JP2014155500A - Method for identifying eukaryote species - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for identifying a species useful for such as identification, evaluation, and classification of an eukaryote.SOLUTION: The method for identifying a species contained in a sample includes the steps of: with a DNA obtained from the sample as templates, amplifying a DNA region encoding an amino acid sequence present in a C-terminal region of the largest subunit of RNA polymerase I and sandwiched between two regions having conservativeness in the process of evolution, by using primers prepared on the basis of base sequences each encoding the two regions; determining the amino acid sequence encoded by a base sequence of the produced amplification product; and comparing the determined amino acid sequence with an amino acid sequence of a PolA1 gene of the eukaryote having been registered in a computer database.

Description

The present invention relates to a method for identifying a eukaryotic species. More particularly, the present invention relates to RNA polymerase I
The present invention relates to a method for identifying a eukaryotic species, characterized by analyzing an amino acid sequence encoded by a base sequence of a specific DNA region of a maximum subunit gene (sometimes abbreviated as PolA1 gene) as an index. More specifically, the present invention uses DNA obtained from a sample as a template, and DNA encoding an amino acid sequence sandwiched between two regions that are conserved in the process of evolution existing in the C-terminal region of RNA polymerase I largest subunit. The region is amplified using a primer prepared based on the base sequence encoding each of the two regions, the amino acid sequence encoded by the base sequence of the generated amplification product is determined, and the determined amino acid sequence is The present invention relates to a method for identifying a eukaryotic species, characterized by identifying a eukaryotic species as a sample by comparing with the amino acid sequence of a eukaryotic PolA1 gene registered in a computer database.

There are 3 to 10 million species listed on the earth, and it is estimated that there are actually more than 10 times this species. The scientific name of the biological species is Carl von Linne
) Has been described and systematized in two nomenclatures (genus name and species name) based on established morphological characteristics. About 250 years later, even though the study of genetics and molecular biology has advanced significantly, it has become possible to analyze the relationship between species in detail, but a new organism that replaces Linne's two nomenclature The species classification method has not been established yet.

In Rinne's two nomenclature, species of organisms are identified based on differences in external morphology,
In many cases, species cannot be identified unless they are specialists of each organism. In addition, the classification standards of species differ among taxonologists. Furthermore, since the number of researchers specializing in morphological taxonomy is currently declining significantly, it is expected that a situation where species cannot be identified based on morphology in the near future can occur sufficiently.

In recent years, with the progress of DNA analysis technology, researches to characterize organism species and strains by DNA base sequences have been actively conducted (Non-patent Document 1). We have identified rpl16 and r on chloroplast DNA.
The pl14 intergenic region was found to correspond well with the plant affinity, and the sequence of this region was named as a plastic subtype identity (PSID) sequence (Non-patent Document 2). Although this PSID sequence is as short as about 100 to 300 bases, it is effective for identifying plant genera and species (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6,
Non-patent document 7 and Non-patent document 8). Recently, Herbert et al. Proposed an attempt to distinguish species of all organisms (DNAbarcoding) using the nucleotide sequence of mitochondrial cytochrome c oxidase I (CO1) gene (Non-Patent Document 9 and Non-patent document 10). DNA barcoding using the CO1 gene is known for insects (Non-Patent Document 11) and birds (Non-Patent Documents).
12), fish (Non-patent document 13), red algae (Non-patent document 14), fungi (Non-patent document 15) have been reported in many organisms, and there are also evaluations with renaissance in biotaxonomy (Non-patent document) 16).

On the other hand, since the evolution rate of the CO1 gene is slow in plants, DNA barcoding using a gene sequence of chloroplast DNA has been proposed (Non-patent Documents 17 and 18). In addition, since mitochondrial and chloroplast DNA is transmitted by cytoplasmic inheritance, it is impossible to distinguish interspecific hybrids and diploid species that are abundant in plants. Furthermore, since the transfer of mitochondrial DNA fragments to the nuclear genome has been reported in animals (Non-patent document 19), plants (Non-patent document 20), etc., there are many critical opinions on DNA barcoding using the CO1 gene ( Non-patent document 21 and Non-patent document 22). For this reason, DNA barcoding using a plurality of nuclear genes has been studied (Non-patent Document 23 and Non-Patent Document 24).

Ribosomal RNA as an effective DNA base sequence for classifying species in various organisms
The (rRNA) gene is known (Non-patent document 25 and Non-patent document 26). It is known that the base sequences of 16S and 23S rRNA genes are effective for identifying prokaryotic genera and species (Non-patent Document 27). On the other hand, in eukaryotes, 18S rRNA and 5.8S r
ITS1 (internal transcribed spacer 1) region between RNA genes and 5.8S rRNA and 28S rRN
It has been reported that the base sequence of the ITS2 region between the A genes is effective for identification of genera and species (
Non-patent document 28). In contrast, eukaryotic rRNA genes are not suitable for distinguishing genera and species because hundreds of copies are present in the nuclear genome in tandem and interspersed and show intraspecific variation. There is also a report (Non-patent document 29).

Therefore, research on unique genes contained in one to several copies in the nuclear genome is also being conducted. For example, glycerol-3-phosphate acyltransferase (Gly
cerol-3-phosphateacyltransferase, Non-Patent Document 30), alcohol dehydrogenase (
Alcohol dehydrogenase, Non-Patent Document 31), Glutamine synthet
Since housekeeping genes having important functions such as ase and non-patent literature 32) are contained in all living organisms, they are effective for species identification and estimation of affinity. However, since mutation occurs only in a region that does not affect each enzyme activity, there is a known case where distant relations do not correspond to similar relations (Non-patent Document 33).

In general, in order to elucidate the speciation of an organism, it is common knowledge to analyze a large number of DNA markers contained in the genome and create a molecular phylogenetic tree. Then, the idea called phylocode (Phylocode) which tries to use this molecular phylogenetic tree as a species name is proposed (nonpatent literature 3).
4 and Non-Patent Document 35) require a large amount of money to analyze a large number of DNA markers for all species and strains. In addition, many common DNAs in all eukaryotes
It seems difficult to analyze using markers. In addition, Phlylocode
There is also a problem that the classification system based on the two nomenclature and the accumulation of knowledge are completely independent concepts (Non-patent Document 36).

Godfray, H. C. “Challenges for taxonomy”, Nature, 2002, 417, p.17-19. Nakamura, I et al., “A proposal for identifying the short ID sequence which addresses the plastid subtype of higher plants”, Breed Science, 1998, 47, p. 385-388. Hosokawa, K. et al., “Discrimination among three species of medicinal Scutellaria plants using RAPD markers.”, Planta Medica, 2000, Vol. 66, p.270-272. Hosokawa, K. et al., “The sequences of the plastid gene rpl16 and therpl16-rpl14 spacer region allow discrimination among six species of Scutellaria.”, Journal of Ethnopharmacology, 2005, Vol. 99, p. .105-108. Ishikawa, R. et al., “Different maternal origins of Japanese lowland and upland rice populations.”, Theoretical and Applied Genetics, 2002, 104, p.976-980. Ishikawa, R. et al., “Origin of cytoplasm substituted rice cultivarsfound in Japan.”, Theoretical and Applied Genetics, 2002, Vol. 105, p.608-613. 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Miller, SE, “DNA barcoding and the renaissance of taxonomy.”, Proceedings of the National Academy of Sciences of the United States of America, 2007, 104, p.4775-4776. Kress, WJ, "Use of DNA barcodes to identify floweringplants.", Proceedings of the National Academy of Sciences of the United States of America, 2005, Volume 102, p.8369-8374. Taberlet.P. E. et al., “Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.”, Nucleic Acids Research, 2007, Vol. 35, No. 3, e14. Thalmann, O. et al., “Unreliable mtDNA data due to nuclear insertions: A cautionary tale from analysis of humans and other great apes.”, Molecular Ecology, 2004, Vol. 13, p.321- 335. Adams, KL et al., “Intracellular gene transfer in action: Dualtranscription and multiple silencings of nuclear and mitochondrial cox2 genesin legumes.”, Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of The United States of America), 1999, 96, p.13863-13868. Wheeler, Q. D., "Losing the plot: DNA 'barcodes' and taxonomy.", Cladistics, 2005, Vol. 21, p.405-407. Rubinoff, D. et al., “Are plant DNA barcodes a search for the HolyGrail?”, Trends in Ecology and Evolution, 2006, Vol. 21, p.1-2. Dashamapatra, K. K. et al., “DNA barcodes: recent successes and future prospects”, Heredity, 2006, 97, p.254-255. Sonnenberg, R. et al., “An evaluation of LSU-rDNA D1-D2 sequences for the use in species identification”, Frontiersin Zoology, 2007, Vol. 4, p.6. Pace, N. I. et al., “Ribosomal RNA phylogeny and the primary lines of evolutionary descent”, Cell, 1986, Vol. 45, No. 3, p. 325-326. Jorgensen, R. et al., “Modes and tempos in the evolution of nuclearribosomal DNA: new characters for evolutionary studies and new markers forgenetic and population studies”, Annals of the Missouri Botanical Garden, 1988 Year, Volume 75, p.1238-1247. Ludwig, W. et al., “Bacterial phylogeny based on 16S and 23S rRNAsequence analysis”, FEMS Microbiology Reviews, 1994, Vol. 15, No. 2-3, p.155 -173. Baroin, A. et al., “Partial phylogeny of the unicellular eukaryotesbased on rapid sequencing of a portion of 28S ribosomal RNA”, Proceedings of the National Academy of Sciences of The United States of America), 1988, Vol. 85, No. 10, p.3474-3478. Luschnig, C. et al., “Intraspecific length heterogeneity of therDNA-IGR in Arabidopsis thaliana due to homologous recombination”, Plant Molecular Biology, 1993, Vol. 22, No. 3, p. 543 -545. Tank, DC et al., “Phylogenetic Utility of the Glycerol-3-Phosphate Acyltransferase Gene: Evolution and Implications in Paeonia (Paeoniaceae)”, Molecular Phylogenetics and Evolution, 2001, Vol. 19, No. 3, p.421-429. Yokoyama, S. et al., “Molecular Phylogeny and Evolutionary Rates of Alcohol Dehydrogenases in Vertebrates and Plants”, Molecular Biology and Evolution, 1993, Vol. 10, No. 6, p. 1215-1226. Pesole, G. et al., “Glutamine Synthetase Gene Evolution: A GoodMolecular Clock”, Proceedings of the National Academy of Sciences of The United States of America, 1991 Year, Vol. 88, No. 2, p.522-526. Dayhoff, M. O., “Atlas of protein sequence and structure.”, National Biomedical Research Foundation, Maryland USA, 1972.

