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8402, 8403, 8404, 8405, 8406, 8407, 8408, 8409, 8410, 8411, 8412 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 230.
  • SEQ ID NOS: 8413, 8414, 8415, 8416, 8417, 8418, 8419, 8420, 8421, 8422, 8423 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 231.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8424, 8425, 8426, 8427, 8428, 8429, 8430, 8431, 8432, 8433, 8434 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 232.
  • SEQ ID NOS: 8435, 8436, 8437, 8438, 8439, 8440, 8441, 8442, 8443, 8444, 8445 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 233.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8446, 8447, 8448, 8449, 8450, 8451, 8452, 8453, 8454, 8455, 8456 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 234.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8457, 8458, 8459, 8460, 8461, 8462, 8463, 8464, 8465, 8466, 8467 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 235.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8468, 8469, 8470, 8471, 8472, 8473, 8474, 8475, 8476, 8477, 8478 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 236.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8479, 8480, 8481, 8482, 8483, 8484, 8485, 8486, 8487, 8488, 8489 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 237.
  • SEQ ID NOS: 8490, 8491, 8492, 8493, 8494, 8495, 8496, 8497, 8498, 8499, 8500 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 238.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8501, 8502, 8503, 8504, 8505, 8506, 8507, 8508, 8509, 8510, 8511 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 239.
  • SEQ ID NOS: 8512, 8513, 8514, 8515, 8516, 8517, 8518, 8519, 8520, 8521, 8522 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 240.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8523, 8524, 8525, 8526, 8527, 8528, 8529, 8530, 8531, 8532, 8533 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 241.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8534, 8535, 8536, 8537, 8538, 8539, 8540, 8541, 8542, 8543, 8544 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 242.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8545, 8546, 8547, 8548, 8549, 8550, 8551, 8552, 8553, 8554, 8555 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 243.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8556, 8557, 8558, 8559, 8560, 8561, 8562, 8563, 8564, 8565, 8566 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 244.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8556, 8557, 8558, 8559, 8560, 8561, 8562, 8563, 8564, 8565, 8566 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 244.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 244 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 244.
  • SEQ ID NOS: 8567, 8568, 8569, 8570, 8571, 8572, 8573, 8574, 8575, 8576, 8577 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 245.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8578, 8579, 8580, 8581, 8582, 8583, 8584, 8585, 8586, 8587, 8588 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 246.
  • SEQ ID NOS: 8589, 8590, 8591, 8592, 8593, 8594, 8595, 8596, 8597, 8598, 8599 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 247.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8600, 8601, 8602, 8603, 8604, 8605, 8606, 8607, 8608, 8609, 8610 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 248.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8611, 8612, 8613, 8614, 8615, 8616, 8617, 8618, 8619, 8620, 8621 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 249.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8622, 8623, 8624, 8625, 8626, 8627, 8628, 8629, 8630, 8631, 8632 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 250.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8633, 8634, 8635, 8636, 8637, 8638, 8639, 8640, 8641, 8642, 8643 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 251.
  • SEQ ID NOS: 8644, 8645, 8646, 8647, 8648, 8649, 8650, 8651, 8652, 8653, 8654 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 252.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8655, 8656, 8657, 8658, 8659, 8660, 8661, 8662, 8663, 8664, 8665 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 253.
  • SEQ ID NOS: 8666, 8667, 8668, 8669, 8670, 8671, 8672, 8673, 8674, 8675, 8676 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 254.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8677, 8678, 8679, 8680, 8681, 8682, 8683, 8684, 8685, 8686, 8687 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 255.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8688, 8689, 8690, 8691, 8692, 8693, 8694, 8695, 8696, 8697, 8698 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 256.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8699, 8700, 8701, 8702, 8703, 8704, 8705, 8706, 8707, 8708, 8709 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 257.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8710, 8711, 8712, 8713, 8714, 8715, 8716, 8717, 8718, 8719, 8720 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 258.
  • SEQ ID NOS: 8721, 8722, 8723, 8724, 8725, 8726, 8727, 8728, 8729, 8730, 8731 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 259.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8732, 8733, 8734, 8735, 8736, 8737, 8738, 8739, 8740, 8741, 8742 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 260.
  • SEQ ID NOS: 8743, 8744, 8745, 8746, 8747, 8748, 8749, 8750, 8751, 8752, 8753 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 261.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8754, 8755, 8756, 8757, 8758, 8759, 8760, 8761, 8762, 8763, 8764 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 262.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8765, 8766, 8767, 8768, 8769, 8770, 8771, 8772, 8773, 8774, 8775 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 263.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8776, 8777, 8778, 8779, 8780, 8781, 8782, 8783, 8784, 8785, 8786 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 264.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8787, 8788, 8789, 8790, 8791, 8792, 8793, 8794, 8795, 8796, 8797 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 265.
  • SEQ ID NOS: 8798, 8799, 8800, 8801, 8802, 8803, 8804, 8805, 8806, 8807, 8808 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 266.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8809, 8810, 8811, 8812, 8813, 8814, 8815, 8816, 8817, 8818, 8819 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 267.
