Patent Publication Number: US-2013237448-A1

Title: Sugarcane-sugar-yield-related marker and the use thereof

Description:
TECHNICAL FIELD 
     The present invention relates to a sugar-yield-related marker whereby a sugarcane line characterized by an increase in sugar yield can be selected, and a method for use thereof. 
     BACKGROUND ART 
     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 or leaf area, an increase in stalk length or stalk number, and an increase in sugar yield). 
     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/line having distinct characteristics in terms of leaf blade length, leaf area, stalk length, stalk number, 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 line with high accuracy because such characteristics are environmentally susceptible. 
     Further, for creation of a novel sugarcane variety, first, tens of thousands of seedlings are created by 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. 
     CITATION LIST 
     Non Patent Literature 
     
         
         NPL 1: Maydica 48 (2003)319-329 “Molecular genotyping of sugarcane clones with microsatellite DNA markers” 
         NPL 2: Nathalie Piperidis et al., Molecular Breeding, 2008, Vol. 21, 233-247 
       
    
     SUMMARY OF INVENTION 
     Technical Problem 
     In view of the above, an object of the present invention is to provide a marker related to sugar yield, which is a quantitative trait of sugarcane. 
     Solution to Problem 
     In order to achieve the object, the present inventors conducted intensive studies. The present inventors prepared many sugarcane markers and carried out linkage analysis of quantitative traits along with such markers for hybrid progeny lines. Accordingly, the present inventors found markers linked to quantitative traits such as an increase in sugar yield. This has led to the completion of the present invention. 
     The present invention encompasses the following. 
     (1) A sugarcane-sugar-yield-related marker, which consists of 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: 5, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 6 and the nucleotide sequence shown in SEQ ID NO: 24, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 47 of a sugarcane chromosome. 
     (2) The sugarcane-sugar-yield-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 47. 
     (3) The sugarcane-sugar-yield-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: 3 and the nucleotide sequence shown in SEQ ID NO: 5, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 9, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 35 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 sugar yield comprising: a step of extracting a chromosome of a progeny plant obtained from parent plants, at least one of which is sugarcane; and a step of determining the presence or absence of the sugarcane-sugar-yield-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 corresponding to the sugarcane-sugar-yield-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. 
     A part or all of the content disclosed in the description and/or drawings of Japanese Patent Application No. 2010-270801, which is a priority document of the present application, is herein incorporated by reference. 
     Advantageous Effects of Invention 
     According to the present invention, a novel sugarcane-sugar-yield-related marker linked to a sugarcane quantitative trait such as an increase in sugar yield can be provided. With the use of the sugarcane-sugar-yield-related marker of the present invention, the sugar yield of a line obtained by crossing sugarcane lines can be identified. Thus, a sugarcane line characterized by an increase in sugar yield can be identified at a very low cost. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         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 sugar yield data for sugarcane variety/line groups used in the Examples. 
         FIG. 4  is a characteristic chart showing QTL analysis results for the NiF8 sugarcane variety regarding sugar yield (the 12th linkage group). 
         FIG. 5  is a characteristic chart showing QTL analysis results for the Ni9 sugarcane variety regarding sugar yield (the 1st linkage group). 
         FIG. 6  is a characteristic chart showing QTL analysis results for the Ni9 sugarcane variety regarding sugar yield (the 25th linkage group). 
         FIG. 7  is a characteristic chart showing signal levels of N812648 (a marker present in the 12th linkage group of NiF8) for individual lines. 
         FIG. 8  is a characteristic chart showing signal levels of N916035 (a marker present in the 1st linkage group of Ni9) for individual lines. 
         FIG. 9  is a characteristic chart showing signal levels of N913752 (a marker present in the 25th linkage group of Ni9) for individual lines. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The sugarcane-sugar-yield-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-sugar-yield-related marker is described. 
     Sugarcane-Sugar-Yield-Related Markers 
     The sugarcane-sugar-yield-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 sugar yield. Thus, it can be used to identify a trait characterized by an increase in sugarcane sugar yield. 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 sugar yield by confirming the presence of a sugarcane-sugar-yield-related marker in such progeny line. 
     Here, the term “sugar yield” refers to the available sugar yield per unit area (e.g., 1 a (are)). The available sugar yield obtained from sugarcane juice extracted from collected millable stalks is calculated by the following equation. 
       Available sugar yield (kg/a)=Millable stalk weight (kg/a)×Recoverable sugar percent (%)/100
 