Queiroz, K. et al., “Phylogeny as a central principle in taxonomy: Phylogenetic definitions of taxon names”, Systematic Zoology, 1990, Vol. 39, p.307-322. Queiroz, K. et al., “Phylogenetic nomenclature and the PhyloCode.”, Bulletin of Zoological Nomenclature, 2001, Vol. 58, p.254-271. Benton, M. J., “Stems, nodes, crown clades, and rank-freelists: is Linnaeus dead?”, Biological Reviews, 2000, vol. 75, p. 633-648.

Species of organisms are classified using two nomenclatures organized by Linne based on differences in morphological characteristics. With the progress of molecular biology, gene and genome information of many biological species has been obtained, but it has always been collated with the classification results by conventional forms. If the species of an organism can be specified using an amino acid sequence, it will be possible to greatly contribute to identification, identification, classification, and the like.

An object of the present invention is to provide a method for identifying a species useful for identification, identification, classification, and the like of an organism.

In general, speciation of organisms is thought to be caused by the nuclear genome rather than the cytoplasmic genome, so in order to elucidate the speciation, the nucleotide sequence of a gene contained in only one copy in the haploid genome It is considered better to investigate. Moreover, in order to specify a species, it is desirable that it is a gene contained in the nucleus of all eukaryotes.

The present inventor has established a eukaryote in the C-terminal region of RNA polymerase I largest subunit, which is the largest subunit of RNA polymerase I complex involved in the synthesis of ribosomal RNA (rRNA) essential for maintaining life. We found that there is an amino acid sequence that shows a specific mutation in this species, and focused on this. This amino acid sequence consists of about 370 amino acid residues sandwiched between two highly conserved regions and is encoded by a base sequence of about 1.1 kb. Since this amino acid sequence and the base sequence encoding the amino acid sequence can be determined one by one per haploid genome, it can be applied not only to diploids but also to hybrids and polydiploid species. It can be used as an index to identify nuclear species. The present invention has been achieved based on this finding.

That is, the present invention relates to a method for identifying a eukaryotic species comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) a process of amplifying a specific DNA region of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA as a template, wherein the specific DNA region is present in the C-terminal region of the protein encoded by the gene A DNA region that encodes an amino acid sequence sandwiched between two regions that are conserved in the course of evolution.
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention is also the above-described method for identifying a eukaryotic species, wherein one of the two regions that are conserved in the process of evolution existing in the C-terminal region of the protein encoded by the PolA1 gene is A region represented by the 1175th to 1194th amino acid sequence (SEQ ID NO: 1) of the protein, and the other region represented by the 1581st to 1596th amino acid sequence of the protein (SEQ ID NO: 2), wherein the position is associated with a protein encoded by the base sequence of the accession number NM_115626 of the PolA1 gene of Arabidopsis thaliana. About.

The present invention is also the above-described method for identifying a eukaryotic species, wherein one of the two regions that are conserved in the process of evolution existing in the C-terminal region of the protein encoded by the PolA1 gene is The amino acid sequence described in SEQ ID NO: 1 has 40% or more homology, and the other region has 40% or more homology with the amino acid sequence described in SEQ ID NO: 2, and the two regions The present invention relates to a method characterized in that the number of amino acids in between is 300 residues or more.

The present invention is also any one of the above methods for identifying a eukaryotic species, wherein the amplification of a specific DNA region of the PolA1 gene is a process of evolution existing in the C-terminal region of the protein encoded by the gene. By polymerase chain reaction using a primer set consisting of a primer designed based on the base sequence encoding one of the two conservative regions and a primer designed based on the base sequence encoding the other region It relates to a method characterized in that it is performed.

The present invention is also any one of the above methods for identifying a eukaryotic species, wherein the amplification of a partial region of the PolA1 gene is performed by polymerase chain linkage using one or more sets selected from the following primer set group. On a method characterized by being carried out by reaction:
(1) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 8,
(2) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 12,
(3) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 16;
(4) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 20,
(5) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 21 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 22;
(6) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 23 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 24;
(7) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 25 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 26;
(8) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 27 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 28;
(9) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 29 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 30;
(10) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 31 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 32;
(11) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 33 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 34;
(12) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 35 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 36;
(13) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 37 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 38;
(14) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 39 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 40;
(15) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 41 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 42;
(16) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 43 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 44;
(17) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 45 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 46;
(18) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 47 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 48;
(19) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 49 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 50;
(20) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 51 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 52;
(21) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 53 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 54;
(22) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 55 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 56;
(23) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 57 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 58;
(24) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 59 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 60;
(25) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 61 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 62;
(26) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 63 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 64;
(27) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 65 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 66;
(28) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 67 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 68;
(29) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 69 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 70;
(30) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 71 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 72;
(31) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 73 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 74;
(32) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 75 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 76;
(33) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 77 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 78;
(34) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 79 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 80;
(35) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 81 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 82;
(36) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 83 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 84;
(37) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 85 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 86;
(38) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 87 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 88;
(39) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 89 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 90;
(40) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 91 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 92;
(41) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 93 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 94;
(42) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 95 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 96;
(43) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 97 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 98;
(44) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 99 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 100;
(45) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 101 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 102;
(46) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 103 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 104;
(47) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 105 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 106;
(48) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 107 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 108;
(49) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 109 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 110;
(50) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 111 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 112;
(51) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 113 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 114;
(52) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 115 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 116;
(53) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 117 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 118;
(54) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 119 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 120;
(55) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 121 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 122;
(56) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 123 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 124;
(57) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 125 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 126;
(58) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 127 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 128;
(59) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 129 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 130;
(60) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 131 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 132;
(61) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 133 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 134;
(62) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 135 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 136;
(63) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 137 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 138;
(64) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 139 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 140;
(65) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 141 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 142;
(66) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 143 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 144;
(67) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 145 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 146;
(68) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 147 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 148;
(69) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 149 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 150;
(70) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 151 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 152;
(71) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 153 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 154,
(72) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 155 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 156;
(73) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 157 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 158,
(74) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 159 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 160;
(75) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 161 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 162;
(76) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 163 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 164;
(77) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 165 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 166;
(78) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 167 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 168,
(79) A primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 169 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 170,
(80) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 171 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 172,
(81) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 173 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 174,
(82) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 175 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 176,
(83) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 177 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 178,
(84) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 179 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 180,
(85) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 181 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 182;
(86) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 183 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 184,
(87) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 185 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 186;
(88) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 187 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 188,
(89) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 189 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 190,
(90) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 191 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 192,
(91) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 193 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 194;
(92) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 195 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 196;
(93) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 197 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 198;
(94) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 199 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 200;
(95) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 201 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 202;
(96) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 203 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 204;
(97) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 205 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 206;
(98) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 207 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 208;
(99) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 209 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 210;
(100) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 211 and SEQ ID NO: 212
A primer set consisting of an oligonucleotide represented by the base sequence described in
(101) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 213 and SEQ ID NO: 214
A primer set consisting of an oligonucleotide represented by the base sequence described in
(102) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 215 and SEQ ID NO: 216
A primer set consisting of an oligonucleotide represented by the base sequence described in
(103) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 217 and SEQ ID NO: 218
A primer set consisting of an oligonucleotide represented by the base sequence described in
(104) the oligonucleotide represented by the base sequence set forth in SEQ ID NO: 219 and SEQ ID NO: 220
A primer set consisting of an oligonucleotide represented by the base sequence described in
(105) the oligonucleotide represented by the base sequence described in SEQ ID NO: 221 and SEQ ID NO: 222
A primer set consisting of an oligonucleotide represented by the base sequence described in
(106) an oligonucleotide represented by the base sequence described in SEQ ID NO: 223 and SEQ ID NO: 224
A primer set consisting of an oligonucleotide represented by the base sequence described in
(107) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 225 and SEQ ID NO: 226
A primer set consisting of an oligonucleotide represented by the base sequence described in
(108) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 227 and SEQ ID NO: 228
A primer set consisting of an oligonucleotide represented by the base sequence described in
(109) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 229 and SEQ ID NO: 230
A primer set consisting of an oligonucleotide represented by the base sequence described in
(110) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 231 and SEQ ID NO: 232
A primer set consisting of an oligonucleotide represented by the base sequence described in
(111) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 233 and SEQ ID NO: 234
A primer set consisting of an oligonucleotide represented by the base sequence described in
(112) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 235 and SEQ ID NO: 236
A primer set consisting of an oligonucleotide represented by the base sequence described in
(113) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 237 and SEQ ID NO: 238
A primer set consisting of an oligonucleotide represented by the base sequence described in
(114) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 239 and SEQ ID NO: 240
A primer set consisting of an oligonucleotide represented by the base sequence described in
(115) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 241 and SEQ ID NO: 242
A primer set consisting of an oligonucleotide represented by the base sequence described in
(116) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 243 and SEQ ID NO: 244
A primer set consisting of an oligonucleotide represented by the base sequence described in
(117) the oligonucleotide represented by the base sequence set forth in SEQ ID NO: 245 and SEQ ID NO: 246
A primer set consisting of an oligonucleotide represented by the base sequence described in
(118) an oligonucleotide represented by the base sequence described in SEQ ID NO: 247 and SEQ ID NO: 248
A primer set consisting of an oligonucleotide represented by the base sequence described in
(119) an oligonucleotide represented by the base sequence described in SEQ ID NO: 249 and SEQ ID NO: 250
A primer set consisting of an oligonucleotide represented by the base sequence described in
(120) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 251 and SEQ ID NO: 252
A primer set consisting of an oligonucleotide represented by the base sequence described in
(121) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 253 and SEQ ID NO: 254
A primer set consisting of an oligonucleotide represented by the base sequence described in
(122) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 255 and SEQ ID NO: 256
A primer set consisting of an oligonucleotide represented by the base sequence described in
(123) the oligonucleotide represented by the base sequence set forth in SEQ ID NO: 257 and SEQ ID NO: 258
A primer set consisting of an oligonucleotide represented by the base sequence described in
(124) the oligonucleotide represented by the base sequence described in SEQ ID NO: 259 and SEQ ID NO: 260
A primer set consisting of an oligonucleotide represented by the base sequence described in
(125) an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 261 and SEQ ID NO: 262
A primer set consisting of an oligonucleotide represented by the base sequence described in

The present invention also relates to a method for identifying rice species (Oryza sativa) comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention also relates to a method for identifying Zizania latifolia comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention also relates to a method for identifying human wheat (Triticum monococcum), barley (Hordeum vulgaris) or barley (Bromuscatharticus) comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention also includes a petunia axillaris species comprising the following steps:
Regarding the identification method:
(I) a step of preparing DNA from a sample;
(Ii) Using the DNA as a template, the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 13 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 16 as primers, and the RNA polymerase I maximum subunit Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention also relates to a method for identifying Cerasus pendula comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 20 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

The present invention also provides a Fusarium oxysporum species comprising the following steps:
) Identification method:
(I) a step of preparing DNA from a sample;
(Ii) Amplifying a DNA fragment of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA represented by SEQ ID NO: 21 and the oligonucleotide represented by SEQ ID NO: 22 as primers Process,
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

INDUSTRIAL APPLICABILITY According to the present invention, a method for identifying a eukaryotic species can be provided which comprises analyzing an amino acid sequence encoded by the base sequence of a specific DNA region of RNA polymerase I largest subunit gene as an index. The amino acid sequence encoded by the base sequence of the specific DNA region analyzed in the species identification method according to the present invention has two conservative properties in the process of evolution existing in the C-terminal region of the RNA polymerase I largest subunit. Amino acid sequence between regions.
Furthermore, the present invention can provide a primer useful for a method for identifying a eukaryotic species.