  • SEQ ID NOS: 8820, 8821, 8822, 8823, 8824, 8825, 8826, 8827, 8828, 8829, 8830 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 268.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8831, 8832, 8833, 8834, 8835, 8836, 8837, 8838, 8839, 8840, 8841 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 269.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8842, 8843, 8844, 8845, 8846, 8847, 8848, 8849, 8850, 8851, 8852 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 270.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8853, 8854, 8855, 8856, 8857, 8858, 8859, 8860, 8861, 8862, 8863 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 271.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8864, 8865, 8866, 8867, 8868, 8869, 8870, 8871, 8872, 8873, 8874 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 272.
  • SEQ ID NOS: 8875, 8876, 8877, 8878, 8879, 8880, 8881, 8882, 8883, 8884, 8885 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 273.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8886, 8887, 8888, 8889, 8890, 8891, 8892, 8893, 8894, 8895, 8896 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 274.
  • SEQ ID NOS: 8897, 8898, 8899, 8900, 8901, 8902, 8903, 8904, 8905, 8906, 8907 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 275.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8908, 8909, 8910, 8911, 8912, 8913, 8914, 8915, 8916, 8917, 8918 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 276.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8919, 8920, 8921, 8922, 8923, 8924, 8925, 8926, 8927, 8928, 8929 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 277.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8930, 8931, 8932, 8933, 8934, 8935, 8936, 8937, 8938, 8939, 8940 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 278.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8941, 8942, 8943, 8944, 8945, 8946, 8947, 8948, 8949, 8950, 8951 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 279.
  • SEQ ID NOS: 8952, 8953, 8954, 8955, 8956, 8957, 8958, 8959, 8960, 8961, 8962 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 280.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8963, 8964, 8965, 8966, 8967, 8968, 8969, 8970, 8971, 8972, 8973 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 281.
  • SEQ ID NOS: 8974, 8975, 8976, 8977, 8978, 8979, 8980, 8981, 8982, 8983, 8984 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 282.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8985, 8986, 8987, 8988, 8989, 8990, 8991, 8992, 8993, 8994, 8995 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 283.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 8996, 8997, 8998, 8999, 9000, 9001, 9002, 9003, 9004, 9005, 9006 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 284.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9007, 9008, 9009, 9010, 9011, 9012, 9013, 9014, 9015, 9016, 9017 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 285.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 286.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 286.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9018, 9019, 9020, 9021, 9022, 9023, 9024, 9025, 9026, 9027, 9028 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 286.
  • SEQ ID NOS: 9029, 9030, 9031, 9032, 9033, 9034, 9035, 9036, 9037, 9038, 9039 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 287.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9040, 9041, 9042, 9043, 9044, 9045, 9046, 9047, 9048, 9049, 9050 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 288.
  • SEQ ID NOS: 9051, 9052, 9053, 9054, 9055, 9056, 9057, 9058, 9059, 9060, 9061 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 289.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9062, 9063, 9064, 9065, 9066, 9067, 9068, 9069, 9070, 9071, 9072 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 290.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9073, 9074, 9075, 9076, 9077, 9078, 9079, 9080, 9081, 9082, 9083 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 291.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9084, 9085, 9086, 9087, 9088, 9089, 9090, 9091, 9092, 9093, 9094 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 292.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9095, 9096, 9097, 9098, 9099, 9100, 9101, 9102, 9103, 9104, 9105 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 293.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9095, 9096, 9097, 9098, 9099, 9100, 9101, 9102, 9103, 9104, 9105 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 293.
  • SEQ ID NOS: 9106, 9107, 9108, 9109, 9110, 9111, 9112, 9113, 9114, 9115, 9116 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 294.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9117, 9118, 9119, 9120, 9121, 9122, 9123, 9124, 9125, 9126, 9127 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 295.
  • SEQ ID NOS: 9128, 9129, 9130, 9131, 9132, 9133, 9134, 9135, 9136, 9137, 9138 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 296.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9139, 9140, 9141, 9142, 9143, 9144, 9145, 9146, 9147, 9148, 9149 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 297.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9150, 9151, 9152, 9153, 9154, 9155, 9156, 9157, 9158, 9159, 9160 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 298.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9161, 9162, 9163, 9164, 9165, 9166, 9167, 9168, 9169, 9170, 9171 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 299.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9172, 9173, 9174, 9175, 9176, 9177, 9178, 9179, 9180, 9181, 9182 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 300.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9172, 9173, 9174, 9175, 9176, 9177, 9178, 9179, 9180, 9181, 9182 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 300.
  • SEQ ID NOS: 9183, 9184, 9185, 9186, 9187, 9188, 9189, 9190, 9191, 9192, 9193 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 301.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9194, 9195, 9196, 9197, 9198, 9199, 9200, 9201, 9202, 9203, 9204 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 302.
