WO2012017683A1 - Leaf-area-related marker of plant of the genus saccharum and the use thereof - Google Patents

Leaf-area-related marker of plant of the genus saccharum and the use thereof Download PDF

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WO2012017683A1
WO2012017683A1 PCT/JP2011/004461 JP2011004461W WO2012017683A1 WO 2012017683 A1 WO2012017683 A1 WO 2012017683A1 JP 2011004461 W JP2011004461 W JP 2011004461W WO 2012017683 A1 WO2012017683 A1 WO 2012017683A1
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seq
sugarcane
nucleotide sequence
sequence shown
leaf
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PCT/JP2011/004461
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French (fr)
Inventor
Tatsuro Kimura
Hiroyuki Enoki
Shoko Tsuzuki
Satoru Nishimura
Etsuko Hattori
Aya Murakami
Takayoshi Terauchi
Takeo Sakaigaichi
Taiichiro Hattori
Makoto Matsuoka
Yoshifumi Terajima
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Toyota Jidosha Kabushiki Kaisha
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Publication of WO2012017683A1 publication Critical patent/WO2012017683A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/04—Stems
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to a leaf-area-related marker whereby a plant line of the genus Saccharum characterized by an increase in leaf area can be selected, and a method for use thereof.
  • Sugarcane has been cultivated as a raw material for sugar, liquor, and the like for edible use.
  • sugarcane has been used as, for example, a raw material for biofuel in a variety of industrial fields.
  • desirable characteristics e.g., sugar content, enhanced vegetative capacity, sprouting capacity, disease resistance, insect resistance, cold resistance, an increase in leaf-blade-length, an increase in leaf area, and an increase in stalk length.
  • characteristics comparison for comparison of characteristics data
  • “comparison during cultivation” for comparison of plants cultivated under the same conditions
  • DNA assay for DNA analysis.
  • novel sugarcane variety For creation of a novel sugarcane variety, first, tens of thousands of seedlings are created via crossing, followed by seedling selection and stepwise selection of excellent lines. Eventually, 2 or 3 types of novel varieties having desired characteristics can be obtained. As described above, for creation of a novel sugarcane variety, it is necessary to cultivate and evaluate an enormous number of lines, and it is also necessary to prepare a large-scale field and make highly time-consuming efforts. Therefore, it has been required to develop a method for identifying a sugarcane line having desired characteristics with the use of markers present in the sugarcane genome.
  • Non-Patent Document 2 suggests the possibility that a sugarcane genetic map can be created by increasing the number of markers, comparing individual markers in terms of a characteristic relationship, and verifying the results. However, in Non-Patent Document 2, an insufficient number of markers are disclosed and markers linked to desired characteristics have not been found.
  • an object of the present invention is to provide a marker related to leaf area, which is a quantitative trait of sugarcane plants.
  • the present inventors conducted intensive studies.
  • the present inventors prepared many sugarcane plant markers and carried out linkage analysis of quantitative traits along with sugarcane plant markers for hybrid progeny lines. Accordingly, the present inventors found markers linked to quantitative traits such as an increase in leaf area. This has led to the completion of the present invention.
  • the present invention encompasses the following.
  • a sugarcane-leaf-area-related marker which comprises a continuous nucleic acid region existing in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 14, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 15 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 23 and the nucleotide sequence shown in SEQ ID NO: 33, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 34 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 39 of a sugarcane chromosome.
  • the sugarcane-leaf-area-related marker according to (1), wherein the continuous nucleic acid region comprises any nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 39.
  • a method for producing a sugarcane line having an increased leaf area comprising: a step of extracting a chromosome of a progeny plant obtained from parent plants, at least one of which is a sugarcane plant; and a step of determining the presence or absence of the sugarcane-leaf-area-related marker according to any one of (1) to (3) in the obtained sugarcane chromosome.
  • a novel sugarcane-leaf-area-related marker linked to a sugarcane quantitative trait such as an increase in leaf area can be provided.
  • the leaf area of a line obtained by crossing sugarcane lines can be identified.
  • a sugarcane line characterized by an increase in leaf area can be identified at a very low cost.
  • Fig. 1 schematically shows the process of production of a DNA microarray used for acquisition of sugarcane chromosome markers.
  • Fig. 2 schematically shows a step of signal detection with the use of a DNA microarray.
  • Fig. 3 is a characteristic chart showing leaf-area data for sugarcane variety/line groups used in the Examples.
  • Fig. 4 is a characteristic chart showing QTL analysis results regarding leaf area (the 9th linkage group).
  • Fig. 5 is a characteristic chart showing QTL analysis results regarding leaf area (the 11th linkage group).
  • Fig. 6 is a characteristic chart showing QTL analysis results regarding leaf area (the 38th linkage group).
  • Fig. 7 is a characteristic chart showing QTL analysis results regarding leaf area (the 47th linkage group).
  • Fig. 1 schematically shows the process of production of a DNA microarray used for acquisition of sugarcane chromosome markers.
  • Fig. 2 schematically shows a step of signal detection with the use of a DNA microarray.
  • Fig. 8 is a characteristic chart showing QTL analysis results regarding leaf area (the 82nd linkage group).
  • Fig. 9 is a characteristic chart showing N812691 signal levels for individual lines.
  • Fig. 10 is a characteristic chart showing N828503 signal levels for individual lines.
  • Fig. 11 is a characteristic chart showing N812717 signal levels for individual lines.
  • Fig. 12 is a characteristic chart showing N828591 signal levels for individual lines.
  • Fig. 13 is a characteristic chart showing N824402 signal levels for individual lines.
  • the sugarcane-leaf-area-related marker and the method for using the same according to the present invention are described below.
  • a method for producing a sugarcane line using a sugarcane-leaf-area-related marker is described.
  • the sugarcane-leaf-area-related marker of the present invention corresponds to a specific region present on a sugarcane chromosome and is linked to causative genes (i.e., gene group) for a trait that causes an increase in sugarcane leaf area. Thus it can be used to identify a trait characterized by an increase in sugarcane leaf area.
  • a progeny line obtained using a known sugarcane line is a line having a trait characterized by an increase in leaf area by confirming the presence of a sugarcane-leaf-area-related marker in such progeny line.
  • sugarcane used herein refers to a plant belonging to the genus Saccharum of the family Poaceae.
  • the term “sugarcane” includes both so-called noble cane (scientific name: Saccharum officinarum) and wild cane (scientific name: Saccharum spontaneum).
  • noble cane scientific name: Saccharum officinarum
  • wild cane scientific name: Saccharum spontaneum
  • known sugarcane variety/line is not particularly limited. It includes any variety/line capable of being used in Japan and any variety/line used outside Japan.
  • sugarcane varieties cultivated in Japan include, but are not limited to, Ni1, NiN2, NiF3, NiF4, NiF5, Ni6, NiN7, NiF8, Ni9, NiTn10, Ni11, Ni12, Ni14, Ni15, Ni16, Ni17, NiTn19, NiTn20, Ni22, and Ni23.
  • main sugarcane varieties used in Japan described herein include, but are not limited to, NiF8, Ni9, NiTn10, and Ni15.
  • main sugarcane varieties that have been introduced into Japan include, but are not limited to, F177, NCo310, and F172.
  • a progeny line may be a line obtained by crossing a mother plant and a father plant of the same species, each of which is a sugarcane variety/line, or it may be a hybrid line obtained from parent plants when one thereof is a sugarcane variety/line and the other is a closely related variety/line (Erianthus arundinaceus).
  • a progeny line may be obtained via so-called backcrossing.
  • the sugarcane-leaf-area-related marker of the present invention has been newly identified by QTL (Quantitative Trait Loci) analysis using a genetic linkage map containing 3004 markers originally obtained from sugarcane chromosomes and sugarcane leaf-area data.
  • an approximately 18.9-cM (centimorgan) region an approximately 23.4-cM region, an approximately 18.8-cM region, an approximately 12.6-cM region, and an approximately 9.3-cM region having such peaks.
  • morgan (M) used herein refers to a unit representing the relative distance between genes on a chromosome, and it is expressed by the percentage of the crossover rate. In a case of a sugarcane chromosome, 1 cM corresponds to approximately 2000 kb.
  • causative genes i.e., gene group
  • the 18.9-cM region having the above peak is a region that comprises 14 types of markers listed in table 1 below in the order shown in table 1.