  The species identification method according to the present invention is useful for organism identification, identification, classification, and the like.

RNA polymerase I The amino acid sequence between two highly conserved regions existing in the C-terminal region of the largest subunit and the base sequence that encodes the amino acid sequence are not merely a list of amino acids and base sequences, but are related to the strain. In addition to being effective in determining the description of new species and variety patents, etc., management, identification, and identification of resource organisms, rare organisms, disease organisms, food organisms, raw organisms, experimental organisms, etc. It can be applied to diagnosis and investigation. In particular,
It is also useful as a reference for preventing biological strain differences in Genebank, which has recently become a problem.

The present invention relates to a method for identifying eukaryotic species. In this method, species identification is carried out using as an index the amino acid sequence showing a mutation specific to a eukaryotic species present in the C-terminal region of the protein encoded by the eukaryotic RNA polymerase I largest subunit gene. It is characterized by. In the present specification, a region encoding this amino acid sequence in the RNA polymerase I largest subunit gene is referred to as a specific DNA region of the RNA polymerase I largest subunit gene.

“Identification of eukaryotic species” includes both identifying eukaryotic species of different species and identifying eukaryotic subspecies of the same species. “Eukaryote” refers to an organism having a structure called a cell nucleus in cells constituting the body, and includes animals, plants, fungi, protists, and the like. “Species” is the basic unit of biological classification and refers to the category immediately below the genus or subgenus. In general, it can be defined as a population of organisms that can be mated and as a result can leave offspring. “Subspecies” refers to subdivisions of species in a biological taxonomy. In the case of plants, subspecies and variety are used interchangeably.

The eukaryotic species identification method according to the present invention comprises the following steps:
(I) a step of preparing DNA from a sample;
(Ii) a process of amplifying a specific DNA region of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA as a template, wherein the specific DNA region is present in the C-terminal region of the protein encoded by the gene A DNA region that encodes an amino acid sequence sandwiched between two regions that are conserved in the course of evolution.
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

Any sample can be used as long as it is a sample derived from a eukaryote and contains a nucleic acid. Preparation of DNA from a sample can be performed using a known DNA preparation method. For example, the total genomic DNA is prepared by the CTAB (Cetyltrimethil ammmonium bromide) method (Doyle, J.
J.) et al., “A rapid DNA isolation procedure for small quantities of fresh leaftis.
sue ", Phytochemical Bulletin, 1987, Volume 19, p.11-
15) and its modification method. The cDNA can be prepared, for example, by the method of Gubler and Hoffman (Gubler, U. et al., “A simple and very efficient method for gen
erating cDNA libraries ", Gene, 1983, Vol. 25, p.263-269).

“RNA polymerase largest subunit 1 (sometimes abbreviated as POLA1)” is the largest subunit of the RNA polymerase I complex involved in the synthesis of ribosomal RNA (rRNA) that is essential for maintaining life. The gene encoding POLA1 is referred to as RNA polymerase I largest subunit gene (hereinafter sometimes abbreviated as PolA1 gene).

The “specific DNA region of the PolA1 gene” is a region encoding an amino acid sequence showing a mutation specific to a eukaryotic species present in the C-terminal region of POLA1.

The specific DNA region of the PolA1 gene is referred to herein as Ntag. A DNA region encoding an amino acid sequence sandwiched between two regions that are conserved in the process of evolution existing in the C-terminal region of the protein encoded by the PolA1 gene, and consists of about 1.1 kb.

A region present in the C-terminal region of POLA1 encoded by Ntag is referred to as Ptag. The Ptag sequence is an amino acid sequence sandwiched between two regions that are conserved in the process of evolution existing in the C-terminal region of POLA1, and consists of 300 to 500 amino acid residues. The Ptag sequence is an amino acid sequence that shows mutations specific to eukaryotic species.

Of the two regions that are conserved during the evolution process across the Ptag sequence, the region adjacent to the N-terminal side of the Ptag sequence is called PBL (Protein border sequence of left), and the region adjacent to the C-terminal side of the Ptag sequence Is called PBR (Protein border sequence of right). PBL and PBR
The DNA region of the PolA1 gene that encodes each is called NBL and NBR. PBL sequences and PBR
The NBL and NBR sequences corresponding to the sequences are also well conserved during evolution.

Two regions that are conserved in the process of evolution across the Ptag sequence, for example, PBL is the 117th of POLA1.
A region represented by the 5th to 1194th amino acid sequence (SEQ ID NO: 1), and PBR is a region represented by the 1581st to 1596th amino acid sequence (SEQ ID NO: 2) of the protein; Here, this position is associated with a protein encoded by the nucleotide sequence of accession number NM_115626 of the PolA1 gene of Arabidopsis thaliana.

PBL and PBR in POLA1 of each eukaryote are, for example, Arabidopsis Sariana (Arabi
dopsis thaliana) protein encoded by the PolA1 gene can be identified based on the homology with PBL and PBR (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). A region consisting of an amino acid sequence having at least 40% or more, preferably 50% or more, more preferably 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1 can be identified as PBL. A region consisting of an amino acid sequence having at least 40% or more, more preferably 50% or more, more preferably 60% or more, even more preferably 70% or more homology with the amino acid sequence shown in SEQ ID NO: 2 is referred to as PBR Can be identified. Furthermore, since the number of amino acids of Ptag, which is an amino acid sequence sandwiched between PBL and PBR, is 300 to 500 residues, in addition to the above homology, between one region and the other region of the two regions When the number of amino acids between them is 300 residues or more, preferably 300 to 500 residues, the two regions can be identified as PBL and PBR. For example, human PBL and PBR have 95% homology with the amino acid sequence set forth in SEQ ID NO: 1 and 69% with the amino acid sequence set forth in SEQ ID NO: 2, respectively. Giardia PBL and PBR have 70% homology with the amino acid sequence set forth in SEQ ID NO: 1 and 63% with the amino acid sequence set forth in SEQ ID NO: 2, respectively. Malaria protozoa (Malaria) PBL and PBR have 50% homology with the amino acid sequence set forth in SEQ ID NO: 1 and 70% and with the amino acid sequence set forth in SEQ ID NO: 2, respectively. Theileria PBL and PBR have the amino acid sequence set forth in SEQ ID NO: 1 and
85% and 69% homology with the amino acid sequence set forth in SEQ ID NO: 2. Negrelia (Naegl
eria) PBL and PBR have 65% homology with the amino acid sequence set forth in SEQ ID NO: 1 and 56% with the amino acid sequence set forth in SEQ ID NO: 2, respectively.

Ptag and Ntag sequences can be determined one by one per haploid genome, so they can be applied not only to diploid but also to hybrid and polydiploid species, so all eukaryotic species are identified Can be used as an indicator for “Haploid”, also referred to as haploid or monophasic, means a cell or individual having only half the chromosome set. The gametes produced by maturation are only haploid with only one set of genomic chromosomes. “Hybrid” refers to an organism born by mating / crossing between individuals of different strains in genetics. “Diploid” refers to a cell or individual having a chromosome number twice that of the basic number. When the genome is clear, it means an individual with two identical genomes. “Amphidiploid” refers to an allogeneic tetraploid with two different genomes.

Determination of the Ptag sequence of each eukaryote can be performed by decoding the Ntag sequence encoding the Ptag sequence in the genomic DNA or cDNA base sequence. The Ntag sequence can be decoded, for example, by amplifying Ntag and sequencing it.

Ntag amplification can be performed using a known technique capable of amplifying a gene fragment, such as the specific DNA.
Polymerase chain reaction using primers that can amplify the region
on; abbreviated as PCR). PCR is a reaction that specifically amplifies a DNA fragment sandwiched between a set of primers by executing a DNA polymerase reaction continuously and in a chain.

Since the amino acid sequence encoded by the Ntag sequence is an amino acid sequence sandwiched between two regions that are conserved during the evolution process existing in the C-terminal region of the protein encoded by the PolA1 gene, for example, the two Using a primer set consisting of a primer designed based on a base sequence encoding one region of a conservative region and a primer designed based on a base sequence encoding the other region, the two conservation properties Ntag between regions with
The sequence can be amplified.

In other words, using a primer set created based on NBL and NBR sequences, PCR
Can amplify Ntag. As mentioned above, both NBL and NBR sequences are PB
As well as the L and PBR sequences, they are well conserved during the evolution process, so by designing and utilizing primers based on the NBL and NBR sequences, you can use PCR for Ntag sequences.
Amplification and sequencing can be performed.

The base sequence of the amplification product can be determined using a known genetic engineering technique. For example, it can be simply carried out using a commercially available DNA sequencer.

In fungi and nematodes, no intron is found inside the Ntag sequence, so that DNA fragments containing the Ntag sequence can be amplified using not only cDNA but also genomic DNA as a template, and analysis of the Ntag sequence can be easily performed. On the other hand, in eukaryotes such as animals, plants and insects, the Ntag sequence is separated by a plurality of introns. Depending on the species, a transposon or the like may be inserted into the intron and the intron may be extremely long. Therefore, it may be difficult to analyze the Ntag sequence by amplifying a DNA fragment containing the Ntag sequence using genomic DNA as a template. In such a case, analysis of the Ntag sequence can be carried out by amplifying Ntag by PCR using cDNA as a template and decoding the base sequence using direct sequencing.

The amino acid sequence encoded by the amplified DNA fragment, that is, the Ptag sequence can be easily determined from the base sequence of the obtained amplification product.

The amino acid sequence encoded by the amplified DNA fragment is compared with the amino acid sequence of the known eukaryotic POLA1. Comparison of amino acid sequences can be performed using a computer database. NCBI's pblastn web server (Altschul, SF et al., “Basic local alignment search tool”, Journal of Molecular Biology, 1990, 215
Vol., P.403-410) and Clustal W (Thompson, J. D) of DDBJ of the National Institute of Genetics
.) Et al., “CLUSTAL W: improving the sensitivity of progressive multiplesequence al.
ignment through sequence weighting, position-specific gap penaltiesand weight ma
trix choice ", Nucleic Acids Research, 1994, Vol. 22, No. 22, p.4673-4680).