  • SEQ ID NOS: 9205, 9206, 9207, 9208, 9209, 9210, 9211, 9212, 9213, 9214, 9215 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 303.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9216, 9217, 9218, 9219, 9220, 9221, 9222, 9223, 9224, 9225, 9226 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 304.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9227, 9228, 9229, 9230, 9231, 9232, 9233, 9234, 9235, 9236, 9237 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 305.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9238, 9239, 9240, 9241, 9242, 9243, 9244, 9245, 9246, 9247, 9248 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 306.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9249, 9250, 9251, 9252, 9253, 9254, 9255, 9256, 9257, 9258, 9259 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 307.
  • SEQ ID NOS: 9260, 9261, 9262, 9263, 9264, 9265, 9266, 9267, 9268, 9269, 9270 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 308.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9271, 9272, 9273, 9274, 9275, 9276, 9277, 9278, 9279, 9280, 9281 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 309.
  • SEQ ID NOS: 9282, 9283, 9284, 9285, 9286, 9287, 9288, 9289, 9290, 9291, 9292 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 310.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9293, 9294, 9295, 9296, 9297, 9298, 9299, 9300, 9301, 9302, 9303 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 311.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9304, 9305, 9306, 9307, 9308, 9309, 9310, 9311, 9312, 9313, 9314 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 312.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9315, 9316, 9317, 9318, 9319, 9320, 9321, 9322, 9323, 9324, 9325 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 313.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9326, 9327, 9328, 9329, 9330, 9331, 9332, 9333, 9334, 9335, 9336 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 314.
  • SEQ ID NOS: 9337, 9338, 9339, 9340, 9341, 9342, 9343, 9344, 9345, 9346, 9347, 9348, 9349, 9350, 9351, 9352, 9353, 9354, 9355, 9356, 9357, 9358 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 315.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9359, 9360, 9361, 9362, 9363, 9364, 9365, 9366, 9367, 9368, 9369 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 316.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9370, 9371, 9372, 9373, 9374, 9375, 9376, 9377, 9378, 9379, 9380 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 317.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9381, 9382, 9383, 9384, 9385, 9386, 9387, 9388, 9389, 9390, 9391 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 318.
  • SEQ ID NOS: 9392, 9393, 9394, 9395, 9396, 9397, 9398, 9399, 9400, 9401, 9402 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 319.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9403, 9404, 9405, 9406, 9407, 9408, 9409, 9410, 9411, 9412, 9413 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 320.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9414, 9415, 9416, 9417, 9418, 9419, 9420, 9421, 9422, 9423, 9424 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 321.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9425, 9426, 9427, 9428, 9429, 9430, 9431, 9432, 9433, 9434, 9435 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 322.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9436, 9437, 9438, 9439, 9440, 9441, 9442, 9443, 9444, 9445, 9446 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 323.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9447, 9448, 9449, 9450, 9451, 9452, 9453, 9454, 9455, 9456, 9457 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 324.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9458, 9459, 9460, 9461, 9462, 9463, 9464, 9465, 9466, 9467, 9468 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 325.
  • SEQ ID NOS: 9469, 9470, 9471, 9472, 9473, 9474, 9475, 9476, 9477, 9478, 9479 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 326.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9480, 9481, 9482, 9483, 9484, 9485, 9486, 9487, 9488, 9489, 9490 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 327.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9491, 9492, 9493, 9494, 9495, 9496, 9497, 9498, 9499, 9500, 9501 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 328.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9502, 9503, 9504, 9505, 9506, 9507, 9508, 9509, 9510, 9511, 9512 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 329.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9513, 9514, 9515, 9516, 9517, 9518, 9519, 9520, 9521, 9522, 9523 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 330.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9524, 9525, 9526, 9527, 9528, 9529, 9530, 9531, 9532, 9533, 9534 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 331.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9535, 9536, 9537, 9538, 9539, 9540, 9541, 9542, 9543, 9544, 9545, 9546, 9547, 9548, 9549, 9550, 9551, 9552, 9553, 9554, 9555, 9556 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 332.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9557, 9558, 9559, 9560, 9561, 9562, 9563, 9564, 9565, 9566, 9567, 9568, 9569, 9570, 9571, 9572, 9573, 9574, 9575, 9576, 9577, 9578 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 333.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9579, 9580, 9581, 9582, 9583, 9584, 9585, 9586, 9587, 9588, 9589 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 334.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9579, 9580, 9581, 9582, 9583, 9584, 9585, 9586, 9587, 9588, 9589 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 334.
  • SEQ ID NOS: 9590, 9591, 9592, 9593, 9594, 9595, 9596, 9597, 9598, 9599, 9600 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 335.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9601, 9602, 9603, 9604, 9605, 9606, 9607, 9608, 9609, 9610, 9611 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 336..
  • SEQ ID NOS: 9612, 9613, 9614, 9615, 9616, 9617, 9618, 9619, 9620, 9621, 9622 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 337.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9623, 9624, 9625, 9626, 9627, 9628, 9629, 9630, 9631, 9632, 9633 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 338.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9634, 9635, 9636, 9637, 9638, 9639, 9640, 9641, 9642, 9643, 9644 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 339.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9645, 9646, 9647, 9648, 9649, 9650, 9651, 9652, 9653, 9654, 9655 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 340.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9656, 9657, 9658, 9659, 9660, 9661, 9662, 9663, 9664, 9665, 9666 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 341.