  • the 23.4-cM region having the above peak is a region that comprises 8 types of markers listed in table 2 below in the order shown in table 2.
  • the 18.8-cM region having the above peak is a region that comprises 11 types of markers listed in table 3 below in the order shown in table 3.
  • the 12.6-cM region having the above peak is a region that comprises 3 types of markers listed in table 4 below in the order shown in table 4.
  • the 9.3-cM region having the above peak is a region that comprises 3 types of markers listed in table 5 below in the order shown in table 5.
  • Linkage group represents the number given to each group among a plurality of linkage groups specified by QTL analysis.
  • Marker name represents the name given to each marker originally obtained in the present invention.
  • Synignal threshold represents a threshold used for determination of the presence or absence of a marker.
  • the peak contained in the 18.9-cM region is present in a region sandwiched between a marker (N832136) having the nucleotide sequence shown in SEQ ID NO: 4 and a marker (N824951) having the nucleotide sequence shown in SEQ ID NO: 6.
  • the peak contained in the 23.4-cM region is present in a region sandwiched between a marker (N800235) having the nucleotide sequence shown in SEQ ID NO: 17 and a marker (N829030) having the nucleotide sequence shown in SEQ ID NO: 19.
  • the peak contained in the 18.8-cM region is present in a region sandwiched between a marker (N818627) having the nucleotide sequence shown in SEQ ID NO: 28 and a marker (N812717) having the nucleotide sequence shown in SEQ ID NO: 29.
  • the peak contained in the 12.6-cM region is present in a region sandwiched between a marker (N828591) having the nucleotide sequence shown in SEQ ID NO: 35 and a marker (N820986) having the nucleotide sequence shown in SEQ ID NO: 36.
  • the peak contained in the 9.3-cM region is present in a region sandwiched between a marker (N823809) having the nucleotide sequence shown in SEQ ID NO: 37 and a marker (N824402) having the nucleotide sequence shown in SEQ ID NO: 38.
  • nucleic acid region refers to a region having a nucleotide sequence having 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less identity to a different region present on a sugarcane chromosome. If the identity of a nucleic acid region serving as a sugarcane-leaf-area-related marker to a different region falls within the above range, the nucleic acid region can be specifically detected according to a standard method. The identity level described herein can be calculated using default parameters and BLAST or a similar algorithm.
  • the base length of a nucleic acid region serving as a sugarcane-leaf-area-related marker can be at least 8 bases, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases. If the base length of a nucleic acid region serving as a sugarcane-leaf-area-related marker falls within the above range, the nucleic acid region can be specifically detected according to a standard method. In particular, among the 14 types of markers contained in the 18.9-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6.
  • a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19.
  • a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29.
  • a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36.
  • a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38.
  • a nucleic acid region containing a single marker selected from among the 39 types of markers shown in tables 1 to 5 can be used as a sugarcane-leaf-area-related marker.
  • a nucleic acid region containing a marker (N832136) comprising the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 18.9-cM region
  • a nucleic acid region containing a marker (N800241) comprising the nucleotide sequence shown in SEQ ID NO: 18 located closest to the peak position in the 23.4-cM region
  • a nucleic acid region containing a marker (N812717) comprising the nucleotide sequence shown in SEQ ID NO: 29 located closest to the peak position in the 18.8-cM region
  • a nucleic acid region containing a marker (N820986) comprising the nucleotide sequence shown in SEQ
  • the nucleotide sequence of a nucleic acid region containing the marker can be specified by inverse PCR using primers designed based on the nucleotide sequence of such marker.
  • a sugarcane-leaf-area-related marker any of the above 39 types of markers can be directly used. Specifically, one or more type(s) of markers selected from among the 39 types of such markers can be directly used as a sugarcane-leaf-area-related marker.
  • a marker (N832136) consisting of the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 18.9-cM region
  • a marker (N800241) consisting of the nucleotide sequence shown in SEQ ID NO: 18 located closest to the peak position in the 23.4-cM region
  • a marker (N812717) consisting of the nucleotide sequence shown in SEQ ID NO: 29 closest to the peak position in the 18.8-cM region
  • a marker (N820986) consisting of the nucleotide sequence shown in SEQ ID NO: 36 closest to the peak position in the 12.6-cM region
  • a marker (N824402) consisting of the nucleotide sequence shown in SEQ ID NO: 38 closest to the peak position in the 9.3-cM region.
  • ⁇ Sugarcane marker identification>> sugarcane-leaf-area-related markers were identified from among 3004 markers originally obtained from sugarcane chromosomes in the present invention. These 3004 markers are described below. Upon identification of these markers, a DNA microarray can be used according to the method disclosed in JP Patent Application No. 2009-283430. Specifically, 3004 markers originally obtained from sugarcane chromosomes are used with a DNA microarray having probes designed by the method disclosed in JP Patent Application No. 2009-283430. The method for designing probes as shown in fig. 1 is described below. First, genomic DNA is extracted from sugarcane (step 1a).
  • restriction enzymes A and B are used (in the order of A first and then B) to digest genomic DNA.
  • the restriction enzymes used herein are not particularly limited. However, examples of restriction enzymes that can be used include PstI, EcoRI, HindIII, BstNI, HpaII, and HaeIII.
  • restriction enzymes can be adequately selected in consideration of the frequency of appearance of recognition sequences such that a genomic DNA fragment having a base length of 20 to 10000 can be obtained when genomic DNA is completely digested.
  • a genomic DNA fragment obtained after the use of all restriction enzymes it is preferable for a genomic DNA fragment obtained after the use of all restriction enzymes to have a base length of 200 to 6000.
  • the order in which restriction enzymes are subjected to treatment is not particularly limited.
  • a plurality of restriction enzymes may be used in an identical reaction system if they are treated under identical conditions (e.g., solution composition and temperature). Specifically, in the example shown in fig. 1, genomic DNA is digested using restriction enzymes A and B in such order. However, genomic DNA may be digested by simultaneously using restriction enzymes A and B in an identical reaction system. Alternatively, genomic DNA may be digested using restriction enzymes B and A in such order.
  • adapters are bound to a genomic DNA fragment subjected to restriction enzyme treatment (step 1c).
  • the adapter used herein is not particularly limited as long as it can be bound to both ends of a genomic DNA fragment obtained via the above restriction enzyme treatment.
  • an adapter it is possible to use, as an adapter, an adapter having a single strand complementary to a protruding end (sticky end) formed at each end of genomic DNA via restriction enzyme treatment and a primer binding sequence to which a primer used upon amplification treatment as described in detail below can hybridize.
  • an adapter having a single strand complementary to the above protruding end (sticky end) and a restriction enzyme recognition site that is incorporated into a vector upon cloning.
  • a plurality of adapters corresponding to the relevant restriction enzymes can be prepared and used.
  • a plurality of adapters having single strands complementary to different protruding ends formed upon digestion of genomic DNA with a plurality of restriction enzymes may have a common primer binding sequence such that a common primer can hybridize to each such adapter.
  • genomic DNA when genomic DNA is digested using a plurality of restriction enzymes, it is possible to use, as an adaptor, an adapter corresponding to at least one restriction enzyme selected from among a plurality of the used restriction enzymes.
  • an adapter When an adapter having a primer binding sequence is used, the genomic DNA fragment can be amplified using a primer that can hybridize to the primer binding sequence.
  • a genomic DNA fragment to which an adapter has been added is cloned into a vector using the adapter sequence.
  • the genomic DNA fragment can be amplified using primers that can hybridize to specific regions of the vector.
  • PCR can be used for a genomic DNA fragment amplification reaction using primers.
  • the adapters are ligated to all genomic DNA fragments obtained via treatment with a plurality of restriction enzymes. In this case, all the obtained genomic DNA fragments can be amplified by carrying out a nucleic acid amplification reaction using primer binding sequences contained in adapters.
  • genomic DNA is digested using a plurality of restriction enzymes, followed by ligation of adapter(s) corresponding to one or more restriction enzyme(s) selected from among a plurality of the used restriction enzymes to genomic DNA fragments.
  • a genomic DNA fragment to both ends of which the selected restriction enzyme recognition sequences have been bound can be exclusively amplified.
  • the nucleotide sequence of the amplified genomic DNA fragment is determined (step 1e). Then, at least one region, which has a base length shorter than the base length of the genomic DNA fragment and corresponds to at least a partial region of the genomic DNA fragment, is specified.