POL containing the amino acid sequence encoded by the amplified DNA fragment by comparison of amino acid sequences
If A1 is found in a protein database or DNA database, it can be determined that the eukaryotic species or subspecies having POLA1 is the eukaryotic species or subspecies from which the amplified DNA fragment was derived.

For example, in the method for identifying a eukaryotic species according to the present invention, when determining whether a sample is a eukaryote of a specific species or subspecies, the Ntag sequence of the specific species or subspecies can be amplified.
Using a pair of primers, an amplification reaction is performed using the nucleic acid extracted from the sample as a template. The base sequence of the obtained DNA fragment is determined, and the amino acid sequence encoded by the DNA fragment is compared with the amino acid sequence of POLA1 of the specific species or subspecies. An amino acid sequence encoded by the DNA fragment;
If the amino acid sequence of POLA1 of the specific species or subspecies matches, it can be determined that the sample is a eukaryote of the specific species or subspecies.

Further, for example, in the method for identifying a eukaryotic species according to the present invention, when discriminating a plant species or subspecies as a sample, the sample is used by using one or more primers capable of amplifying a plant Ntag sequence. An amplification reaction is performed using the nucleic acid extracted from the template as a template. The base sequence of the obtained DNA fragment is determined, and the amino acid sequence encoded by the DNA fragment is compared with the amino acid sequence of plant POLA1 registered in a protein database or DNA database. If a species or subspecies having POLA1 containing an amino acid sequence that matches the amino acid sequence encoded by the DNA fragment is found in the searched database, the sample can be determined to be a plant of the special species or subspecies.

On the other hand, if POLA1 containing the amino acid sequence encoded by the amplified DNA fragment is not found in the protein database or DNA database, the eukaryotic species or subspecies from which the amplified DNA fragment was derived may be a new species It can be determined that there is sex. In such cases, the amplified
By performing molecular phylogenetic analysis on the amino acid sequence encoded by the DNA fragment, it is possible to predict which species or subspecies the eukaryotic species or subspecies from which the amplified DNA fragment was derived. "Molecular phylogenetic analysis" is a method to estimate the path (system) in which an organism has evolved (by creating a phylogenetic tree) by comparing the amino acid sequence of the protein of the organism and the base sequence of the gene. . Molecular phylogenetic analysis is, for example, MEGA4 (Tamura, K.)
Et al., `` MEGA4: Molecular Evolutionary Genetics Analysis MEGA Software Version 4.0 ''
, Molecular Biology and Evolution
volution), 2007, Vol. 24, p.1596-1599).

Thus, in addition to the steps (i) to (iv) above, the method for identifying a eukaryotic species according to the present invention further comprises (v) a step of performing molecular phylogenetic analysis based on the decoded amino acid sequence. Can be included.

In the method according to the present invention, primer sets used for amplification of Ntag encoding a Ptag sequence are exemplified below. This method can be performed using any one of these primer sets, or two or more sets.

Specific examples of primers used for amplification of Ntag encoding a Ptag sequence include the primers shown in Table 1. The Ntag sequences of the species shown in Table 1 are exons 19 and 19.
NBL and N encoded between exon 20 and exon 21 and sandwiching Ntag sequence
BR exists in exons 19 and 21, respectively. 19ex5P and 21ex3 shown in Table 1
The primer set consisting of P can be used as a primer for amplification of the Ntag of the corresponding species shown in Table 1. Note that 20ex5Pi and 20ex3Pi in Table 1 are sequencing primers used for sequencing amplification products obtained using the amplification primers shown in Table 1.

More specifically, a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 8 is
It can be used to amplify the Ntag of the rice species (Oryza sativa) and Macomo species (Zizania latifolia). A primer set comprising an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 9 and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 12 is a grass family Triticum monococcum species, barley species (Hordeum species) vulgaris
) And Ntag amplification of Bromus catharticus. A primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 16 is Ntag of Petunia axillaris species belonging to the genus Petunia. Can be used for amplification of A primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 20 is Ntag of the Rosaceae genus Cerasus pendula. Can be used for amplification of

In addition, as a primer set used for amplification of Ntag encoding a Ptag sequence, a primer comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 21 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 22 A set can be illustrated. This primer set can be used to amplify Ntag of the plant pathogen Fusarium oxysporum species.

Furthermore, as a primer set used for amplification of Ntag encoding Ptag sequence, Table 2-
The primer sets shown in Table 1, Table 2-2, Table 2-3, Table 2-4, Table 2-5, Table 2-6, and Table 2-7 can be exemplified. Based on the base sequences of primers shown in Table 2-1, Table 2-2, Table 2-3, Table 2-4, Table 2-5, Table 2-6, and Table 2-7, and further suitable for amplification Simple primers can be designed.

The base sequences of these primers are also common primers that amplify the Ntag sequence of all related species, primers that only amplify the Ntag sequence of a specific species, primers that only amplify the Ntag sequence of the infecting pathogen, crops It is useful for designing primers used for genotyping such as varieties. For example, a primer that amplifies an Ntag sequence of a species or subspecies belonging to a grass family without the selectivity of the species or subspecies can be used for identifying a species or subspecies of a grass family by the identification method according to the present invention. . Primers that amplify the Ntag sequence of species or subspecies belonging to the genus Gramineae without the selectivity of the species or subspecies are used to identify the species or subspecies of the genus Gramineae by the identification method according to the present invention. it can. Primers that amplify the Ntag sequence of a species or subspecies belonging to the genus Gramineae without the selectivity of the species or subspecies are used to identify the species or subspecies of the genus Wheat by the identification method according to the present invention it can. A primer that amplifies the Ntag sequence of a species or subspecies belonging to the genus Baraceae without selective species or subspecies is used to identify the species or subspecies of a barley plant by the identification method according to the present invention. it can.

The 5P primer and 3P primer in Table 2-1, Table 2-2, Table 2-3, Table 2-4, Table 2-5, Table 2-6, and Table 2-7 correspond respectively to each table. Created based on Species NBL and NBR sequences. Therefore, a primer set consisting of 5P primer and 3P primer shown in each table can be used as an amplification primer for Ntag of the corresponding species shown in the table. For example, of Ajellomyces capsulatus
Examples of Ntag amplification primers include a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 23 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 24. Also, for example, Ascosphaera
As a primer for Ntag amplification of (apis), a primer set comprising an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 25 and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 26 can be exemplified.

Examples of a method for identifying a eukaryotic species according to the present invention include a method for identifying a rice species (Oryza sativa). The method for identifying rice species (Oryza sativa) according to the present invention includes the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

If the amino acid sequence of the amplified DNA fragment matches the partial sequence of POLA1 of a rice species by comparison of the amino acid sequences, it can be determined that the rice species is contained in the sample. As shown in the examples described later, this method could distinguish between Indian and Japanese subspecies of rice.

As a method for identifying a eukaryotic species according to the present invention, an example of a method for identifying a Zakonia latifolia species can be given. The identification method of the Zosteria latifolia according to the present invention includes the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

If the amino acid sequence encoded by the amplified DNA fragment matches the partial sequence of POLA1 of the Macomo species by comparison of the amino acid sequences, it can be determined that the sample contains the Macomo species.

As a method for identifying eukaryotic species according to the present invention, human wheat seed (Triticummonococcum)
And a method for identifying barley species (Hordeum vulgaris) or barley species (Bromus catharticus). The method for identifying human wheat (Triticum monococcum), barley (Hordeum vulgaris) or barley (Bromus catharticus) according to the present invention comprises the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

If the amino acid sequence of the amplified DNA fragment matches the partial sequence of POLA1 of human wheat wheat species by comparison of the amino acid sequences, it can be determined that the sample contains the human wheat wheat species. When the amino acid sequence encoded by the amplified DNA fragment matches the partial sequence of POLA1 of barley species, it can be determined that the sample contains the barley species. In addition, POLA1 of the barley species with the amino acid sequence encoded by the amplified DNA fragment
Can be determined to contain the barley species.

As a method for identifying a eukaryotic species according to the present invention, Petunia axialis species (Petunia ax
illaris) can be exemplified. Petunia axillaris species (Petunia) according to the present invention
axillaris) identification method comprises the following steps:
(I) a step of preparing DNA from a sample;
(Ii) Using the DNA as a template, the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 13 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 16 as primers, and the RNA polymerase I maximum subunit Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

When the amino acid sequence is compared with the partial sequence of POLA1 of Petunia axillaris species having an amino acid sequence encoded by the amplified DNA fragment, it can be determined that the sample contains the Petunia axillaris species.

As a method for identifying a eukaryotic species according to the present invention, Edahigan cherry (Cerasus pendula)
The identification method can be illustrated. The identification method of the edible cherry tree species (Cerasus pendula) according to the present invention is characterized by including the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 20 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

If the amino acid sequence of the amplified DNA fragment matches the partial sequence of POLA1 of Edoganzakura species as a result of the comparison of the amino acid sequences, it can be determined that the Edohirazakura species are contained in the sample.

As a method for identifying a eukaryotic species according to the present invention, Fusarium oxysporum species (Fusarium
The identification method of oxysporum) can be illustrated. Fusarium oxysporum species (Fu) according to the present invention
sarium oxysporum) identification method is characterized by the following steps:
(I) a step of preparing DNA from a sample;
(Ii) Amplifying a DNA fragment of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA represented by SEQ ID NO: 21 and the oligonucleotide represented by SEQ ID NO: 22 as primers Process,
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. Po
The step of comparing with the amino acid sequence of the lA1 gene.

When the amino acid sequence is compared with the partial sequence of POLA1 of the Fusarium oxysporum species having an amino acid sequence encoded by the amplified DNA fragment, it can be determined that the sample contains the Fusarium oxysporum species. As shown in the examples described later, the present method was able to distinguish between Race 1-2 and Race 4 of the Fusarium oxysporum species.

EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

(1) Examination of homology of the largest subunits of three types of RNA polymerases in plants Each of the three types of RNA polymerase complexes in Arabidopsis: RNA polymerase I complex, RNA polymerase II complex, and RNA polymerase III complex The homology of the largest subunits POLA1, POLB1, and POLC1 with the β ′ largest subunit of the E. coli RNA polymerase complex was compared.

As a result, although they were generally similar to each other, a specific CTR (C-terminal repeat) sequence was contained at the C-terminus of the POLB1 subunit. In addition, POLA1 subunit
Compared to the two subunits, about 180 amino acid sequences were inserted in the N-terminal and C-terminal regions, respectively (FIG. 1).