  • SEQ ID NOS: 9667, 9668, 9669, 9670, 9671, 9672, 9673, 9674, 9675, 9676, 9677 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 342.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9678, 9679, 9680, 9681, 9682, 9683, 9684, 9685, 9686, 9687, 9688 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 343.
  • SEQ ID NOS: 9689, 9690, 9691, 9692, 9693, 9694, 9695, 9696, 9697, 9698, 9699 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 344.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9700, 9701, 9702, 9703, 9704, 9705, 9706, 9707, 9708, 9709, 9710 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 345.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9711, 9712, 9713, 9714, 9715, 9716, 9717, 9718, 9719, 9720, 9721 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 346.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9722, 9723, 9724, 9725, 9726, 9727, 9728, 9729, 9730, 9731, 9732 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 347.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9733, 9734, 9735, 9736, 9737, 9738, 9739, 9740, 9741, 9742, 9743 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 348.
  • SEQ ID NOS: 9744, 9745, 9746, 9747, 9748, 9749, 9750, 9751, 9752, 9753, 9754 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 349.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9755, 9756, 9757, 9758, 9759, 9760, 9761, 9762, 9763, 9764, 9765 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 350.
  • SEQ ID NOS: 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 351.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9777, 9778, 9779, 9780, 9781, 9782, 9783, 9784, 9785, 9786, 9787 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 352.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9788, 9789, 9790, 9791, 9792, 9793, 9794, 9795, 9796, 9797, 9798 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 353.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9799, 9800, 9801, 9802, 9803, 9804, 9805, 9806, 9807, 9808, 9809 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 354.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9810, 9811, 9812, 9813, 9814, 9815, 9816, 9817, 9818, 9819, 9820 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 355.
  • SEQ ID NOS: 9821, 9822, 9823, 9824, 9825, 9826, 9827, 9828, 9829, 9830, 9831 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 356.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9832, 9833, 9834, 9835, 9836, 9837, 9838, 9839, 9840, 9841, 9842 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 357.
  • SEQ ID NOS: 9843, 9844, 9845, 9846, 9847, 9848, 9849, 9850, 9851, 9852, 9853 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 358.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9854, 9855, 9856, 9857, 9858, 9859, 9860, 9861, 9862, 9863, 9864 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 359.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9865, 9866, 9867, 9868, 9869, 9870, 9871, 9872, 9873, 9874, 9875 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 360.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9876, 9877, 9878, 9879, 9880, 9881, 9882, 9883, 9884, 9885, 9886 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 361.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9887, 9888, 9889, 9890, 9891, 9892, 9893, 9894, 9895, 9896, 9897 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 362.
  • SEQ ID NOS: 9898, 9899, 9900, 9901, 9902, 9903, 9904, 9905, 9906, 9907, 9908 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 363.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9909, 9910, 9911, 9912, 9913, 9914, 9915, 9916, 9917, 9918, 9919 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 364.
  • SEQ ID NOS: 9920, 9921, 9922, 9923, 9924, 9925, 9926, 9927, 9928, 9929, 9930 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 365.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9931, 9932, 9933, 9934, 9935, 9936, 9937, 9938, 9939, 9940, 9941 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 366.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9942, 9943, 9944, 9945, 9946, 9947, 9948, 9949, 9950, 9951, 9952 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 367.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9953, 9954, 9955, 9956, 9957, 9958, 9959, 9960, 9961, 9962, 9963 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 368.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9964, 9965, 9966, 9967, 9968, 9969, 9970, 9971, 9972, 9973, 9974 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 369.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9964, 9965, 9966, 9967, 9968, 9969, 9970, 9971, 9972, 9973, 9974 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 369.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9964, 9965, 9966, 9967, 9968, 9969, 9970, 9971, 9972, 9973, 9974 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 369.
  • SEQ ID NOS: 9975, 9976, 9977, 9978, 9979, 9980, 9981, 9982, 9983, 9984, 9985 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 370.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9986, 9987, 9988, 9989, 9990, 9991, 9992, 9993, 9994, 9995, 9996 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 371.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 9997, 9998, 9999, 10000, 10001, 10002, 10003, 10004, 10005, 10006, 10007 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 372.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10008, 10009, 10010, 10011, 10012, 10013, 10014, 10015, 10016, 10017, 10018 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 373.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10019, 10020, 10021, 10022, 10023, 10024, 10025, 10026, 10027, 10028, 10029 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 374.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10030, 10031, 10032, 10033, 10034, 10035, 10036, 10037, 10038, 10039, 10040 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 375.