  • Sugarcane probes are designed using at least one of the thus specified regions (step 1f).
  • a method for determining the nucleotide sequence of a genomic DNA fragment is not particularly limited.
  • a conventionally known method using a DNA sequencer applied to the Sanger method or the like can be used.
  • a region to be designed herein has a 20- to 100-base length, preferably a 30- to 90-base length, and more preferably a 50- to 75-base length as described above.
  • a DNA microarray can be produced by designing many probes using genomic DNA extracted from sugarcane and synthesizing an oligonucleotide having a desired nucleotide sequence on a support based on the nucleotide sequence of the designed probe.
  • 3004 markers including the above 39 types of sugarcane-leaf-area-related markers shown in SEQ ID NOS: 1 to 39, can be identified. More specifically, the present inventors obtained signal data of known sugarcane varieties (NiF8 and Ni9) and a progeny line (line 191) obtained by crossing the varieties with the use of the DNA microarray described above. Then, genotype data were obtained based on the obtained signal data.
  • chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993). As a result, 3004 markers, including the aforementioned 39 types of sugarcane-leaf-area-related markers shown in SEQ ID NOS: 1 to 39, were identified.
  • sugarcane-leaf-area-related markers makes it possible to determine whether a sugarcane progeny line or the like, which has a phenotype exhibiting unknown leaf area, is a line having a phenotype showing an increase in leaf area.
  • the expression "the use of sugarcane-leaf-area-related markers" used herein indicates the use of a DNA microarray having probes corresponding to sugarcane-leaf-area-related markers in one embodiment.
  • probes corresponding to sugarcane-leaf-area-related markers indicates oligonucleotides that can specifically hybridize under stringent conditions to sugarcane-leaf-area-related markers defined as above.
  • oligonucleotides can be designed as partial or whole regions with base lengths of at least 10 continuous bases, 15 continuous bases, 20 continuous bases, 25 continuous bases, 30 continuous bases, 35 continuous bases, 40 continuous bases, 45 continuous bases, or 50 or more continuous bases of the nucleotide sequences or complementary strands thereof of sugarcane-leaf-area-related markers defined as above.
  • a DNA microarray having such probes may be any type of microarray, such as a microarray having a planar substrate comprising glass, silicone, or the like, a bead array comprising microbeads as carriers, or a three-dimensional microarray having an inner wall comprising hollow fibers to which probes are fixed.
  • a DNA microarray prepared as described above makes it possible to determine whether a sugarcane line such as a progeny line or the like, which has a phenotype exhibiting unknown leaf-area, is a line having a phenotype showing an increase in leaf area.
  • a sugarcane line which has a phenotype exhibiting unknown leaf area
  • a line having a trait characterized by an increase in leaf area by detecting the above sugarcane-leaf-area-related markers by a conventionally known method.
  • the method involving the use of a DNA microarray is described in more detail. As shown in fig. 2, first, genomic DNA is extracted from a sugarcane sample.
  • a sugarcane sample is a sugarcane line such as a sugarcane progeny line, which has a phenotype exhibiting unknown leaf area, and thus which can be used as a subject to be determined whether to have a trait characterized by an increase in leaf area or not.
  • a plurality of genomic DNA fragments are prepared by digesting the extracted genomic DNA with restriction enzymes used for preparing the DNA microarray. Then, the obtained genomic DNA fragments are ligated to adapters used for preparation of the DNA microarray. Subsequently, the genomic DNA fragments, to both ends of which adapters have been added, are amplified using primers employed for preparation of the DNA microarray.
  • sugarcane-sample-derived genomic DNA fragments corresponding to the genomic DNA fragments amplified in step 1d upon preparation of the DNA microarray can be amplified.
  • specific genomic DNA fragments may be selectively amplified. For instance, in a case in which a plurality of adapters corresponding to a plurality of restriction enzymes are used, genomic DNA fragments to which specific adapters have been added can be selectively amplified.
  • genomic DNA fragments to which adapters have been added can be selectively amplified by adding adapters only to genomic DNA fragments that have protruding ends corresponding to specific restriction enzymes among the obtained genomic DNA fragments.
  • DNA fragment concentration can be increased by selectively amplifying specific genomic DNA fragments.
  • amplified genomic DNA fragments are labeled. Any conventionally known substance may be used as a labeling substance. Examples of a labeling substance that can be used include fluorescent molecules, dye molecules, and radioactive molecules.
  • this step can be omitted using a labeled nucleotide in the step of amplifying genomic DNA fragments.
  • amplified DNA fragments can be labeled.
  • labeled genomic DNA fragments are allowed to come into contact with the DNA microarray under certain conditions such that probes fixed to the DNA microarray hybridize to the labeled genomic DNA fragments.
  • highly stringent conditions are provided for hybridization. Under highly stringent conditions, it becomes possible to determine with high accuracy whether or not sugarcane-leaf-area-related markers are present in a sugarcane sample.
  • stringent conditions can be adjusted based on reaction temperature and salt concentration. That is, an increase in temperature or a decrease in salt concentration results in more stringent conditions.
  • hybridization conditions 40 degrees C to 44 degrees C; 0.21SDS; and 6 x SSC.
  • hybridization between labeled genomic DNA fragments and probes can be confirmed by detecting a labeling substance. Specifically, after the above hybridization reaction of labeled genomic DNA fragments and probes, unreacted genomic DNA fragments and the like are washed, and the labeling substance bound to each genomic DNA fragment specifically hybridizing to a probe is observed. For instance, in a case in which the labeling substance is a fluorescent material, the fluorescence wavelength is detected. In a case in which the labeling substance is a dye molecule, the dye wavelength is detected.
  • apparatuses such as fluorescent detectors and image analyzers used for conventional DNA microarray analysis can be used.
  • a sugarcane sample has the above sugarcane-leaf-area-related marker(s) with the use of a DNA microarray.
  • the area of a field used for cultivation of a sugarcane sample and other factors such as cost of cultivation can be significantly reduced with the use of the sugarcane-leaf-area-related marker(s).
  • a novel sugarcane variety when a novel sugarcane variety is created, it is preferable to produce several tens of thousands of hybrid varieties via crossing and then to identify a novel sugarcane variety using sugarcane-leaf-area-related markers prior to or instead of seedling selection.
  • the use of such sugarcane-leaf-area-related marker(s) makes it possible to significantly reduce the number of excellent lines that need to be cultivated in an actual field. This allows drastic reduction of time-consuming efforts and the cost required to create a novel sugarcane variety.
  • Genomic DNAs (750 ng each) were treated with a PstI restriction enzyme (NEB; 25 units) at 37 degrees C for 2 hours.
  • a BstNI restriction enzyme (NEB; 25 units) was added thereto, followed by treatment at 60 degrees C for 2 hours.
  • the adapters were selectively added to genomic DNA fragments having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
  • a PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3'(SEQ ID NO: 42)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adaptors obtained in (3).
  • the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C for 10 seconds, 55 degrees C for 15 seconds, and 72 degrees C for 1 minute for 30 cycles, and then at 72 degrees C for 3 minutes, followed by storage at 4 degrees C).
  • Genome sequence acquisition The nucleotide sequence of the genomic DNA fragment subjected to PCR amplification in (4) was determined by the Sanger method. In addition, information on a nucleotide sequence sandwiched between PstI recognition sequences was obtained based on the total sorghum genome sequence information contained in the genome database (Gramene: http://www.gramene.org/). (6) Probe design and DNA microarray production 50- to 75-bp probes were designed based on the genome sequence information in (5). Based on the nucleotide sequence information of the designed probes, a DNA microarray having the probes was produced. 2.
  • Adapter ligation PstI sequence adapters (5'-CACGATGGATCCAGTGCA-3' (SEQ ID NO: 40) and 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 41)) and T4 DNA Ligase (NEB; 800 units) were added to the genomic DNA fragments treated in (2) (120 ng each), and the obtained mixtures were treated at 16 degrees C for a full day.
  • the adaptors were selectively added to a genomic DNA fragment having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
  • PCR amplification A PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3' (SEQ ID NO: 42)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adapters obtained in (3). Then, the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C for 10 seconds, 55 degrees C for 15 seconds, 72 degrees C for 1 minute for 30 cycles, and then at 72 degrees C for 3 minutes, followed by storage at 4 degrees C). (5) Labeling The PCR amplification fragment obtained in (4) above was purified with a column (Qiagen). Cy3-labeled 9mers (TriLink; 1 O.D.) was added thereto.