     Here, the term “millable stalk weight” refers to the millable stalk weight per unit area (e.g., 1 a (are)). The term “millable stalk” refers to a stalk used as a raw material for production of crude sugar or the like, which is obtained by removing low-sugar-content portions such as a cane top, leaves, and roots from an untreated sugarcane stalk. In general, the length of a millable stalk is 1 m or longer. In addition, “recoverable sugar percent” can be determined using a conventionally known calculation method (e.g., the CCS method (the Australia method)). 
     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 by so-called backcrossing. 
     The sugarcane-sugar-yield-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 chromosomes of the NiF8 sugarcane variety, a genetic linkage map containing 4569 markers originally obtained from chromosomes of the Ni9 sugarcane variety, and sugarcane sugar yield data. In addition, many genes are presumably associated with sugarcane sugar yield, which is a quantitative trait characterized by a continuous distribution of sugar yield 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, N.C.) 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., 3.0) have been found in 3 regions included in the above genetic linkage maps by QTL analysis described above. That is, the following 3 regions having such peaks have been specified: an approximately 12.4-cM (centimorgan) region (the NiF8 sugarcane variety); and an approximately 32.0-cM region and an approximately 31.7-cM region (the Ni9 sugarcane variety). 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 sugar yield could be present at the peak positions or in the vicinity thereof. 
     The 12.4-cM region having the above peak of the NiF8 sugarcane variety is a region that comprises 5 types of markers listed in table 1 below in the order shown in table 1. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Linkage 
                 Marker 
                   
                 Signal 
                   
               
               
                 group 
                 name 
                 Nucleotide sequence information 
                 threshold 
                 SEQ ID NO 
               
               
                   
               
             
            
               
                 NiF8_12 
                 N812648 
                 TTGACCTAATTCGCTTCACACTGTCGTCGTCGTTGTTGTTTT 
                 1000 
                 SEQ ID NO 1 
               
               
                   
                   
                 TGCTTACAAACAAAATG 
                   
                   
               
               
                   
               
               
                   
                 N817248 
                 GTGCAGCAGTGGGCATCGGCACAACTAGTTGCCCTTGGCATC 
                 1000 
                 SEQ ID NO 2 
               
               
                   
                   
                 ACTAGCCA 
                   
                   
               
               
                   
               
               
                   
                 N827148 
                 TGGAATAAAAAAAGAGCTCTAATATAAATTCTGCGGATTCGT 
                 1000 
                 SEQ ID NO 3 
               
               
                   
                   
                 TGACTTGTGCAGTGTCTGATTTCGATTG 
                   
                   
               
               
                   
               
               
                   
                 N823594 
                 ATACTTGTTTGTGCTATCCTGTTGTGCTTGCCTGTTCCTCTG 
                 1000 
                 SEQ ID NO 4 
               
               
                   
                   
                 TAGTGTTGACAAAAAAAATATAG 
                   
                   
               
               
                   
               
               
                   
                 N820026 
                 ATCAGGGTAGCAAGGTTAGTATTCTGCGGTTCAATCTTTCTT 
                 1000 
                 SEQ ID NO 5 
               
               
                   
                   
                 TTGTTTTGTAATTCATGGTTAGCAAA 
               
               
                   
               
            
           
         
       
     