  • SEQ ID NOS: 10052, 10053, 10054, 10055, 10056, 10057, 10058, 10059, 10060, 10061, 10062 were selected from the
  • SEQ ID NOS: 10063, 10064, 10065, 10066, 10067, 10068, 10069, 10070, 10071, 10072, 10073 were selected from the
  • SEQ ID NOS: 10074, 10075, 10076, 10077, 10078, 10079, 10080, 10081, 10082, 10083, 10084 were selected from the
  • SEQ ID NOS: 10085, 10086, 10087, 10088, 10089, 10090, 10091, 10092, 10093, 10094, 10095 were selected from the
  • SEQ ID NOS: 10096, 10097, 10098, 10099, 10100, 10101, 10102, 10103, 10104, 10105, 10106 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10107, 10108, 10109, 10110, 10111, 10112, 10113, 10114, 10115, 10116, 10117 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 382.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10118, 10119, 10120, 10121, 10122, 10123, 10124, 10125, 10126, 10127, 10128 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 383.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10129, 10130, 10131, 10132, 10133, 10134, 10135, 10136, 10137, 10138, 10139 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 384.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10140, 10141, 10142, 10143, 10144, 10145, 10146, 10147, 10148, 10149, 10150 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 385.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10151, 10152, 10153, 10154, 10155, 10156, 10157, 10158, 10159, 10160, 10161 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 386.
  • SEQ ID NOS: 10173, 10174, 10175, 10176, 10177, 10178, 10179, 10180, 10181, 10182, 10183 were selected from the
  • SEQ ID NOS: 10184, 10185, 10186, 10187, 10188, 10189, 10190, 10191, 10192, 10193, 10194 were selected from the
  • SEQ ID NOS: 10195, 10196, 10197, 10198, 10199, 10200, 10201, 10202, 10203, 10204, 10205 were selected from the
  • SEQ ID NOS: 10206, 10207, 10208, 10209, 10210, 10211, 10212, 10213, 10214, 10215, 10216 were selected from the
  • SEQ ID NOS: 10217, 10218, 10219, 10220, 10221, 10222, 10223, 10224, 10225, 10226, 10227 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10228, 10229, 10230, 10231, 10232, 10233, 10234, 10235, 10236, 10237, 10238 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 393.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10239, 10240, 10241, 10242, 10243, 10244, 10245, 10246, 10247, 10248, 10249 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 394.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10250, 10251, 10252, 10253, 10254, 10255, 10256, 10257, 10258, 10259, 10260 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 395.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10261, 10262, 10263, 10264, 10265, 10266, 10267, 10268, 10269, 10270, 10271 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 396.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10272, 10273, 10274, 10275, 10276, 10277, 10278, 10279, 10280, 10281, 10282 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 397.
  • SEQ ID NOS: 10294, 10295, 10296, 10297, 10298, 10299, 10300, 10301, 10302, 10303, 10304 were selected from the
  • SEQ ID NOS: 10305, 10306, 10307, 10308, 10309, 10310, 10311, 10312, 10313, 10314, 10315 were selected from the
  • SEQ ID NOS: 10316, 10317, 10318, 10319, 10320, 10321, 10322, 10323, 10324, 10325, 10326 were selected from the
  • SEQ ID NOS: 10327, 10328, 10329, 10330, 10331, 10332, 10333, 10334, 10335, 10336, 10337 were selected from the
  • SEQ ID NOS: 10338, 10339, 10340, 10341, 10342, 10343, 10344, 10345, 10346, 10347, 10348 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10349, 10350, 10351, 10352, 10353, 10354, 10355, 10356, 10357, 10358, 10359 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 404.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10360, 10361, 10362, 10363, 10364, 10365, 10366, 10367, 10368, 10369, 10370 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 405.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10371, 10372, 10373, 10374, 10375, 10376, 10377, 10378, 10379, 10380, 10381 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 406.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10382, 10383, 10384, 10385, 10386, 10387, 10388, 10389, 'l0390, 10391, 10392 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 407.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10393, 10394, 10395, 10396, 10397, 10398, 10399, 10400, 10401, 10402, 10403 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 408.
  • SEQ ID NOS: 10415, 10416, 10417, 10418, 10419, 10420, 10421, 10422, 10423, 10424, 10425 were selected from the
  • SEQ ID NOS: 10426, 10427, 10428, 10429, 10430, 10431, 10432, 10433, 10434, 10435, 10436 were selected from the
  • SEQ ID NOS: 10437, 10438, 10439, 10440, 10441, 10442, 10443, 10444, 10445, 10446, 10447 were selected from the
  • SEQ ID NOS: 10448, 10449, 10450, 10451, 10452, 10453, 10454, 10455, 10456, 10457, 10458 were selected from the
  • SEQ ID NOS: 10459, 10460, 10461, 10462, 10463, 10464, 10465, 10466, 10467, 10468, 10469 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10470, 10471, 10472, 10473, 10474, 10475, 10476, 10477, 10478, 10479, 10480 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 415.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10481, 10482, 10483, 10484, 10485, 10486, 10487, 10488, 10489, 10490, 10491 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 416.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10492, 10493, 10494, 10495, 10496, 10497, 10498, 10499, 10500, 10501, 10502 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 417.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10503, 10504, 10505, 10506, 10507, 10508, 10509, 10510, 10511, 10512, 10513 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 418.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10514, 10515, 10516, 10517, 10518, 10519, 10520, 10521, 10522, 10523, 10524 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 419.