  • Genotype data of possible 3004 markers were obtained based on the signal data detected in 2 above of the NiF8 and Ni9 sugarcane varieties and the progeny line (line 191). Based on the obtained genotype data, chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993).
  • peaks exceeding the LOD threshold were observed in the following ranges for the NiF8 sugarcane variety: the range between markers N812691 and N803255 present in the 9th linkage group; the range between markers N803305 and N826149 present in the 11th linkage group; the range between markers N828907 and N803226 present in the 38th linkage group; the range between markers N825608 and N820986 present in the 47th linkage group; and the range between markers N823809 and N821676 present in the 82nd linkage group. It was possible to specify the obtained peaks as shown in table 6, suggesting the presence of causative genes (i.e., gene group) each having the function of causing an increase in leaf area at relevant peak positions.
  • causative genes i.e., gene group
  • markers located in the vicinity of the relevant peaks are inherited in linkage with causative genes (i.e., gene group) each having the function of causing an increase in leaf area.
  • causative genes i.e., gene group
  • the markers can be used as sugarcane-leaf-area-related markers.
  • 39 types of markers shown in figs. 4 to 8 can be used as sugarcane-leaf-area-related markers.
  • table 7 shows signal levels of 14 types of markers among markers N812691 to N803255 present in the 9th linkage group for NiF8 and Ni9 and their 17 progeny lines (F1_1 to F1_17).
  • the signal levels of N812691 are shown in fig. 9. Signal levels of 14 types of markers were found to be very high for NiF8 and the progeny lines such as F1_1 and F1_5 with relatively large leaf areas. These results also revealed that 14 types of markers among markers N812691 to N803255 present in the 9th linkage group can be used as sugarcane-leaf-area-related markers.
  • table 8 shows signal levels of 8 types of markers for NiF8 and Ni9, and 17 progeny lines (F1_1 to F1_17) among markers N803305 to N826149 present in the 11th linkage group.
  • fig. 10 shows the signal levels of N828503.

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Abstract

According to the present invention, a novel sugarcane-leaf-area-related marker linked to a sugarcane quantitative trait such as an increase in leaf area is provided. Such sugarcane-leaf-area-related marker is a sugarcane-leaf-area-related marker, which comprises a continuous nucleic acid region existing in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 14, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 15 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 23 and the nucleotide sequence shown in SEQ ID NO: 33, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 34 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 39 of a sugarcane chromosome.

Description

LEAF-AREA-RELATED MARKER OF PLANT OF THE GENUS SACCHARUM AND THE USE THEREOF
The present invention relates to a leaf-area-related marker whereby a plant line of the genus Saccharum characterized by an increase in leaf area can be selected, and a method for use thereof.
Sugarcane has been cultivated as a raw material for sugar, liquor, and the like for edible use. In addition, sugarcane has been used as, for example, a raw material for biofuel in a variety of industrial fields. Under such circumstances, there is a need to develop novel sugarcane varieties having desirable characteristics (e.g., sugar content, enhanced vegetative capacity, sprouting capacity, disease resistance, insect resistance, cold resistance, an increase in leaf-blade-length, an increase in leaf area, and an increase in stalk length).
In general, the following three ways may be used for identification of a plant variety/line: "characteristics comparison" for comparison of characteristics data, "comparison during cultivation" for comparison of plants cultivated under the same conditions, and "DNA assay" for DNA analysis. There are many problems in line identification with characteristics comparison or comparison during cultivation, including reduction of precision due to differences in cultivation conditions, lengthy duration of field research that requires a number of steps, and the like. In particular, since sugarcane plants are much larger than other graminaceous crops such as rice and maize, it has been difficult to conduct line identification based on field research. In addition, in order to identify a variety having morphologically distinct characteristics in terms of leaf-blade-length, leaf area, stalk length, and the like, it is necessary to collect such characteristic data after long-term cultivation of sugarcane. In addition, even after long-term cultivation of sugarcane, it is difficult to identify such variety with high accuracy because morphological characteristics are environmentally susceptible.
Further, for creation of a novel sugarcane variety, first, tens of thousands of seedlings are created via crossing, followed by seedling selection and stepwise selection of excellent lines. Eventually, 2 or 3 types of novel varieties having desired characteristics can be obtained. As described above, for creation of a novel sugarcane variety, it is necessary to cultivate and evaluate an enormous number of lines, and it is also necessary to prepare a large-scale field and make highly time-consuming efforts.
Therefore, it has been required to develop a method for identifying a sugarcane line having desired characteristics with the use of markers present in the sugarcane genome. In particular, upon creation of a novel sugarcane variety, if excellent markers could be used to examine a variety of characteristics, the above problems particular to sugarcane would be resolved, and the markers would be able to serve as very effective tools. However, since sugarcane plants have a large number of chromosomes (approximately 100 to 130) due to higher polyploidy, the development of marker technology has been slow. In the case of sugarcane, although the USDA reported genotyping with the use of SSR markers (Non-Patent Document 1), the precision of genotyping is low because of the small numbers of markers and polymorphisms in each marker. In addition, the above genotyping is available only for American/Australian varieties, and therefore it cannot be used for identification of the major varieties cultivated in Japan, Taiwan, India, and other countries or lines that serve as useful genetic resources.
In addition, Non-Patent Document 2 suggests the possibility that a sugarcane genetic map can be created by increasing the number of markers, comparing individual markers in terms of a characteristic relationship, and verifying the results. However, in Non-Patent Document 2, an insufficient number of markers are disclosed and markers linked to desired characteristics have not been found.
Maydica 48(2003)319-329 "Molecular genotyping of sugarcane clones with microsatellite DNA markers" Nathalie Piperidis et al., Molecular Breeding, 2008, Vol. 21, 233-247
In view of the above, an object of the present invention is to provide a marker related to leaf area, which is a quantitative trait of sugarcane plants.
In order to achieve the object, the present inventors conducted intensive studies. The present inventors prepared many sugarcane plant markers and carried out linkage analysis of quantitative traits along with sugarcane plant markers for hybrid progeny lines. Accordingly, the present inventors found markers linked to quantitative traits such as an increase in leaf area. This has led to the completion of the present invention.
The present invention encompasses the following.
(1) A sugarcane-leaf-area-related marker, which comprises a continuous nucleic acid region existing in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 14, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 15 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 23 and the nucleotide sequence shown in SEQ ID NO: 33, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 34 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 39 of a sugarcane chromosome.
(2) The sugarcane-leaf-area-related marker according to (1), wherein the continuous nucleic acid region comprises any nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 39.
(3) The sugarcane-leaf-area-related marker according to (1), wherein the continuous nucleic acid region is located at a position in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38 of a sugarcane chromosome.
(4) A method for producing a sugarcane line having an increased leaf area comprising: a step of extracting a chromosome of a progeny plant obtained from parent plants, at least one of which is a sugarcane plant; and a step of determining the presence or absence of the sugarcane-leaf-area-related marker according to any one of (1) to (3) in the obtained sugarcane chromosome.
(5) The method for producing a sugarcane line according to (4), wherein a DNA chip provided with probes each serving as the sugarcane-leaf-area-related marker is used in the determination step.
(6) The method for producing a sugarcane line according to (4), wherein the progeny plant is in the form of seeds or a young seedling and the chromosome is extracted from the seeds or the young seedling.
According to the present invention, a novel sugarcane-leaf-area-related marker linked to a sugarcane quantitative trait such as an increase in leaf area can be provided. With the use of the sugarcane-leaf-area-related marker of the present invention, the leaf area of a line obtained by crossing sugarcane lines can be identified. Thus, a sugarcane line characterized by an increase in leaf area can be identified at a very low cost.