(2) Comparison of plant POLA1 subunits In order to compare the homology of plant POLA1 subunits, the rice genome database was searched to identify the PolA1 gene. The rice PolA1 gene is approximately 15 kb long and is divided into 21 exons and encodes a large protein of 1673 amino acid residues (approximately 188 kDa).

When comparing the amino acid sequences encoded by each exon of the PolA1 gene between rice and Arabidopsis, as shown in Fig. 2, 15 out of 21 exons showed high homology of 40% or more. However, amino acid sequence homology was found to be significantly lower in exons 4-5 and 20 corresponding to the two inserted sequences of the N-terminal and C-terminal regions specific for the POLA1 subunit described above. It was. This 20th exon was the longest exon in the plant PolA1 gene.

(3) Comparison of plant PolA1 gene exon 20 For the rice plants Makomo, barley, barley, single wheat, petunia and cherry, a DNA fragment containing the 20th exon of the PolA1 gene was amplified by PCR, and the direct sequence was used. The nucleotide sequence was analyzed by the method. Primers used for amplification and sequencing of DNA fragments were designed based on the nucleotide sequences of rice, wheat, tomato and almond EST (expression sequence tag) (Table 1).

In addition, DNA sequences were searched to collect base sequences of exon 20 of plant PolA1 gene. When the base sequences of the 13th exon thus obtained were converted to amino acid sequences and molecular phylogenetic analysis was performed using MEGA4, it was revealed that the mutations closely matched the plant affinity (Fig. 3). ).

(4) Ptag sequence setting In order to compare the amino acid sequences of the C-terminal region of all eukaryotic POLA1 proteins, the base sequence of the whole genome from five taxonomic groups of fungi, nematodes, insects, animals and plants Has been deciphered 8
Species (Arabidopsis thaliana, Oryza sativa, Homo sapiens, Mus musculus, Xenopus laev
was, Danio rerio, Caenorhabditis elegans, Saccharomyces cerevisiae) PolA1 gene sequences were searched to identify PBR and PBL sequences that are highly conserved among POLA1 proteins.

The identified PBR and PBL sequences were about 20 and 16 residues, respectively (Figure 4). Ptag to characterize the eukaryotic haploid genome with the amino acid sequence between these two conserved sequences
Set as an array. When these 8 Ptag sequences were compared, no homology was found between the 5 taxa.

(5) Structure of the eukaryotic PolA1 gene When comparing the structure of the PolA1 gene of representative species belonging to different eukaryotic taxonomic groups, it was found that the exon structure is greatly different. FIG. 5 schematically shows the structure of fungal, nematode, insect and animal genes in the region corresponding to exons 19 to 21 of the plant PolA1 gene. Within this region, there are 2 introns in plants, 3 in nematodes, 4 in insects, and 8 in animals, but no introns were found in fungi and protists. In addition, exon length and intron insertion position in each taxon were well preserved, but intron length varied greatly depending on species and strains due to insertion and deletion of transposon and the like.

(6) Analysis of Ntag sequence and Ptag sequence Ntag sequence of each organism can be obtained by querying Ptag sequence in DNA database such as NCBI.
Searched and collected using blastn server. Convert collected base sequences to amino acid sequences
PBR and PBL sequences were identified. NBR and N corresponding to the identified PBR and PBL sequences, respectively
The BL sequence was analyzed (FIG. 6), and the base sequence between the two was checked against the EST database. But,
In most species, EST clones containing full-length Ntag sequences could not be detected, so Ntag and Ptag sequences were determined using partial EST information. In vertebrates and the like, the Ptag sequence was analyzed using the base sequence attached with the annotation of the PolA1 gene.

So far, a total of 120 Ptag sequences have been determined, including 57 fungi, 18 insects, 2 nematodes, 14 animals, 15 plants, and 14 protists (other eukaryotes). The average length of the sequence was 366 residues. The shortest Ptag sequence was 301 of the microsporidia Encephalitozoon cuniculi
Residues, the longest being significantly different from the 457 residues of Piroplasma parasite Babesia bovis (Tables 3-1 to 3-7). The Ptag sequence contained two types of acidic amino acids (glutamic acid and aspartic acid) in a total of 14 to 25% (Tables 3-1 to 3-7). Since the content of the two acidic amino acids in a normal protein is about 9%, the content of acidic amino acids in this Ptag sequence appears to be abnormally high. Although the function of this Ptag sequence is not yet understood, it is possible that it is involved in binding to the DNA strand because of its high acidic amino acid content. Further, the DNA fragment encoding the full-length Ptag sequence could not be cloned using an E. coli plasmid. Although it is not known whether this is associated with a high content of acidic amino acids, it is thought to be consistent with the fact that EST clones containing the full-length Ntag sequence cannot be detected from the DNA database. Furthermore, since the Ptag sequence contains many unique amino acid sequences that are not found in ordinary proteins, it is considered that synthetic peptide antibodies can be produced and applied to species determination.

(7) Molecular phylogenetic tree created from eukaryotic Ptag sequences
Most of the Ptag sequences collected from DNA databases are of fungi. The reason is
This is because the Ptag sequence could be determined directly from the genomic DNA base sequence since the Ntag region of fungi did not contain introns. In order to accurately align various Ptag sequences, common (eg, rice) PBR and PBL sequences were added to both ends of the Ptag sequence. Collected
Data of 120 Ptag sequences were aligned by Clustal W, input to MEGA4 software, and a molecular phylogenetic tree was created using the neighborhood distance binding method (FIGS. 7-1 and 7-2).

In the obtained molecular phylogenetic tree, fungi, animals (nematodes, insects, vertebrates) and plants are
Each formed three large groups. Species belonging to the fungus Ascomycota are divided into three main groups: Aspergillus group, such as filamentous fungi group, Shizosaccharomyces group, and Saccharomycesis unicellular group. Divided. The Aspergillus genus and Neosartorya (Aspergillus tereomorph) genus formed one clade, and the various fungal groups were in good agreement with the conventional classification. On the other hand, in the yeast genus group, there were some differences from the conventional classification system, including two species of the genus Saccharomyces and one species of the genus Candida included in different clades. Basidiomycota
The genus Laccaria, Malassezia, Cryptococcus and Ustilago4 of diomycota formed a clade. In addition, the genus Batrachochytrium of Chitridiomycota and the Rhizopus genus of Zygomycota belonged to the same clade as Shizosaccharomyces, which seems to be due to the fact that there was only one test strain. It is.

Monosiga brevicollis, the origin of metacellularization of metazoans, diverged from a common ancestor of fungi, animals and plants. The diatom (Thalassiosira)
It was contained in the same clade as three species of genus a. Nematoda and insects (Insect) belonging to the animal kingdom
a) and various species of vertebrates (Vertebrata) formed a large clade.
ylocentrotus purpuratus) was located between nematodes and vertebrates. In insects, Dr
It was consistent with the conventional classification system, including 12 species of the genus osophila included in the same group. In vertebrates, the positions of amphibians and birds were reversed from the conventional classification system, but various other relationships were consistent with the conventional theory. 12 terrestrial plants are unicellular green algae Chlamydomona
The same clade was formed with one species of genus s (C. reinhardtii) and two species of genus Ostreococcus (O. lucimarinus, O. tauri). Nine dicotyledons and three monocotyledons were clearly branched.

Protozoan Shigella Amoeba (E. histolytica, E. dispar), slime mold (Di
ctyosterium discoidem), ciliate Paramecium tetraurelia and GiardiaLamblia formed a group. Three species of Piroplasma parasites (Theileria annulata, T. parva, Babesia bovis) and microsporidia (Encephalitoz)
oonconiculi) belonged to the fastest branched clade.

(8) Comparison of Ptag and Ntag sequences of strains classified into the same species Although there are not many examples of Ptag sequences determined so far in strains classified into the same species, two species of the fungus Coccidioides genus In C. immitis and C. poradasii, 3 and 4 Ptag sequences could be determined, respectively (Table 4). Two residue substitutions were observed between the two Ptag sequences, but strains belonging to the same species each showed the same Ptag sequence. Meanwhile, Ntag
The sequence showed 8 base substitutions between both species, and one C. poradasii strain (2133) had 2 substitutions with the other 3 strains of the same species. In Candida albicans, two Ptags
And the Ntag sequence could be determined. There were 4 base substitutions in the Ntag sequence between the two lines, but the Ptag sequence was the same. In two strains of Cryptococcus neoformans, Ptag and Nt
Both ag sequences were the same.

In Table 4, the numerical values shown in the items of the degree of change of Ptag and the degree of change of Ntag indicate the length of each amino acid sequence and base sequence for each line described in the top row, and for other lines Indicates the number of amino acids and nucleotides different from those of the outer strain.

Next, the Ntag and Ptag sequences of each of the three lines classified into two ecotypes (Indian and Japanese) of cultivated rice (Oryza sativa) were analyzed. As a result, although no mutation was observed in each ecotype, substitution of 2 to 4 bases in the Ntag sequence and 1 residue in the Ptag sequence was observed between the Indian and Japanese types (Table 4).

(9) Identification of subspecies of cultivated rice using Ptag sequence and Ntag sequence as indicators. The Indian type and Japanese type of cultivated rice (Oryza sativa) were used as samples, and they were identified. Genomic DNA was extracted from each sample according to the DNA extraction method described below. As a primer for amplifying rice Ntag, an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 (FIG. 9) were used. Rice Ntag was amplified by PCR using the genomic DNA as a template and the above-mentioned pair of primers. The amplified DNA fragment was purified and the nucleotide sequence was decoded. Big Dye Termi
After performing the reaction using nator Cycle Sequencing Kit (AppliedBiosystems) and sequence primer (SEQ ID NO: 6 and SEQ ID NO: 7), ABI3100 sequencer (Appl
ied Biosystems).

The analyzed Ntag sequence was searched using the amino acid sequence of POLA1 protein as a query using NCBI's pblastn web server. As a result, the amino acid sequence encoded by the base sequence of the DNA fragment amplified as described above using Indian rice as a sample matched the amino acid sequence of Indian rice registered in the computer database. In addition, the amino acid sequence encoded by the base sequence of the DNA fragment amplified as described above using Japanese rice as a sample matched the amino acid sequence of Japanese rice registered in the computer database. Thus, using the amino acid sequence encoded by Ntag as an index, we were able to distinguish between Indian and Japanese subspecies of rice.

(10) Discrimination of phytopathogenic fungus Fusarium using Ptag and Ntag sequences as indicators. Among the Fusarium oxysporum species, Race1-2 and Race4 that infect bananas were identified. Genomic DNA was extracted from each sample according to the DNA extraction method described below. As primers for amplifying Fusarium Ntag, an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 21 and an oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 22 (FIG. 9) were used. Using the genomic DNA as a template, the above 1
Fusarium Ntag was amplified by PCR using a pair of primers. The amplified DNA fragment was purified and the nucleotide sequence was decoded.