  • SEQ ID NOS: 10536, 10537, 10538, 10539, 10540, 10541, 10542, 10543, 10544, 10545, 10546 were selected from the
  • SEQ ID NOS: 10547, 10548, 10549, 10550, 10551, 10552, 10553, 10554, 10555, 10556, 10557 were selected from the
  • SEQ ID NOS: 10558, 10559, 10560, 10561, 10562, 10563, 10564, 10565, 10566, 10567, 10568 were selected from the
  • SEQ ID NOS: 10569, 10570, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579 were selected from the
  • SEQ ID NOS: 10580, 10581, 10582, 10583, 10584, 10585, 10586, 10587, 10588, 10589, 10590 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10591, 10592, 10593, 10594, 10595, 10596, 10597, 10598, 10599, 10600, 10601 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 426.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10602, 10603, 10604, 10605, 10606, 10607, 10608, 10609, 10610, 10611, 10612 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 427.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10613, 10614, 10615, 10616, 10617, 10618, 10619, 10620, 10621, 10622, 10623 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 428.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10624, 10625, 10626, 10627, 10628, 10629, 10630, 10631, 10632, 10633, 10634 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 429.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10635, 10636, 10637, 10638, 10639, 10640, 10641, 10642, 10643, 10644, 10645 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 430.
  • SEQ ID NOS: 10657, 10658, 10659, 10660, 10661, 10662, 10663, 10664, 10665, 10666, 10667 were selected from the
  • SEQ ID NOS: 10668, 10669, 10670, 10671, 10672, 10673, 10674, 10675, 10676, 10677, 10678 were selected from the
  • SEQ ID NOS: 10679, 10680, 10681, 10682, 10683, 10684, 10685, 10686, 10687, 10688, 10689 were selected from the
  • SEQ ID NOS: 10690, 10691, 10692, 10693, 10694, 10695, 10696, 10697, 10698, 10699, 10700 were selected from the
  • SEQ ID NOS: 10701, 10702, 10703, 10704, 10705, 10706, 10707, 10708, 10709, 10710, 10711 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10712, 10713, 10714, 10715, 10716, 10717, 10718, 10719, 10720, 10721, 10722 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 437.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10723, 10724, 10725, 10726, 10727, 10728, 10729, 10730, 10731, 10732, 10733 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 438.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10734, 10735, 10736, 10737, 10738, 10739, 10740, 10741, 10742, 10743, 10744 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 439.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10745, 10746, 10747, 10748, 10749, 10750, 10751, 10752, 10753, 10754, 10755 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 440.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 441.
  • SEQ ID NOS: 10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788 were selected from the
  • SEQ ID NOS: 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799 were selected from the
  • SEQ ID NOS: 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810 were selected from the
  • SEQ ID NOS: 10811, 10812, 10813, 10814, 10815, 10816, 10817, ⁇ 0818, 10819, 10820, 10821 were selected from the
  • SEQ ID NOS: 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 448.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10844, 10845, 10846, 10847, 10848, 10849, 10850, 10851, 10852, 10853, 10854 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 449.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10855, 10856, 10857, 10858, 10859, 10860, 10861, 10862, 10863, 10864, 10865 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 450.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10866, 10867, 10868, 10869, 10870, 10871, 10872, 10873, 10874, 10875, 10876 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 451.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10877, 10878, 10879, 10880, 10881, 10882, 10883, 10884, 10885, 10886, 10887 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 452.
  • SEQ ID NOS: 10899, 10900, 10901, 10902, 10903, 10904, 10905, 10906, 10907, 10908, 10909 were selected from the
  • SEQ ID NOS: 10910, 10911, 10912, 10913, 10914, 10915, 10916, 10917, 10918, 10919, 10920 were selected from the
  • SEQ ID NOS: 10921, 10922, 10923, 10924, 10925, 10926, 10927, 10928, 10929, 10930, 10931 were selected from the
  • SEQ ID NOS: 10932, 10933, 10934, 10935, 10936, 10937, 10938, 10939, 10940, 10941, 10942 were selected from the
  • SEQ ID NOS: 10943, 10944, 10945, 10946, 10947, 10948, 10949, 10950, 10951, 10952, 10953 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10954, 10955, 10956, 10957, 10958, 10959, 10960, 10961, 10962, 10963, 10964 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 459.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10965, 10966, 10967, 10968, 10969, 10970, 10971, 10972, 10973, 10974, 10975 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 460.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10976, 10977, 10978, 10979, 10980, 10981, 10982, 10983, 10984, 10985, 10986 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 461.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10987, 10988, 10989, 10990, 10991, 10992, 10993, 10994, 10995, 10996, 10997 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 462.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 10998, 10999, 11000, 11001, 11002, 11003, 11004, 11005, 11006, 11007, 11008 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 463.