     The 32.0-cM region having the above peak of the Ni9 sugarcane variety is a region that comprises 19 types of markers listed in table 2 below in the order shown in table 2. 
                                     TABLE 2               Linkage   Marker       Signal           group   name   Nucleotide sequence information   threshold   SEQ ID NO                  Ni9_1   N915070   ATAGTCTACCTATACTGGTGCCACAAGTCAACAAGTGATGGC   1000   SEQ ID NO 6               AATACCCATTCAAATT                           N915209   TGGCAATACCCATTCAAATTGCGTCAAATGTGAATAAATGGA   1500   SEQ ID NO 7               GGTAGATGACTAACACCTTTGTTTCAAAA                           N916186   CTGCAATACAATGCGGTGGAAGCGGATTGGTGGAAGGCATGC   1500   SEQ ID NO 8               ATGCATCA                           N902342   CCAAATACCTAAGTGCACTTTTTTCTGAGGCCAAATACCTAG   1000   SEQ ID NO 9               GTTCGAAAGATTCGT                           N919949   CCGCCTCAAAAGGAAGTAACACAGGAACATGATCATACGGAG   1000   SEQ ID NO 10               TAGTACTAT                           N920597   CTTGCCGGCCGGGACCCTGCTGGCACGATCAAGCGACTACAG   1500   SEQ ID NO 11               TACAATGC                           N916081   CAAAGAAAGCACATTACCGCGTATGTTACCAACTTCCTATGT   1000   SEQ ID NO 12               TGACTATCCAAATACTG                           N902047   GGATTGGTCTAGTACAATCTTTATTGAAGACGAAAGATTTAT   1500   SEQ ID NO 13               GCATGGTGATTAGTTGAGCCTGT                           N916874   CAAATATGACGATGGAAATATATAGTACTATTAATAAGACAT   1000   SEQ ID NO 14               AACTTGCAGCATATATTAATTTCATAGGATAAG                           N918161   CTAGTTAGAGCATCTCCAAGCGTACTCAGAAGAGTCGCCCAA   1000   SEQ ID NO 15               TCTAGCAA                           N918536   CAGAGAAACTGGGAACGAAACAGGACAATACATCTGTACGTT   1000   SEQ ID NO 16               TGGCTTGT                           N901676   TCCCTGTACTGTATGGTCGCCACAAATGCATATTGATAGACA   1000   SEQ ID NO 17               TGTTTATGATGTAGAATTTGATGTTTACA                           N919743   AAATCAATAAAGAAAGGCACGCTGAAAATAAGATGGTCTGAT   1000   SEQ ID NO 18               CGAGCTCCTGTGTTTAGTACAA                           N901176   ATTCCAATGAACTAAGGGTAAGTAGAGATTATTATATATAAA   1500   SEQ ID NO 19               TCAATGATACACAAACTGATCAATCAACTAA                           N916035   GCCTTCTTGATCTCTCAGACTAAGAACATAGGCCCAGAGTGA   1000   SEQ ID NO 20               GGGGAAAC                           N921010   CGTTCGCTTGAGCTTATTAGATAAAATCAATCAGCAATAAAA   1500   SEQ ID NO 21               TAATATTTTTTTCTAATAAAAATCAGCA                           N915635   TTTATCAGCTTCGGAAATCAGCTTGAGCTGACGAAGACATCA   1500   SEQ ID NO 22               ATCTTCTACATCAGAT                           N901348   ACATGTATGTGCAAAATATCTTGAGACCCTCTGCTTTAACAT   1000   SEQ ID NO 23               GCATGTCCTTCACATGT                           N920207   CAGCTCTGTCATTGCCGCCAAACACATATGCGCCTTCATGCC   1000   SEQ ID NO 24               CTTCTCCC                    
The 31.7-cM region having the above peak of the Ni9 sugarcane variety is a region that comprises 23 types of markers listed in table 3 below in the order shown in table 3.
 