Fig. 1 schematically shows the process of production of a DNA microarray used for acquisition of sugarcane chromosome markers. Fig. 2 schematically shows a step of signal detection with the use of a DNA microarray. Fig. 3 is a characteristic chart showing leaf-area data for sugarcane variety/line groups used in the Examples. Fig. 4 is a characteristic chart showing QTL analysis results regarding leaf area (the 9th linkage group). Fig. 5 is a characteristic chart showing QTL analysis results regarding leaf area (the 11th linkage group). Fig. 6 is a characteristic chart showing QTL analysis results regarding leaf area (the 38th linkage group). Fig. 7 is a characteristic chart showing QTL analysis results regarding leaf area (the 47th linkage group). Fig. 8 is a characteristic chart showing QTL analysis results regarding leaf area (the 82nd linkage group). Fig. 9 is a characteristic chart showing N812691 signal levels for individual lines. Fig. 10 is a characteristic chart showing N828503 signal levels for individual lines. Fig. 11 is a characteristic chart showing N812717 signal levels for individual lines. Fig. 12 is a characteristic chart showing N828591 signal levels for individual lines. Fig. 13 is a characteristic chart showing N824402 signal levels for individual lines.
The sugarcane-leaf-area-related marker and the method for using the same according to the present invention are described below. In particular, a method for producing a sugarcane line using a sugarcane-leaf-area-related marker is described.
<<Sugarcane-leaf-area-related markers>>
The sugarcane-leaf-area-related marker of the present invention corresponds to a specific region present on a sugarcane chromosome and is linked to causative genes (i.e., gene group) for a trait that causes an increase in sugarcane leaf area. Thus it can be used to identify a trait characterized by an increase in sugarcane leaf area. Specifically, it is possible to determine that a progeny line obtained using a known sugarcane line is a line having a trait characterized by an increase in leaf area by confirming the presence of a sugarcane-leaf-area-related marker in such progeny line.
The term "sugarcane" used herein refers to a plant belonging to the genus Saccharum of the family Poaceae. In addition, the term "sugarcane" includes both so-called noble cane (scientific name: Saccharum officinarum) and wild cane (scientific name: Saccharum spontaneum). The term "known sugarcane variety/line" is not particularly limited. It includes any variety/line capable of being used in Japan and any variety/line used outside Japan. Examples of sugarcane varieties cultivated in Japan include, but are not limited to, Ni1, NiN2, NiF3, NiF4, NiF5, Ni6, NiN7, NiF8, Ni9, NiTn10, Ni11, Ni12, Ni14, Ni15, Ni16, Ni17, NiTn19, NiTn20, Ni22, and Ni23. Examples of main sugarcane varieties used in Japan described herein include, but are not limited to, NiF8, Ni9, NiTn10, and Ni15. In addition, examples of main sugarcane varieties that have been introduced into Japan include, but are not limited to, F177, NCo310, and F172.
In addition, a progeny line may be a line obtained by crossing a mother plant and a father plant of the same species, each of which is a sugarcane variety/line, or it may be a hybrid line obtained from parent plants when one thereof is a sugarcane variety/line and the other is a closely related variety/line (Erianthus arundinaceus). In addition, a progeny line may be obtained via so-called backcrossing.
The sugarcane-leaf-area-related marker of the present invention has been newly identified by QTL (Quantitative Trait Loci) analysis using a genetic linkage map containing 3004 markers originally obtained from sugarcane chromosomes and sugarcane leaf-area data. In addition, many genes are presumably associated with sugarcane leaf area, which is a quantitative trait characterized by a continuous distribution of leaf-area values. For QTL analysis, the QTL Cartographer gene analysis software (Wang S., C. J. Basten, and Z.-B. Zeng (2010); Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) is used, and the analysis is carried out by the composite interval mapping (CIM) method.
Specifically, peaks with LOD scores equivalent to or exceeding a given threshold (e.g., 2.5) have been found in 5 regions included in the above genetic linkage map by QTL analysis described above. That is, the following 5 regions have been specified: an approximately 18.9-cM (centimorgan) region, an approximately 23.4-cM region, an approximately 18.8-cM region, an approximately 12.6-cM region, and an approximately 9.3-cM region having such peaks. The term "morgan (M)" used herein refers to a unit representing the relative distance between genes on a chromosome, and it is expressed by the percentage of the crossover rate. In a case of a sugarcane chromosome, 1 cM corresponds to approximately 2000 kb. In addition, it is suggested that causative genes (i.e., gene group) for a trait that causes an increase in leaf area could be present at the peak positions or in the vicinity thereof.
The 18.9-cM region having the above peak is a region that comprises 14 types of markers listed in table 1 below in the order shown in table 1.
Figure JPOXMLDOC01-appb-T000001
 The 23.4-cM region having the above peak is a region that comprises 8 types of markers listed in table 2 below in the order shown in table 2.
Figure JPOXMLDOC01-appb-T000002
 The 18.8-cM region having the above peak is a region that comprises 11 types of markers listed in table 3 below in the order shown in table 3.
Figure JPOXMLDOC01-appb-T000003
 The 12.6-cM region having the above peak is a region that comprises 3 types of markers listed in table 4 below in the order shown in table 4.
Figure JPOXMLDOC01-appb-T000004
 The 9.3-cM region having the above peak is a region that comprises 3 types of markers listed in table 5 below in the order shown in table 5.
Figure JPOXMLDOC01-appb-T000005
 In addition, in tables 1 to 5, "Linkage group" represents the number given to each group among a plurality of linkage groups specified by QTL analysis. In tables 1 to 5, "Marker name" represents the name given to each marker originally obtained in the present invention. In tables 1 to 5, "Signal threshold" represents a threshold used for determination of the presence or absence of a marker.
In addition, the peak contained in the 18.9-cM region is present in a region sandwiched between a marker (N832136) having the nucleotide sequence shown in SEQ ID NO: 4 and a marker (N824951) having the nucleotide sequence shown in SEQ ID NO: 6. The peak contained in the 23.4-cM region is present in a region sandwiched between a marker (N800235) having the nucleotide sequence shown in SEQ ID NO: 17 and a marker (N829030) having the nucleotide sequence shown in SEQ ID NO: 19. The peak contained in the 18.8-cM region is present in a region sandwiched between a marker (N818627) having the nucleotide sequence shown in SEQ ID NO: 28 and a marker (N812717) having the nucleotide sequence shown in SEQ ID NO: 29. The peak contained in the 12.6-cM region is present in a region sandwiched between a marker (N828591) having the nucleotide sequence shown in SEQ ID NO: 35 and a marker (N820986) having the nucleotide sequence shown in SEQ ID NO: 36. The peak contained in the 9.3-cM region is present in a region sandwiched between a marker (N823809) having the nucleotide sequence shown in SEQ ID NO: 37 and a marker (N824402) having the nucleotide sequence shown in SEQ ID NO: 38.
A continuous nucleic acid region existing in any of 5 regions containing markers shown in tables 1 to 5 can be used as a sugarcane-leaf-area-related marker. The term "nucleic acid region" used herein refers to a region having a nucleotide sequence having 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less identity to a different region present on a sugarcane chromosome. If the identity of a nucleic acid region serving as a sugarcane-leaf-area-related marker to a different region falls within the above range, the nucleic acid region can be specifically detected according to a standard method. The identity level described herein can be calculated using default parameters and BLAST or a similar algorithm.
In addition, the base length of a nucleic acid region serving as a sugarcane-leaf-area-related marker can be at least 8 bases, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases. If the base length of a nucleic acid region serving as a sugarcane-leaf-area-related marker falls within the above range, the nucleic acid region can be specifically detected according to a standard method.
In particular, among the 14 types of markers contained in the 18.9-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6. In addition, among the 8 types of markers contained in the 23.4-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19. Further, among the 11 types of markers contained in the 18.8-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29. Furthermore, among the 3 types of markers contained in the 12.6-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36. Even furthermore, among the 3 types of markers contained in the 9.3-cM region, a sugarcane-leaf-area-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38.
In addition, a nucleic acid region containing a single marker selected from among the 39 types of markers shown in tables 1 to 5 can be used as a sugarcane-leaf-area-related marker. For example, it is preferable to use, as a sugarcane-leaf-area-related marker, a nucleic acid region containing a marker (N832136) comprising the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 18.9-cM region, a nucleic acid region containing a marker (N800241) comprising the nucleotide sequence shown in SEQ ID NO: 18 located closest to the peak position in the 23.4-cM region, a nucleic acid region containing a marker (N812717) comprising the nucleotide sequence shown in SEQ ID NO: 29 located closest to the peak position in the 18.8-cM region, a nucleic acid region containing a marker (N820986) comprising the nucleotide sequence shown in SEQ ID NO: 36 located closest to the peak position in the 12.6-cM region, or a nucleic acid region containing a marker (N824402) comprising the nucleotide sequence shown in SEQ ID NO: 38 located closest to the peak position in the 9.3-cM region. In such case, the nucleotide sequence of a nucleic acid region containing the marker can be specified by inverse PCR using primers designed based on the nucleotide sequence of such marker.