  • SEQ ID NOS: 11020, 11021,' 11022, 11023, 11024, 11025, 11026, 11027, 11028, 11029, 11030 were selected from the
  • SEQ ID NOS: 11031, 11032, 11033, 11034, 11035, 11036, 11037, 11038, 11039, 11040, 11041 were selected from the
  • SEQ ID NOS: 11042, 11043, 11044, 11045, 11046, 11047, 11048, 11049, 11050, 11051, 11052 were selected from the
  • SEQ ID NOS: 11053, 11054, 11055, 11056, 11057, 11058, 11059, 11060, 11061, 11062, 11063 were selected from the
  • SEQ ID NOS: 11064, 11065, 11066, 11067, 11068, 11069, 11070, 11071, 11072, 11073, 11074 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11075, 11076, 11077, 11078, 11079, 11080, 11081, 11082, 11083, 11084, 11085 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 470.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11086, 11087, 11088, 11089, 11090, 11091, 11092, 11093, 11094, 11095, 11096 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 471.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11097, 11098, 11099, 11100, 11101, 11102, 11103, 11104, 11105, 11106, 11107 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 472.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11108, 11109, 11110, 11111, 11112, 11113, 11114, 11115, 11116, 11117, 11118 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 473.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11119, 11120, 11121, 11122, 11123, 11124, 11125, 11126, 11127, 11128, 11129 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 474.
  • SEQ ID NOS: 11141, 11142, 11143, 11144, 11145, 11146, 11147, 11148, 11149, 11150, 11151 were selected from the
  • SEQ ID NOS: 11152, 11153, 11154, 11155, 11156, 11157, 11158, 11159, 11160, 11161, 11162 were selected from the
  • SEQ ID NOS: 11163, 11164, 11165, 11166, 11167, 11168, 11169, 11170, 11171, 11172, 11173 were selected from the
  • SEQ ID NOS: 11174, 11175, 11176, 11177, 11178, 11179, 11180, 11181, 11182, 11183,.11184 were selected from the
  • SEQ ID NOS: 11185, 11186, 11187, 11188, 11189, 11190, 11191, 11192, 11193, 11194, 11195 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11196, 11197, 11198, 11199, 11200, 11201, 11202, 11203, 11204, 11205, 11206 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 481.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11207, 11208, 11209, 11210, 11211, 11212, 11213, 11214, 11215, 11216, 11217 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 482.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11218, 11219, 11220, 11221, 11222, 11223, 11224, 11225, 11226, 11227, 11228 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 483.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11229, 11230, 11231, 11232, 11233, 11234, 11235, 11236, 11237, 11238, 11239 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 484.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11240, 11241, 11242, 11243, 11244, 11245, 11246, 11247, 11248, 11249, 11250 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 485.
  • SEQ ID NOS: 11262, 11263, 11264, 11265, 11266, 11267, 11268, 11269, 11270, 11271, 11272 were selected from the
  • SEQ ID NOS: 11273, 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283 were selected from the
  • SEQ ID NOS: 11284, 11285, 11286, 11287, 11288, 11289, 11290, 11291, 11292, 11293, 11294 were selected from the
  • SEQ ID NOS: 11295, 11296, 11297, 11298, 11299, 11300, 11301, 11302, 11303, 11304, 11305 were selected from the
  • SEQ ID NOS: 11306, 11307, 11308, 11309, 11310, 11311, 11312, 11313, 11314, 11315, 11316 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11317, 11318, 11319, 11320, 11321, 11322, 11323, 11324, 11325, 11326, 11327 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 492.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11328, 11329, 11330, 11331, 11332, 11333, 11334, 11335, 11336, 11337, 11338 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 493.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11339, 11340, 11341, 11342, 11343, 11344, 11345, 11346, 11347, 11348, 11349 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 494.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11350, 11351, 11352, 11353, 11354, 11355, 11356, 11357, 11358, 11359, 11360 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 495.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11361, 11362, 11363, 11364, 11365, 11366, 11367, 11368, 11369, 11370, 11371 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 496.
  • SEQ ID NOS: 11383, 11384, 11385, 11386, 11387, 11388, 11389, 11390, 11391, 11392, 11393 were selected from the
  • SEQ ID NOS: 11394, 11395, 11396, 11397, 11398, 11399, 11400, 11401, 11402, 11403, 11404 were selected from the
  • SEQ ID NOS: 11405, 11406, 11407, 11408, 11409, 11410, 11411, 11412, 11413, 11414, 11415 were selected from the
  • SEQ ID NOS: 11416, 11417, 11418, 11419, 11420, 11421, 11422, 11423, 11424, 11425, 11426 were selected from the
  • SEQ ID NOS: 11427, 11428, 11429, 11430, 11431, 11432, 11433, 11434, 11435, 11436, 11437 were selected from the
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11438, 11439, 11440, 11441, 11442, 11443, 11444, 11445, 11446, 11447, 11448 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 503.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11449, 11450, 11451, 11452, 11453, 11454, 11455, 11456, 11457, 11458, 11459 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 504.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11460, 11461, 11462, 11463, 11464, 11465, 11466, 11467, 11468, 11469, 11470 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 505.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11471, 11472, 11473, 11474, 11475, 11476, 11477, 11478, 11479, 11480, 11481 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 506.