The analyzed Ntag sequence was searched using the amino acid sequence of POLA1 protein as a query using NCBI's pblastn web server. As a result, the amino acid sequence encoded by the base sequence of the DNA fragment amplified as described above using Fusarium Race1-2 was identical to the Fusarium Race1-2 amino acid sequence registered in the computer database. Further, the amino acid sequence encoded by the base sequence of the DNA fragment amplified as described above using Fusarium Race4 as a sample matched the amino acid sequence of Fusarium Race4 registered in the computer database. Thus, using the amino acid sequence encoded by Ntag as an index, Fusarium could identify Race1-2 and Race4 that infect bananas.

(11) Homology between PBL and PBR sequences In different species, PBL and PBR sequences were compared and their homology was examined. As shown in FIG. 10, the PBL and PBR sequences were compared between human and Arabidopsis thaliana.
aliana) showed 95% and 69% homology, respectively. PBL and PBR sequences can be transferred to Giardia and Arabidopsis thaliana
) Showed 70% and 63% homology, respectively. PBL sequences and PBR
The sequence is composed of Malaria and Arabidopsis thaliana.
) Showed 70% and 50% homology, respectively. PBL sequences and PBR
The sequence is divided into Theileria and Arabidopsis thali.
ana) showed 85% and 69% homology, respectively. PBL sequences and
PBR sequences can be obtained from Naegleria and Arabidopsis thalia.
Comparison between na) showed 65% and 56% homology, respectively. PBL sequences and P
The BR sequence was compared between human and flagellate (Giardia) and showed 65% and 69% homology, respectively. The PBL and PBR sequences were compared between human and malaria parasites and showed 70% and 56% homology, respectively. PBL and PBR sequences were compared between rice and malaria, showing 70% and 63% homology, respectively.

As described above, eukaryotic species or subspecies can be identified by using the Ptag sequence as an index. In order to elucidate the speciation of an organism, it is important to analyze a large number of DNA markers contained in the genome and proceed with the comprehensive analysis.
Analyzing the A marker requires cost and effort. In order to use the DNA base sequence analysis method as an auxiliary means for determining the species of an organism, it is effective to use a small number of base sequences as proposed in DNA barcoding. If it is a small number of base sequences, it can be easily analyzed and it is considered easy to construct a database. But DN
When the A base sequence is used for species determination, it is ambiguous where to delimit the base sequence homology boundary. On the other hand, when an amino acid sequence is used, only non-synonymous base substitutions are reflected in the amino acid sequence, so that the boundary of molecular biological classification can be clearly determined. In this example, as a result of analysis of several strains belonging to the same species, in Candida albicans and Cryptococcus neoformans, which belong to the genus Coccidioides, the strains belonging to the same species are the same P
It had a tag array (Table 3). On the other hand, in cultivated rice (Oryza sativa), 2-residue amino acid substitutions were found in the Indian and Japanese Ptag sequences, which are classified into two ecotypes (Table 4).
. I don't know how much the classification of the Linne species corresponds to the mutation of the Ptag sequence, but grouping the strains with the same Ptag sequence and classifying one molecular biological classification: ipsum (identical
protein sequence unit member).

  The materials and methods used in this example are described below.

1) Extraction of plant material and genomic DNA The materials shown in Table 1 above were used as test materials such as rice genus, barley genus and cherry genus. 100 mg of the test material leaves are placed in a 2 ml tube and frozen with liquid nitrogen.
The product was pulverized using Yasui Kikai Co., Ltd. Total genomic DNA is CTAB (Cetyltrimethil ammm
onium bromide method (Doyle, JJ et al., “A rapid DNA isolation procedure f
or small quantities of fresh leaftissue ", Phytochemic
al Bulletin), 1987, Vol. 19, p.11-15).

2) PCR amplification and direct sequencing The DNA fragment containing the plant's PolA1 gene exon 20 is PC using a pair of primers (Table 1).
Amplified by R method. Primer sequences were designed with reference to EST data such as Arabidopsis, rice, wheat and almonds. Amplification of DNA fragments was performed using PCR plate PTC200 (MJ Res
40 cycles were performed under the conditions of denaturation 94 ° C. for 1 minute, annealing 58 ° C. for 1 minute and extension at 72 ° C. for 2 minutes. ExTaq DNA polymerase (manufactured by TaKaRa Co., Ltd.) was used as the thermostable DNA polymerase.

The amplified DNA fragment was subjected to 1.2% agarose gel electrophoresis to confirm its amplification, and QIAq
Purification was performed using a uick PCR purification kit (Qiagen). The purified DNA fragment is Big
Reaction was performed using Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and sequence primer (Table 1, Figure 8), and ABI3100 sequencer (Applied Biosyst
The base sequence was decoded using EMS.

3) Nucleotide sequence analysis The Ntag sequences of various eukaryotes were searched using the NCBI pblastn web server as a query for the amino acid sequence of POLA1 protein (Altschul, SF et al., “Bas
ic local alignmentsearch tool ", Journal of Molecular Biology (J
ournal of Molecular Biology), 1990, Vol. 215, p.403-410). Full length Ntag sequence is EST
Since it could not be detected from the database, the Ntag sequence was identified using the base sequence data extracted from the full-length genome or genome survey sequence database. Detailed analysis of the base sequence was performed using DNASIS (manufactured by Hitachi) and Genentyx (manufactured by Softoware). Nucleotide sequence alignment is based on Clustal W (Thompson, J
D.) et al., “CLUSTAL W: improving the sensitivity of progressive multiplesequence.
alignment through sequence weighting, position-specific gap penaltiesand weight
matrix choice ", Nucleic Acids Research, 199
4 years, Vol. 22, No. 22, p.4673-4680).

4) Creation of molecular phylogenetic tree The molecular phylogenetic analysis of the amino acid sequence of exon 20 of the PolA1 gene in plants and various Ptag sequences of eukaryotic organisms was performed by MEGA4 (Tamura, K. et al., “MEGA4: Molecular Evolutionary Geneti”.
cs Analysis MEGA Software Version 4.0 ”, Molecular Biology and Evolution, 2007, Vol. 24, pp.1596-1599
). Data aligned by Clustal W was input to MEGA4 and a molecular phylogenetic tree was created using the neighborhood distance coupling method. Create a phylogenetic tree using 500 data
The tstrap value was obtained.

The accession numbers of various eukaryotic base sequence data used in this study are listed in Table 3 above.

The three RNA polymerase complexes in Arabidopsis, namely the largest subunits of each of the RNA polymerase I, RNA polymerase II, and RNA polymerase III complexes, POLA1, POLB1, and POLC1, and the RNA polymerase complex of E. coli It is a figure which shows the result of having compared the homology with (beta) 'largest subunit (RpoC). The part surrounded by a frame shows an insertion sequence specific to POLA1. It is a figure which shows the result of having compared the amino acid sequence encoded by each exon of PolA1 gene between rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana). The arrow indicates the position of the insertion sequence specific for POLA1 shown in FIG. It is a figure which shows that the homology of the amino acid sequence encoded by the 20th exon of the PolA1 gene of a plant corresponds well with the plant affinity. FIG. 3 shows PBL and PBR sequences that are highly conserved among the eight POLA1 proteins of five taxa of fungi, nematodes, insects, animals, and plants. The species compared are: Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), human (Homo sapiens), mouse (Musmusculus), Xenopus (Xenopuslaevis), zebrafish (Danio rerio), nematoda (Caenorhabditis elegans), yces (Sacchary) It is. In the figure, the symbol “*” indicates an amino acid residue that is completely conserved among these species. It is a schematic diagram showing the structure of the 3 ′ region of the PolA1 gene in fungi, nematodes, insects, animals, and plants. It is a figure which shows Ptag arrangement | sequence and the Ntag arrangement | sequence which codes it as an example for Arabidopsis thaliana. FIG. 3 is a diagram showing a molecular phylogenetic tree prepared by aligning Ptag sequences and analyzing the data with MEGA4 software. This figure is the asterisk marked in the figure and is connected to Figure 7-2. FIG. 3 is a diagram showing a molecular phylogenetic tree prepared by aligning Ptag sequences and analyzing the data with MEGA4 software. This figure is the asterisk marked in the figure and is connected to Figure 7-1. FIG. 3 is a schematic diagram showing the position of the nucleotide sequence of a primer for amplification of a DNA fragment containing plant 20th exon of the PolA1 gene in the gene. It is a schematic diagram which shows the position in the rice gene and a fusarium gene of the base sequence of the Ntag amplification primer of rice and a fusarium. It is a figure which shows the homology of a PBL sequence and a PBR sequence. In the figure, the symbol “*” indicates an amino acid residue that is completely conserved.