Further, as a sugarcane-leaf-area-related marker, any of the above 39 types of markers can be directly used. Specifically, one or more type(s) of markers selected from among the 39 types of such markers can be directly used as a sugarcane-leaf-area-related marker. For example, it is preferable to use, as a sugarcane-leaf-area-related marker, a marker (N832136) consisting of the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 18.9-cM region, a marker (N800241) consisting of the nucleotide sequence shown in SEQ ID NO: 18 located closest to the peak position in the 23.4-cM region, a marker (N812717) consisting of the nucleotide sequence shown in SEQ ID NO: 29 closest to the peak position in the 18.8-cM region, a marker (N820986) consisting of the nucleotide sequence shown in SEQ ID NO: 36 closest to the peak position in the 12.6-cM region, or a marker (N824402) consisting of the nucleotide sequence shown in SEQ ID NO: 38 closest to the peak position in the 9.3-cM region.
<<Sugarcane marker identification>>
As described above, sugarcane-leaf-area-related markers were identified from among 3004 markers originally obtained from sugarcane chromosomes in the present invention. These 3004 markers are described below. Upon identification of these markers, a DNA microarray can be used according to the method disclosed in JP Patent Application No. 2009-283430.
Specifically, 3004 markers originally obtained from sugarcane chromosomes are used with a DNA microarray having probes designed by the method disclosed in JP Patent Application No. 2009-283430. The method for designing probes as shown in fig. 1 is described below. First, genomic DNA is extracted from sugarcane (step 1a). Next, the extracted genomic DNA is digested with a single or a plurality of restriction enzyme(s) (step 1b). In addition, in the example shown in fig. 1, 2 types of restriction enzymes illustrated as restriction enzymes A and B are used (in the order of A first and then B) to digest genomic DNA. The restriction enzymes used herein are not particularly limited. However, examples of restriction enzymes that can be used include PstI, EcoRI, HindIII, BstNI, HpaII, and HaeIII. In particular, restriction enzymes can be adequately selected in consideration of the frequency of appearance of recognition sequences such that a genomic DNA fragment having a base length of 20 to 10000 can be obtained when genomic DNA is completely digested. In addition, when a plurality of restriction enzymes are used, it is preferable for a genomic DNA fragment obtained after the use of all restriction enzymes to have a base length of 200 to 6000. Further, when a plurality of restriction enzymes are used, the order in which restriction enzymes are subjected to treatment is not particularly limited. In addition, a plurality of restriction enzymes may be used in an identical reaction system if they are treated under identical conditions (e.g., solution composition and temperature). Specifically, in the example shown in fig. 1, genomic DNA is digested using restriction enzymes A and B in such order. However, genomic DNA may be digested by simultaneously using restriction enzymes A and B in an identical reaction system. Alternatively, genomic DNA may be digested using restriction enzymes B and A in such order. Further, 3 or more restriction enzymes may be used.
Next, adapters are bound to a genomic DNA fragment subjected to restriction enzyme treatment (step 1c). The adapter used herein is not particularly limited as long as it can be bound to both ends of a genomic DNA fragment obtained via the above restriction enzyme treatment. For example, it is possible to use, as an adapter, an adapter having a single strand complementary to a protruding end (sticky end) formed at each end of genomic DNA via restriction enzyme treatment and a primer binding sequence to which a primer used upon amplification treatment as described in detail below can hybridize. In addition, it is also possible to use, as an adapter, an adapter having a single strand complementary to the above protruding end (sticky end) and a restriction enzyme recognition site that is incorporated into a vector upon cloning.
In addition, when genomic DNA is digested using a plurality of restriction enzymes, a plurality of adapters corresponding to the relevant restriction enzymes can be prepared and used. Specifically, it is possible to use a plurality of adapters having single strands complementary to different protruding ends formed upon digestion of genomic DNA with a plurality of restriction enzymes. Here, a plurality of adapters corresponding to a plurality of restriction enzymes each may have a common primer binding sequence such that a common primer can hybridize to each such adapter. Alternatively, they may have different primer binding sequences such that different primers can separately hybridize thereto.
Further, when genomic DNA is digested using a plurality of restriction enzymes, it is possible to use, as an adaptor, an adapter corresponding to at least one restriction enzyme selected from among a plurality of the used restriction enzymes.
Next, a genomic DNA fragment to both ends of which adapters have been added is amplified (step 1d). When an adapter having a primer binding sequence is used, the genomic DNA fragment can be amplified using a primer that can hybridize to the primer binding sequence. Alternatively, a genomic DNA fragment to which an adapter has been added is cloned into a vector using the adapter sequence. The genomic DNA fragment can be amplified using primers that can hybridize to specific regions of the vector. In addition, as an example, PCR can be used for a genomic DNA fragment amplification reaction using primers.
In addition, when genomic DNA is digested using a plurality of restriction enzymes and a plurality of adapters corresponding to the relevant restriction enzymes are ligated to genomic DNA fragments, the adapters are ligated to all genomic DNA fragments obtained via treatment with a plurality of restriction enzymes. In this case, all the obtained genomic DNA fragments can be amplified by carrying out a nucleic acid amplification reaction using primer binding sequences contained in adapters.
Alternatively, genomic DNA is digested using a plurality of restriction enzymes, followed by ligation of adapter(s) corresponding to one or more restriction enzyme(s) selected from among a plurality of the used restriction enzymes to genomic DNA fragments. In such case, among the obtained genomic DNA fragments, a genomic DNA fragment to both ends of which the selected restriction enzyme recognition sequences have been bound can be exclusively amplified.
Next, the nucleotide sequence of the amplified genomic DNA fragment is determined (step 1e). Then, at least one region, which has a base length shorter than the base length of the genomic DNA fragment and corresponds to at least a partial region of the genomic DNA fragment, is specified. Sugarcane probes are designed using at least one of the thus specified regions (step 1f). A method for determining the nucleotide sequence of a genomic DNA fragment is not particularly limited. A conventionally known method using a DNA sequencer applied to the Sanger method or the like can be used. For example, a region to be designed herein has a 20- to 100-base length, preferably a 30- to 90-base length, and more preferably a 50- to 75-base length as described above.
As described above, a DNA microarray can be produced by designing many probes using genomic DNA extracted from sugarcane and synthesizing an oligonucleotide having a desired nucleotide sequence on a support based on the nucleotide sequence of the designed probe. With the use of a DNA microarray prepared as described above, 3004 markers, including the above 39 types of sugarcane-leaf-area-related markers shown in SEQ ID NOS: 1 to 39, can be identified.
More specifically, the present inventors obtained signal data of known sugarcane varieties (NiF8 and Ni9) and a progeny line (line 191) obtained by crossing the varieties with the use of the DNA microarray described above. Then, genotype data were obtained based on the obtained signal data. Based on the obtained genotype data, chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993). As a result, 3004 markers, including the aforementioned 39 types of sugarcane-leaf-area-related markers shown in SEQ ID NOS: 1 to 39, were identified.
<<Use of sugarcane-leaf-area-related markers>>
The use of sugarcane-leaf-area-related markers makes it possible to determine whether a sugarcane progeny line or the like, which has a phenotype exhibiting unknown leaf area, is a line having a phenotype showing an increase in leaf area. The expression "the use of sugarcane-leaf-area-related markers" used herein indicates the use of a DNA microarray having probes corresponding to sugarcane-leaf-area-related markers in one embodiment. The expression "probes corresponding to sugarcane-leaf-area-related markers" indicates oligonucleotides that can specifically hybridize under stringent conditions to sugarcane-leaf-area-related markers defined as above. For instance, such oligonucleotides can be designed as partial or whole regions with base lengths of at least 10 continuous bases, 15 continuous bases, 20 continuous bases, 25 continuous bases, 30 continuous bases, 35 continuous bases, 40 continuous bases, 45 continuous bases, or 50 or more continuous bases of the nucleotide sequences or complementary strands thereof of sugarcane-leaf-area-related markers defined as above. In addition, a DNA microarray having such probes may be any type of microarray, such as a microarray having a planar substrate comprising glass, silicone, or the like, a bead array comprising microbeads as carriers, or a three-dimensional microarray having an inner wall comprising hollow fibers to which probes are fixed.