  • the probes having the nucleotide sequence represented by SEQ ID NOS: 11482, 11483, 11484, 11485, 11486, 11487, 11488, 11489, 11490, 11491, 11492 were selected from the ORFs having the nucleotide sequence represented by SEQ ID NO: 507.
  • SEQ ID NOS: 11504, 11505, 11506, 11507, 11508, 11509, 11510, 11511, 11512, 11513, 11514 were selected from the
  • SEQ ID NOS: 11515, 11516, 11517, 11518, 11519, 11520, 11521, 11522, 11523, 11524, 11525 were selected from the
  • SEQ ID NOS: 11526, 11527, 11528, 11529, 11530, 11531, 11532, 11533, 11534, 11535, 11536 were selected from the
  • SEQ ID NOS: 11537, 11538, 11539, 11540, 11541, 11542, 11543, 11544, 11545, 11546, 11547 were selected from the

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne notamment une technique permettant d'analyser un gène de levure industrielle. Cette technique englobe les opérations suivantes: (a) analyse de la séquence génomique de la levure industrielle; et (c-1) sélection dans la levure industrielle d'un gène codant pour une séquence d'acides aminés identique à 70- 97 % à une séquence d'acides aminés codée par le gène de Saccharomyces cerevisiae, ou (c-2) sélection dans la levure industrielle d'un gène constitué d'une séquence de nucléotides identique à 60-94 % à la séquence de nucléotides du gène de Saccharomyces cerevisiae.-
EP05783945A 2004-09-02 2005-08-30 Technique d'analyse pour genes de levures industrielles Ceased EP1784507A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/IB2004/002873 WO2006024892A1 (fr) 2004-09-02 2004-09-02 Methode d'analyse de genes de levures industrielles
PCT/IB2005/002908 WO2006024951A2 (fr) 2004-09-02 2005-08-30 Technique d'analyse pour genes de levures industrielles

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KR (1) KR20070083594A (fr)
CN (1) CN101052729B (fr)
AU (1) AU2005278901A1 (fr)
CA (1) CA2621202A1 (fr)
WO (2) WO2006024892A1 (fr)

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EP1913134A1 (fr) * 2005-08-12 2008-04-23 Suntory Limited Gène d'adényltransférase de sulfate et son utilisation
US20100129491A1 (en) * 2005-09-01 2010-05-27 Yoshihiro Nakao Tryptophan transporter gene and use thereof
DK1869172T3 (da) * 2005-09-13 2010-10-11 Suntory Holdings Ltd Forgrenet aminosyre-aminotransferasegen og anvendelse deraf
JP2007228956A (ja) * 2006-02-28 2007-09-13 Suntory Ltd 醸造用酵母由来の有用タンパク質同定方法
EP1874924B1 (fr) * 2006-02-28 2012-08-22 Suntory Holdings Limited Utilisation des gènes de catalase por évaluer ou modifier la capacité de production de sulfites dans les levures
EP2085472B1 (fr) * 2006-10-18 2013-08-21 National University Corporation Nagoya University D-sérine deshydratase et son utilisation
JP5158944B2 (ja) * 2007-03-02 2013-03-06 アサヒビール株式会社 酵母ゲノム解析による酵母菌株判定法
US20100311065A1 (en) * 2009-06-01 2010-12-09 Ubersax Jeffrey A Genetically modified microbes producing isoprenoids
US8357527B2 (en) 2009-06-01 2013-01-22 Amyris, Inc. Method for generating a genetically modified microbe
EP2658994B1 (fr) 2010-12-28 2017-07-19 Qiagen Hamburg GmbH Sonde oligonucléotidique pour la détection d'un adénovirus
WO2015002916A1 (fr) * 2013-07-03 2015-01-08 Butamax Advanced Biofuels Llc Régulation post-transcriptionnelle des voies biosynthétiques
JP7113682B2 (ja) * 2017-08-25 2022-08-05 サントリーホールディングス株式会社 酵母の亜硫酸生成能向上方法
CN117126898B (zh) * 2023-10-26 2023-12-22 内蒙古阜丰生物科技有限公司 一种通过生物技术制备缬氨酸的工艺

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DK1599605T3 (da) * 2003-03-04 2011-06-06 Suntory Holdings Ltd S. pastorianus SSU1-gen, polypeptid og deres anvendelse i alkoholfremstilling

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See references of WO2006024951A3 *

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AU2005278901A1 (en) 2006-03-09
WO2006024951A2 (fr) 2006-03-09
CN101052729A (zh) 2007-10-10
JP2008511305A (ja) 2008-04-17
WO2006024951A3 (fr) 2006-06-22
KR20070083594A (ko) 2007-08-24
WO2006024892A1 (fr) 2006-03-09
CN101052729B (zh) 2011-09-14

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