SEQ ID NO: 5: oligonucleotide designed for primer.
SEQ ID NO: 6: oligonucleotide designed for primer.
SEQ ID NO: 7: oligonucleotide designed for primer.
SEQ ID NO: 8: oligonucleotide designed for primer.
SEQ ID NO: 9: oligonucleotide designed for primer.
SEQ ID NO: 10: oligonucleotide designed for primer.
SEQ ID NO: 11: oligonucleotide designed for primer.
SEQ ID NO: 12: oligonucleotide designed for primer.
SEQ ID NO: 13: oligonucleotide designed for primer.
SEQ ID NO: 14: oligonucleotide designed for primer.
SEQ ID NO: 15: oligonucleotide designed for primer.
SEQ ID NO: 16: oligonucleotide designed for primer.
SEQ ID NO: 17: oligonucleotide designed for primer.
SEQ ID NO: 18: oligonucleotide designed for primer.
SEQ ID NO: 19: oligonucleotide designed for primer.
SEQ ID NO: 20: oligonucleotide designed for primer.
SEQ ID NO: 21: oligonucleotide designed for primer.
SEQ ID NO: 22: oligonucleotide designed for primer.
SEQ ID NO: 23: oligonucleotide designed for primer.
SEQ ID NO: 24: oligonucleotide designed for primer.
SEQ ID NO: 25: oligonucleotide designed for primer.
SEQ ID NO: 26: oligonucleotide designed for primer.
SEQ ID NO: 27: oligonucleotide designed for primer.
SEQ ID NO: 28: oligonucleotide designed for primer.
SEQ ID NO: 29: oligonucleotide designed for primer.
SEQ ID NO: 30: oligonucleotide designed for primer.
SEQ ID NO: 31: oligonucleotide designed for primer.
SEQ ID NO: 32: oligonucleotide designed for primer.
SEQ ID NO: 33: oligonucleotide designed for primer.
SEQ ID NO: 34: oligonucleotide designed for primer.
SEQ ID NO: 35: oligonucleotide designed for primer.
SEQ ID NO: 36: oligonucleotide designed for primer.
SEQ ID NO: 37: oligonucleotide designed for primer.
SEQ ID NO: 38: oligonucleotide designed for primer.
SEQ ID NO: 39: oligonucleotide designed for primer.
SEQ ID NO: 40: oligonucleotide designed for primer.
SEQ ID NO: 41: oligonucleotide designed for primer.
SEQ ID NO: 42: oligonucleotide designed for primer.
SEQ ID NO: 43: oligonucleotide designed for primer.
SEQ ID NO: 44: oligonucleotide designed for primer.
SEQ ID NO: 45: oligonucleotide designed for primer.
SEQ ID NO: 46: oligonucleotide designed for primer.
SEQ ID NO: 47: oligonucleotide designed for primer.
SEQ ID NO: 48: oligonucleotide designed for primer.
SEQ ID NO: 49: oligonucleotide designed for primer.
SEQ ID NO: 50: oligonucleotide designed for primer.
SEQ ID NO: 51: oligonucleotide designed for primer.
SEQ ID NO: 52: oligonucleotide designed for primer.
SEQ ID NO: 53: oligonucleotide designed for primer.
SEQ ID NO: 54: oligonucleotide designed for primer.
SEQ ID NO: 55: oligonucleotide designed for primer.
SEQ ID NO: 56: oligonucleotide designed for primer.
SEQ ID NO: 57: oligonucleotide designed for primer.
SEQ ID NO: 58: oligonucleotide designed for primer.
SEQ ID NO: 59: oligonucleotide designed for primer.
SEQ ID NO: 60: oligonucleotide designed for primer.
SEQ ID NO: 61: oligonucleotide designed for primer.
SEQ ID NO: 62: oligonucleotide designed for primer.
SEQ ID NO: 63: oligonucleotide designed for primer.
SEQ ID NO: 64: oligonucleotide designed for primer.
SEQ ID NO: 65: oligonucleotide designed for primer.
SEQ ID NO: 66: oligonucleotide designed for primer.
SEQ ID NO: 67: oligonucleotide designed for primer.
SEQ ID NO: 68: oligonucleotide designed for primer.
SEQ ID NO: 69: oligonucleotide designed for primer.
SEQ ID NO: 70: oligonucleotide designed for primer.
SEQ ID NO: 71: oligonucleotide designed for primer.
SEQ ID NO: 72: oligonucleotide designed for primer.
SEQ ID NO: 73: oligonucleotide designed for primer.
SEQ ID NO: 74: oligonucleotide designed for primer.
SEQ ID NO: 75: oligonucleotide designed for primer.
SEQ ID NO: 76: oligonucleotide designed for primer.
SEQ ID NO: 77: oligonucleotide designed for primer.
SEQ ID NO: 78: oligonucleotide designed for primer.
SEQ ID NO: 79: oligonucleotide designed for primer.
SEQ ID NO: 80: oligonucleotide designed for primer.
SEQ ID NO: 81: oligonucleotide designed for primer.
SEQ ID NO: 82: oligonucleotide designed for primer.
SEQ ID NO: 83: oligonucleotide designed for primer.
SEQ ID NO: 84: oligonucleotide designed for primer.
SEQ ID NO: 85: oligonucleotide designed for primer.
SEQ ID NO: 86: oligonucleotide designed for primer.
SEQ ID NO: 87: oligonucleotide designed for primer.
SEQ ID NO: 88: oligonucleotide designed for primer.
SEQ ID NO: 89: oligonucleotide designed for primer.
SEQ ID NO: 90: oligonucleotide designed for primer.
SEQ ID NO: 91: oligonucleotide designed for primer.
SEQ ID NO: 92: oligonucleotide designed for primer.
SEQ ID NO: 93: oligonucleotide designed for primer.
SEQ ID NO: 94: oligonucleotide designed for primer.
SEQ ID NO: 95: oligonucleotide designed for primer.
SEQ ID NO: 96: oligonucleotide designed for primer.
SEQ ID NO: 97: oligonucleotide designed for primer.
SEQ ID NO: 98: oligonucleotide designed for primer.
SEQ ID NO: 99: oligonucleotide designed for primer.
SEQ ID NO: 100: oligonucleotide designed for primer.
SEQ ID NO: 101: oligonucleotide designed for primer.
SEQ ID NO: 102: oligonucleotide designed for primer.
SEQ ID NO: 103: oligonucleotide designed for primer.
SEQ ID NO: 104: oligonucleotide designed for primer.
SEQ ID NO: 105: oligonucleotide designed for primer.
SEQ ID NO: 106: oligonucleotide designed for primer.
SEQ ID NO: 107: oligonucleotide designed for primer.
SEQ ID NO: 108: oligonucleotide designed for primer.
SEQ ID NO: 109: oligonucleotide designed for primer.
SEQ ID NO: 110: oligonucleotide designed for primer.
SEQ ID NO: 111: oligonucleotide designed for primer.
SEQ ID NO: 112: oligonucleotide designed for primer.
SEQ ID NO: 113: oligonucleotide designed for primer.
SEQ ID NO: 114: oligonucleotide designed for primer.
SEQ ID NO: 115: oligonucleotide designed for primer.
SEQ ID NO: 116: oligonucleotide designed for primer.
SEQ ID NO: 117: oligonucleotide designed for primer.
SEQ ID NO: 118: oligonucleotide designed for primer.
SEQ ID NO: 119: oligonucleotide designed for primer.
SEQ ID NO: 120: oligonucleotide designed for primer.
SEQ ID NO: 121: oligonucleotide designed for primer.
SEQ ID NO: 122: oligonucleotide designed for primer.
SEQ ID NO: 123: oligonucleotide designed for primer.
SEQ ID NO: 124: oligonucleotide designed for primer.
SEQ ID NO: 125: oligonucleotide designed for primer.
SEQ ID NO: 126: oligonucleotide designed for primer.
SEQ ID NO: 127: oligonucleotide designed for primer.
SEQ ID NO: 128: oligonucleotide designed for primer.
SEQ ID NO: 129: oligonucleotide designed for primer.
SEQ ID NO: 130: oligonucleotide designed for primer.
SEQ ID NO: 131: oligonucleotide designed for primer.
SEQ ID NO: 132: oligonucleotide designed for primer.
SEQ ID NO: 133: oligonucleotide designed for primer.
SEQ ID NO: 134: oligonucleotide designed for primer.
SEQ ID NO: 135: oligonucleotide designed for primer.
SEQ ID NO: 136: oligonucleotide designed for primer
SEQ ID NO: 137: oligonucleotide designed for primer.
SEQ ID NO: 138: oligonucleotide designed for primer.
SEQ ID NO: 139: oligonucleotide designed for primer.
SEQ ID NO: 140: oligonucleotide designed for primer.
SEQ ID NO: 141: oligonucleotide designed for primer.
SEQ ID NO: 142: oligonucleotide designed for primer.
SEQ ID NO: 143: oligonucleotide designed for primer.
SEQ ID NO: 144: oligonucleotide designed for primer.
SEQ ID NO: 145: oligonucleotide designed for primer.
SEQ ID NO: 146: oligonucleotide designed for primer.
SEQ ID NO: 147: oligonucleotide designed for primer.
SEQ ID NO: 148: oligonucleotide designed for primer.
SEQ ID NO: 149: oligonucleotide designed for primer.
SEQ ID NO: 150: oligonucleotide designed for primer.
SEQ ID NO: 151: oligonucleotide designed for primer.
SEQ ID NO: 152: oligonucleotide designed for primer.
SEQ ID NO: 153: oligonucleotide designed for primer.
SEQ ID NO: 154: oligonucleotide designed for primer.
SEQ ID NO: 155: oligonucleotide designed for primer.
SEQ ID NO: 156: oligonucleotide designed for primer.
SEQ ID NO: 157: oligonucleotide designed for primer.
SEQ ID NO: 158: oligonucleotide designed for primer.
SEQ ID NO: 159: oligonucleotide designed for primer.
SEQ ID NO: 160: oligonucleotide designed for primer.
SEQ ID NO: 161: oligonucleotide designed for primer.
SEQ ID NO: 162: oligonucleotide designed for primer.
SEQ ID NO: 163: oligonucleotide designed for primer.
SEQ ID NO: 164: oligonucleotide designed for primer.
SEQ ID NO: 165: oligonucleotide designed for primer.
SEQ ID NO: 166: oligonucleotide designed for primer.
SEQ ID NO: 167: oligonucleotide designed for primer.
SEQ ID NO: 168: oligonucleotide designed for primer.
SEQ ID NO: 169: oligonucleotide designed for primer.
SEQ ID NO: 170: oligonucleotide designed for primer.
SEQ ID NO: 171: oligonucleotide designed for primer.
SEQ ID NO: 172: oligonucleotide designed for primer.
SEQ ID NO: 173: oligonucleotide designed for primer.
SEQ ID NO: 174: oligonucleotide designed for primer.
SEQ ID NO: 175: oligonucleotide designed for primer.
SEQ ID NO: 176: oligonucleotide designed for primer.
SEQ ID NO: 177: oligonucleotide designed for primer.
SEQ ID NO: 178: oligonucleotide designed for primer.
SEQ ID NO: 179: oligonucleotide designed for primer.
SEQ ID NO: 180: oligonucleotide designed for primer.
SEQ ID NO: 181: oligonucleotide designed for primer.
SEQ ID NO: 182: oligonucleotide designed for primer.
SEQ ID NO: 183: oligonucleotide designed for primer.
SEQ ID NO: 184: oligonucleotide designed for primer.
SEQ ID NO: 185: oligonucleotide designed for primer.
SEQ ID NO: 186: oligonucleotide designed for primer.
SEQ ID NO: 187: oligonucleotide designed for primer.
SEQ ID NO: 188: oligonucleotide designed for primer.
SEQ ID NO: 189: oligonucleotide designed for primer.
SEQ ID NO: 190: oligonucleotide designed for primer.
SEQ ID NO: 191: oligonucleotide designed for primer.
SEQ ID NO: 192: oligonucleotide designed for primer.
SEQ ID NO: 193: oligonucleotide designed for primer.
SEQ ID NO: 194: oligonucleotide designed for primer.
SEQ ID NO: 195: oligonucleotide designed for primer.
SEQ ID NO: 196: oligonucleotide designed for primer.
SEQ ID NO: 197: oligonucleotide designed for primer.
SEQ ID NO: 198: oligonucleotide designed for primer.
SEQ ID NO: 199: oligonucleotide designed for primer.
SEQ ID NO: 200: oligonucleotide designed for primer.
SEQ ID NO: 201: oligonucleotide designed for primer.
SEQ ID NO: 202: oligonucleotide designed for primer.
SEQ ID NO: 203: oligonucleotide designed for primer.
SEQ ID NO: 204: oligonucleotide designed for primer.
SEQ ID NO: 205: oligonucleotide designed for primer.
SEQ ID NO: 206: oligonucleotide designed for primer.
SEQ ID NO: 207: oligonucleotide designed for primer.
SEQ ID NO: 208: oligonucleotide designed for primer.
SEQ ID NO: 209: oligonucleotide designed for primer.
SEQ ID NO: 210: oligonucleotide designed for primer.
SEQ ID NO: 211: oligonucleotide designed for primer.
SEQ ID NO: 212: oligonucleotide designed for primer.
SEQ ID NO: 213: oligonucleotide designed for primer.
SEQ ID NO: 214: oligonucleotide designed for primer.
SEQ ID NO: 215: oligonucleotide designed for primer.
SEQ ID NO: 216: oligonucleotide designed for primer.
SEQ ID NO: 217: oligonucleotide designed for primer.
SEQ ID NO: 218: oligonucleotide designed for primer.
SEQ ID NO: 219: oligonucleotide designed for primer.
SEQ ID NO: 220: oligonucleotide designed for primer.
SEQ ID NO: 221: oligonucleotide designed for primer.
SEQ ID NO: 222: oligonucleotide designed for primer.
SEQ ID NO: 223: oligonucleotide designed for primer.
SEQ ID NO: 224: oligonucleotide designed for primer.
SEQ ID NO: 225: oligonucleotide designed for primer.
SEQ ID NO: 226: oligonucleotide designed for primer.
SEQ ID NO: 227: oligonucleotide designed for primer.
SEQ ID NO: 228: oligonucleotide designed for primer.
SEQ ID NO: 229: oligonucleotide designed for primer.
SEQ ID NO: 230: oligonucleotide designed for primer.
SEQ ID NO: 231: oligonucleotide designed for primer.
SEQ ID NO: 232: oligonucleotide designed for primer.
SEQ ID NO: 233: oligonucleotide designed for primer.
SEQ ID NO: 234: oligonucleotide designed for primer.
SEQ ID NO: 235: oligonucleotide designed for primer.
SEQ ID NO: 236: oligonucleotide designed for primer.
SEQ ID NO: 237: oligonucleotide designed for primer.
SEQ ID NO: 238: oligonucleotide designed for primer.
SEQ ID NO: 239: oligonucleotide designed for primer.
SEQ ID NO: 240: oligonucleotide designed for primer.
SEQ ID NO: 241: oligonucleotide designed for primer.
SEQ ID NO: 242: oligonucleotide designed for primer.
SEQ ID NO: 243: oligonucleotide designed for primer.
SEQ ID NO: 244: oligonucleotide designed for primer.
SEQ ID NO: 245: oligonucleotide designed for primer.
SEQ ID NO: 246: oligonucleotide designed for primer.
SEQ ID NO: 247: oligonucleotide designed for primer.
SEQ ID NO: 248: oligonucleotide designed for primer.
SEQ ID NO: 249: oligonucleotide designed for primer.
SEQ ID NO: 250: oligonucleotide designed for primer.
SEQ ID NO: 251: oligonucleotide designed for primer.
SEQ ID NO: 252: oligonucleotide designed for primer.
SEQ ID NO: 253: oligonucleotide designed for primer.
SEQ ID NO: 254: oligonucleotide designed for primer.
SEQ ID NO: 255: oligonucleotide designed for primer.
SEQ ID NO: 256: oligonucleotide designed for primer.
SEQ ID NO: 257: oligonucleotide designed for primer.
SEQ ID NO: 258: oligonucleotide designed for primer.
SEQ ID NO: 259: oligonucleotide designed for primer.
SEQ ID NO: 260: oligonucleotide designed for primer.
SEQ ID NO: 261: oligonucleotide designed for primer.
SEQ ID NO: 262: oligonucleotide designed for primer.