The use of a DNA microarray prepared as described above makes it possible to determine whether a sugarcane line such as a progeny line or the like, which has a phenotype exhibiting unknown leaf-area, is a line having a phenotype showing an increase in leaf area. In addition, in the case of a method other than the above method involving the use of a DNA microarray, it is also possible to determine whether a sugarcane line, which has a phenotype exhibiting unknown leaf area, is a line having a trait characterized by an increase in leaf area by detecting the above sugarcane-leaf-area-related markers by a conventionally known method.
The method involving the use of a DNA microarray is described in more detail. As shown in fig. 2, first, genomic DNA is extracted from a sugarcane sample. In this case, a sugarcane sample is a sugarcane line such as a sugarcane progeny line, which has a phenotype exhibiting unknown leaf area, and thus which can be used as a subject to be determined whether to have a trait characterized by an increase in leaf area or not. Next, a plurality of genomic DNA fragments are prepared by digesting the extracted genomic DNA with restriction enzymes used for preparing the DNA microarray. Then, the obtained genomic DNA fragments are ligated to adapters used for preparation of the DNA microarray. Subsequently, the genomic DNA fragments, to both ends of which adapters have been added, are amplified using primers employed for preparation of the DNA microarray. Accordingly, sugarcane-sample-derived genomic DNA fragments corresponding to the genomic DNA fragments amplified in step 1d upon preparation of the DNA microarray can be amplified.
In this step, among the genomic DNA fragments to which adapters have been added, specific genomic DNA fragments may be selectively amplified. For instance, in a case in which a plurality of adapters corresponding to a plurality of restriction enzymes are used, genomic DNA fragments to which specific adapters have been added can be selectively amplified. In addition, when genomic DNA is digested with a plurality of restriction enzymes, genomic DNA fragments to which adapters have been added can be selectively amplified by adding adapters only to genomic DNA fragments that have protruding ends corresponding to specific restriction enzymes among the obtained genomic DNA fragments. Thus, DNA fragment concentration can be increased by selectively amplifying specific genomic DNA fragments.
Thereafter, amplified genomic DNA fragments are labeled. Any conventionally known substance may be used as a labeling substance. Examples of a labeling substance that can be used include fluorescent molecules, dye molecules, and radioactive molecules. In addition, this step can be omitted using a labeled nucleotide in the step of amplifying genomic DNA fragments. This is because when genomic DNA fragments are amplified using a labeled nucleotide in the amplification step, amplified DNA fragments can be labeled.
Next, labeled genomic DNA fragments are allowed to come into contact with the DNA microarray under certain conditions such that probes fixed to the DNA microarray hybridize to the labeled genomic DNA fragments. At such time, preferably, highly stringent conditions are provided for hybridization. Under highly stringent conditions, it becomes possible to determine with high accuracy whether or not sugarcane-leaf-area-related markers are present in a sugarcane sample. In addition, stringent conditions can be adjusted based on reaction temperature and salt concentration. That is, an increase in temperature or a decrease in salt concentration results in more stringent conditions. For example, when a probe having a length of 50 to 75 bases is used, the following more stringent conditions can be provided as hybridization conditions: 40 degrees C to 44 degrees C; 0.21SDS; and 6 x SSC.
In addition, hybridization between labeled genomic DNA fragments and probes can be confirmed by detecting a labeling substance. Specifically, after the above hybridization reaction of labeled genomic DNA fragments and probes, unreacted genomic DNA fragments and the like are washed, and the labeling substance bound to each genomic DNA fragment specifically hybridizing to a probe is observed. For instance, in a case in which the labeling substance is a fluorescent material, the fluorescence wavelength is detected. In a case in which the labeling substance is a dye molecule, the dye wavelength is detected. More specifically, apparatuses such as fluorescent detectors and image analyzers used for conventional DNA microarray analysis can be used.
As described above, it is possible to determine whether or not a sugarcane sample has the above sugarcane-leaf-area-related marker(s) with the use of a DNA microarray. In particular, according to the method described above, it is not necessary to cultivate a sugarcane sample to such an extent that the actual leaf area thereof becomes measurable. For instance, seeds of a progeny line or a young seedling obtained as a result of germination of such seeds can be used. Therefore, the area of a field used for cultivation of a sugarcane sample and other factors such as cost of cultivation can be significantly reduced with the use of the sugarcane-leaf-area-related marker(s).
In particular, when a novel sugarcane variety is created, it is preferable to produce several tens of thousands of hybrid varieties via crossing and then to identify a novel sugarcane variety using sugarcane-leaf-area-related markers prior to or instead of seedling selection. The use of such sugarcane-leaf-area-related marker(s) makes it possible to significantly reduce the number of excellent lines that need to be cultivated in an actual field. This allows drastic reduction of time-consuming efforts and the cost required to create a novel sugarcane variety.
The present invention is hereafter described in greater detail with reference to the following examples, although the technical scope of the present invention is not limited thereto.
1. Production of DNA microarray probes
(1) Materials
The following varieties were used: sugarcane varieties: NiF8, Ni9, US56-15-8, POJ2878, Q165, R570, Co290 and B3439; closely-related sugarcane wild-type varieties: Glagah Kloet, Chunee, Natal Uba, and Robustum 9; and Erianthus varieties: IJ76-349 and JW630.
(2) Restriction enzyme treatment
Genomic DNA was extracted from each of the above sugarcane varieties, closely-related sugarcane wild-type varieties, and Erianthus varieties using DNeasy Plant Mini Kits (Qiagen). Genomic DNAs (750 ng each) were treated with a PstI restriction enzyme (NEB; 25 units) at 37 degrees C for 2 hours. A BstNI restriction enzyme (NEB; 25 units) was added thereto, followed by treatment at 60 degrees C for 2 hours.
(3) Adapter ligation
PstI sequence adapters (5'-CACGATGGATCCAGTGCA-3'(SEQ ID NO: 40) and 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 41)) and T4 DNA Ligase (NEB; 800 units) were added to the genomic DNA fragments treated in (2) (120 ng each), and the obtained mixtures were subjected to treatment at 16 degrees C for a full day. Thus, the adapters were selectively added to genomic DNA fragments having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
(4) PCR amplification
A PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3'(SEQ ID NO: 42)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adaptors obtained in (3). Then, the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C for 10 seconds, 55 degrees C for 15 seconds, and 72 degrees C for 1 minute for 30 cycles, and then at 72 degrees C for 3 minutes, followed by storage at 4 degrees C).
(5) Genome sequence acquisition
The nucleotide sequence of the genomic DNA fragment subjected to PCR amplification in (4) was determined by the Sanger method. In addition, information on a nucleotide sequence sandwiched between PstI recognition sequences was obtained based on the total sorghum genome sequence information contained in the genome database (Gramene: http://www.gramene.org/).
(6) Probe design and DNA microarray production
50- to 75-bp probes were designed based on the genome sequence information in (5). Based on the nucleotide sequence information of the designed probes, a DNA microarray having the probes was produced.
2. Acquisition of signal data using a DNA microarray
(1) Materials
Sugarcane varieties/lines (NiF8 and Ni9) and a the progeny line (line 191) were used.
(2) Restriction enzyme treatment
Genomic DNAs were extracted from NiF8, Ni9, and the progeny line (line 191) using DNeasy Plant Mini Kits (Qiagen). Genomic DNAs (750 ng each) were treated with a PstI restriction enzyme (NEB; 25 units) at 37 degrees C for 2 hours. Then, a BstNI restriction enzyme (NEB; 25 units) was added thereto, followed by treatment at 60 degrees C for 2 hours.
(3) Adapter ligation
PstI sequence adapters (5'-CACGATGGATCCAGTGCA-3' (SEQ ID NO: 40) and 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 41)) and T4 DNA Ligase (NEB; 800 units) were added to the genomic DNA fragments treated in (2) (120 ng each), and the obtained mixtures were treated at 16 degrees C for a full day. Thus, the adaptors were selectively added to a genomic DNA fragment having PstI recognition sequences at both ends thereof among the genomic DNA fragments treated in (2).
(4) PCR amplification
A PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3' (SEQ ID NO: 42)) and Taq polymerase (TAKARA; PrimeSTAR; 1.25 units) were added to the genomic DNA fragment (15 ng) having the adapters obtained in (3). Then, the genomic DNA fragment was amplified by PCR (treatment at 98 degrees C for 10 seconds, 55 degrees C for 15 seconds, 72 degrees C for 1 minute for 30 cycles, and then at 72 degrees C for 3 minutes, followed by storage at 4 degrees C).
(5) Labeling
The PCR amplification fragment obtained in (4) above was purified with a column (Qiagen). Cy3-labeled 9mers (TriLink; 1 O.D.) was added thereto. The resultant was treated at 98 degrees C for 10 minutes and allowed to stand still on ice for 10 minutes. Then, Klenow (NEB; 100 units) was added thereto, followed by treatment at 37 degrees C for 2 hours. Thereafter, a labeled sample was prepared by ethanol precipitation.
(6) Hybridization/signal detection
The labeled sample obtained in (5) was subjected to hybridization using the DNA microarray prepared in 1 above in accordance with the NimbleGen Array User's Guide. Signals from the label were detected.
3. Identification of QTL for sugarcane leaf area and development of markers
(1) Creation of genetic map datasheet
Genotype data of possible 3004 markers were obtained based on the signal data detected in 2 above of the NiF8 and Ni9 sugarcane varieties and the progeny line (line 191). Based on the obtained genotype data, chromosomal marker position information was obtained by calculation using the gene distance function (Kosambi) and the AntMap genetic map creation software (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm, Breed Sci 56: 371-378). Further, a genetic map datasheet was created based on the obtained marker position information using Mapmaker/EXP ver. 3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993).
(2) Acquisition of leaf-area data
The tested sugarcane varieties (NiF8 and Ni9) and the progeny line (191 lines) were subjected to cultivation with 2 replicates (13 individuals per replicate). In each replicate of cultivation, 5 individuals were subjected to measurement of the length between the dewlap and the leaf blade tip. The mean values obtained from the results for separate replicates of cultivation were used as leaf-blade-length data. Similarly, in each replicate of cultivation, 5 individuals were subjected to measurement of the maximum leaf-blade-width. The measurement results were used as leaf-blade-width data. Based on the obtained leaf-blade-length data and leaf-blade-width data, the leaf area was calculated by the following equation. In addition, the calculated leaf-area values are summarized in fig. 3.
[Leaf-area] = 0.643 x [Leaf-blade-length] x [Leaf-blade-width]
(3) Quantitative trait (quantitative trait loci: QTL) analysis
Based on the genetic map datasheet obtained in (1) above and the leaf-area data obtained in (2) above, QTL analysis was carried out by the composite interval mapping (CIM) method using the QTL Cartographer gene analysis software (Wang S., C. J. Basten, and Z.-B. Zeng (2010), Windows QTL Cartographer 2.5, Department of Statistics, North Carolina State University, Raleigh, NC). Upon analysis, the LOD threshold was determined to be 2.5. As a result, as shown in figs. 4 to 8, peaks exceeding the LOD threshold were observed in the following ranges for the NiF8 sugarcane variety: the range between markers N812691 and N803255 present in the 9th linkage group; the range between markers N803305 and N826149 present in the 11th linkage group; the range between markers N828907 and N803226 present in the 38th linkage group; the range between markers N825608 and N820986 present in the 47th linkage group; and the range between markers N823809 and N821676 present in the 82nd linkage group. It was possible to specify the obtained peaks as shown in table 6, suggesting the presence of causative genes (i.e., gene group) each having the function of causing an increase in leaf area at relevant peak positions.
Figure JPOXMLDOC01-appb-T000006
 As shown in figs. 4 to 8, markers located in the vicinity of the relevant peaks are inherited in linkage with causative genes (i.e., gene group) each having the function of causing an increase in leaf area. This shows that the markers can be used as sugarcane-leaf-area-related markers. Specifically, it has been revealed that the 39 types of markers shown in figs. 4 to 8 can be used as sugarcane-leaf-area-related markers. In addition, as examples of signals detected in 2 (6) above, table 7 shows signal levels of 14 types of markers among markers N812691 to N803255 present in the 9th linkage group for NiF8 and Ni9 and their 17 progeny lines (F1_1 to F1_17). In particular, the signal levels of N812691 are shown in fig. 9.
Figure JPOXMLDOC01-appb-T000007
 Signal levels of 14 types of markers were found to be very high for NiF8 and the progeny lines such as F1_1 and F1_5 with relatively large leaf areas. These results also revealed that 14 types of markers among markers N812691 to N803255 present in the 9th linkage group can be used as sugarcane-leaf-area-related markers.
Similarly, table 8 shows signal levels of 8 types of markers for NiF8 and Ni9, and 17 progeny lines (F1_1 to F1_17) among markers N803305 to N826149 present in the 11th linkage group. In particular, fig. 10 shows the signal levels of N828503.
Figure JPOXMLDOC01-appb-T000008
 Signal levels of 8 types of markers were found to be very high for NiF8 and the progeny lines such as F1_3 and F1_7 with relatively large leaf areas. These results also revealed that 8 types of markers among markers N803305 to N826149 present in the 11th linkage group can be used as sugarcane-leaf-area-related markers.
Similarly, table 9 shows signal levels of 11 types of markers for NiF8 and Ni9, and 17 progeny lines (F1_1 to F1_17) among markers N828907 to N803226 present in the 38th linkage group. In particular, fig. 11 shows the signal levels of N812717.
Figure JPOXMLDOC01-appb-T000009
 Signal levels of 11 types of markers were found to be very high for NiF8 and the progeny lines such as F1_3 and F1_4 with relatively large leaf areas. These results also revealed that 11 types of markers among markers N828907 to N803226 present in the 38th linkage group can be used as sugarcane-leaf-area-related markers.
Similarly, table 10 shows signal levels of 3 types of markers for NiF8 and Ni9, and 17 progeny lines (F1_1 to F1_17) among markers N825608 to N820986 present in the 47th linkage group. In particular, fig. 12 shows the signal levels of N828591.
Figure JPOXMLDOC01-appb-T000010
 Signal levels of 3 types of markers were found to be very high for NiF8 and the progeny lines such as F1_1 and F1_4 with relatively large leaf-areas. These results also revealed that 3 types of markers among markers N825608 to N820986 present in the 47th linkage group can be used as sugarcane-leaf-area-related markers.
Similarly, table 11 shows signal levels of 3 types of markers for NiF8 and Ni9, and 17 progeny lines (F1_1 to F1_17) included among markers N823809 to N821676 present in the 82nd linkage group. In particular, fig. 13 shows the signal levels of N824402.
Figure JPOXMLDOC01-appb-T000011
 Signal levels of 3 types of markers were found to be very high for NiF8 and the progeny lines such as F1_2 and F1_3 with relatively large leaf areas. These results also revealed that 3 types of the markers included in markers N823809 to N821676 present in the 82nd linkage group can be used as sugarcane-leaf-area-related markers.

Claims (6)

  1. A sugarcane-leaf-area-related marker, which comprises a continuous nucleic acid region existing in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 14, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 15 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 23 and the nucleotide sequence shown in SEQ ID NO: 33, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 34 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 39 of a sugarcane chromosome.
  2. The sugarcane-leaf-area-related marker according to claim 1, wherein the continuous nucleic acid region comprises any nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 39.
  3. The sugarcane-leaf-area-related marker according to claim 1, wherein the continuous nucleic acid region is located at a position in a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 19, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 28 and the nucleotide sequence shown in SEQ ID NO: 29, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 and the nucleotide sequence shown in SEQ ID NO: 36, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 37 and the nucleotide sequence shown in SEQ ID NO: 38 of a sugarcane chromosome.
  4. A method for producing a sugarcane line having an increased leaf area comprising: a step of extracting a chromosome of a progeny plant obtained from parent plants, at least one of which is a sugarcane plant; and a step of determining the presence or absence of the sugarcane-leaf-area-related marker according to any one of claims 1 to 3 in the obtained sugarcane chromosome.
  5. The method for producing a sugarcane line according to claim 4, wherein a DNA chip provided with probes each serving as the sugarcane-leaf-area-related marker is used in the determination step.
  6. The method for producing a sugarcane line according to claim 4, wherein the progeny plant is in the form of seeds or a young seedling and the chromosome is extracted from the seeds or the young seedling.
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