Claims (11)

A method for identifying a eukaryotic species comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) a process of amplifying a specific DNA region of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA as a template, wherein the specific DNA region is present in the C-terminal region of the protein encoded by the gene A DNA region that encodes an amino acid sequence sandwiched between two regions that are conserved in the course of evolution.
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. Two regions that are conserved in the process of evolution existing in the C-terminal region of the protein encoded by the PolA1 gene, one of which is the amino acid sequence from 1175th to 1194th amino acid sequence (SEQ ID NO: 1) And the other region is a region (SEQ ID NO: 2) represented by the amino acid sequence of the 1581st to 1596th amino acids of the protein, wherein this position is the position of Arabidopsis thaliana The method according to claim 1, wherein the method is attached in association with a protein encoded by the nucleotide sequence of the accession number NM_115626 of the PolA1 gene. Two regions that are conserved in the process of evolution existing in the C-terminal region of the protein encoded by the PolA1 gene, one region has 40% or more homology with the amino acid sequence described in SEQ ID NO: 1, The other region has 40% or more homology with the amino acid sequence set forth in SEQ ID NO: 2, and the number of amino acids between the two regions is 300 residues or more, Item 2. The method according to Item 1. Amplification of a specific DNA region of the PolA1 gene was designed based on the base sequence encoding one of the two regions that are conservative during the evolution process present in the C-terminal region of the protein encoded by the gene. 4. The method according to any one of claims 1 to 3, wherein the method is carried out by a polymerase chain reaction using a primer set comprising a primer and a primer designed based on a base sequence encoding the other region. The amplification of the partial region of PolA1 gene is performed by polymerase chain reaction using one set or two or more sets selected from the following primer set group: Method:
(1) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 5 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 8,
(2) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 9 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 12,
(3) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 13 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 16;
(4) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 17 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 20,
(5) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 21 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 22;
(6) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 23 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 24;
(7) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 25 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 26;
(8) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 27 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 28;
(9) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 29 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 30;
(10) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 31 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 32;
(11) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 33 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 34;
(12) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 35 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 36;
(13) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 37 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 38;
(14) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 39 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 40;
(15) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 41 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 42;
(16) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 43 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 44;
(17) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 45 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 46;
(18) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 47 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 48;
(19) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 49 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 50;
(20) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 51 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 52;
(21) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 53 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 54;
(22) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 55 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 56;
(23) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 57 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 58;
(24) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 59 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 60;
(25) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 61 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 62;
(26) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 63 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 64;
(27) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 65 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 66;
(28) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 67 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 68;
(29) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 69 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 70;
(30) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 71 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 72;
(31) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 73 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 74;
(32) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 75 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 76;
(33) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 77 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 78;
(34) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 79 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 80;
(35) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 81 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 82;
(36) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 83 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 84;
(37) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 85 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 86;
(38) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 87 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 88;
(39) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 89 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 90;
(40) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 91 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 92;
(41) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 93 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 94;
(42) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 95 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 96;
(43) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 97 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 98;
(44) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 99 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 100;
(45) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 101 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 102;
(46) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 103 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 104;
(47) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 105 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 106;
(48) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 107 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 108;
(49) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 109 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 110;
(50) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 111 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 112;
(51) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 113 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 114;
(52) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 115 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 116;
(53) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 117 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 118;
(54) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 119 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 120;
(55) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 121 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 122;
(56) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 123 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 124;
(57) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 125 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 126;
(58) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 127 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 128;
(59) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 129 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 130;
(60) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 131 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 132;
(61) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 133 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 134;
(62) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 135 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 136;
(63) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 137 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 138;
(64) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 139 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 140;
(65) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 141 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 142;
(66) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 143 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 144;
(67) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 145 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 146;
(68) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 147 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 148;
(69) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 149 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 150;
(70) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 151 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 152;
(71) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 153 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 154,
(72) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 155 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 156;
(73) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 157 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 158,
(74) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 159 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 160;
(75) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 161 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 162;
(76) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 163 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 164;
(77) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 165 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 166;
(78) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 167 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 168,
(79) A primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 169 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 170,
(80) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 171 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 172,
(81) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 173 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 174,
(82) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 175 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 176,
(83) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 177 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 178,
(84) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 179 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 180,
(85) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 181 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 182;
(86) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 183 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 184,
(87) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 185 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 186;
(88) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 187 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 188,
(89) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 189 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 190,
(90) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 191 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 192,
(91) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 193 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 194;
(92) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 195 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 196;
(93) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 197 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 198;
(94) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 199 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 200;
(95) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 201 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 202;
(96) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 203 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 204;
(97) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 205 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 206;
(98) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 207 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 208;
(99) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 209 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 210;
(100) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 211 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 212;
(101) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 213 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 214;
(102) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 215 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 216;
(103) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 217 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 218,
(104) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 219 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 220;
(105) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 221 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 222;
(106) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 223 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 224,
(107) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 225 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 226;
(108) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 227 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 228;
(109) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 229 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 230,
(110) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 231 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 232;
(111) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 233 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 234;
(112) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 235 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 236;
(113) A primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 237 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 238,
(114) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 239 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 240;
(115) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 241 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 242;
(116) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 243 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 244,
(117) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 245 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 246;
(118) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 247 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 248,
(119) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 249 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 250;
(120) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 251 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 252;
(121) a primer set comprising an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 253 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 254,
(122) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 255 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 256;
(123) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 257 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 258,
(124) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 259 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 260;
(125) a primer set consisting of an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 261 and an oligonucleotide represented by the base sequence set forth in SEQ ID NO: 262, A method for identifying rice species (Oryza sativa) comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. A method for identification of Zizania latifolia, including the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 5 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. A method for identifying human wheat (Triticum monococcum), barley (Hordeum vulgaris) or barley (Bromuscatharticus) comprising the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 9 and the oligonucleotide represented by the nucleotide sequence of SEQ ID NO: 12 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. A method for identifying Petunia axillaris, including the following steps:
(I) a step of preparing DNA from a sample;
(Ii) Using the DNA as a template, the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 13 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 16 as primers, and the RNA polymerase I maximum subunit Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. A method for identifying Cerasus pendula, including the following steps:
(I) a step of preparing DNA from a sample;
(Ii) RNA polymerase I maximum subunit using the DNA as a template and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 17 and the oligonucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 20 as primers Amplifying a DNA fragment of the gene (PolA1 gene);
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene. A method for identifying Fusarium oxysporum, including the following steps:
(I) a step of preparing DNA from a sample;
(Ii) Amplifying a DNA fragment of RNA polymerase I largest subunit gene (PolA1 gene) using the DNA represented by SEQ ID NO: 21 and the oligonucleotide represented by SEQ ID NO: 22 as primers Process,
(Iii) determining the base sequence of the obtained amplification product, and decoding the amino acid sequence encoded by the base sequence; and (iv) converting the decoded amino acid sequence into a eukaryotic organism registered in a computer database. A step of comparing with the amino acid sequence of the PolA1 gene.

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