                                     TABLE 3               Linkage   Marker       Signal           group   name   Nucleotide sequence information   threshold   SEQ ID NO                  Ni9_25   N902029   CCTTACATTGCCGGCGGGTGCCAAGGTTAGTTACCACTGCAT   1000   SEQ ID NO 25               CCTGTTAA                           N917675   TCTGCAAGAGCGAGCACAGCGAATGTTTTGCCACGTACACGG   1000   5E0 ID NO 26               GCTACGCG                           N915680   TACGGATGTTCCAAAAGTAGATCTAGATGTTAGATATGTTGC   1500   SEQ ID NO 27               AATGACTATACACGAATGTTGTAAGTACCTAT                           N917310   AAGAGCGAGCACAGCGAATGTTTTGCCACGTACACGGGCTAC   1000   SEQ ID NO 28               GCGTGCAA                           N900440   CCACGTACCCGGGCTACGCGTGCAAATGCAAGGATGGTTACG   1000   SEQ ID NO 29               ACGGCAAC                           N901219   GTTGCAGTTACCATGAAATCCATGCTTGTTGGTCAATGGTCA   1500   SEQ ID NO 30               TGCTTAATATAATACTGAAGATAAGCAAATATA                           N920418   CAAGACCGCCATTAGTGTAGCAATACCGCTGTTACTGTAGCA   1500   SEQ ID NO 31               AAACCACC                           N919541   CCCACTCCATAGACATTGACTGTGGATGAAACAAGGACCAGC   1000   SEQ ID NO 32               AATCTGCA                           N900579   TTAACAAGATCCATGACACGAGATTGATATGATCGGCATTGG   1000   SEQ ID NO 33               CCAACAAGGT                           N900152   AGGCGAGGGGAAGACGCTTGTTTCCACACTTGCAGGTTATCT   1000   SEQ ID NO 34               AAATGCCC                           N919576   ACTCCTCGCAACCTGAAATTCGTGCAGATCCTTCCACCCCCT   1000   SEQ ID NO 35               GCCCCTTG                           N911604   GGTGGCCTCCCATGGGAAGTTGATGCTGCTTGCAGCTTTGGC   1000   SEQ ID NO 36               TTCACGAT                           N911151   TGAGAAATGGAAATTCAAGTAAGTGTGACCTGCCGAGTATCT   1000   SEQ ID NO 37               GGAAAAACTAAACAAAATCTTACAAGA                           N914100   CCATAAAACTGATAAAGATGCCTAGCGGAACATAGGAAATAC   1000   SEQ ID NO 38               TTGAACATCGAACCAATTTCAACATTAT                           N914316   ATACAGTTATGGGCATTAGACCCATGAATCCATTATATAGTG   1000   SEQ ID NO 39               TCTCCAATGCAAGGACAAGAT                           N912566   ACAGCGATATAGATGTGGAGGAGGATGAGAATGAGGATGATG   1000   SEQ ID NO 40               ATGAGAAG                           N913492   ACGAGAATGAGGACAGTGAGGAAGAGGATGACAGCGATATAG   1000   SEQ ID NO 41               ATGTGGAG                           N913359   TGCACCACATGGTACTTGATATGATTAAGTGCAAGTCCAAAG   1000   SEQ ID NO 42               AAGCGAACTTCA                           N920944   ACGTGCTTCCGATCCTGTATGAAAAGATTATTCAAGGTCACA   1000   SEQ ID NO 43               TAGCATGCTATCT                           N918183   AGCTAGGAGTATCTGGCATCAACAAGAAAAACTGCAAGGAGT   1000   SEQ ID NO 44               TCTTCTGTGCAATTT                           N919525   TGCTAAGGCTTACTTGGAAGCTAATAAGATATATACTTACAA   1500   SEQ ID NO 45               TAATCCTCCCCTGCTTTGTAGATTTGCAA                           N913752   GCAGATAAAACCCTCAGCTATCCATCGCCTAATCAAAGCAGT   1000   SEQ ID NO 46               CTTTGAGATTATGTAA                           N918557   ACTCTTGCACTCATGTCTGTCATGTTTTCGTCTTTTGCTTAT   1000   SEQ ID NO 47               GGATACATGCTAAAATTAGGACAA                    
In addition, in tables 1 to 3, “Linkage group” represents the number given to each group among a plurality of linkage groups specified by QTL analysis. In tables 1 to 3, “Marker name” represents the name given to each marker originally obtained in the present invention. In tables 1 to 3, “Signal threshold” represents a threshold used for determination of the presence or absence of a marker.
 
The peak contained in the 12.4-cM region of the NiF8 sugarcane variety is present in a region sandwiched between a marker (N827148) consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a marker (N820026) consisting of the nucleotide sequence shown in SEQ ID NO: 5.
 
In addition, the peak contained in the 32.0-cM region of the Ni9 sugarcane variety is present in a region sandwiched between a marker (N915209) consisting of the nucleotide sequence shown in SEQ ID NO: 7 and a marker (N902342) consisting of the nucleotide sequence shown in SEQ ID NO: 9.
 
Further, the peak contained in the 31.7-cM region of the Ni9 sugarcane variety is present in a region sandwiched between a marker (N919576) consisting of the nucleotide sequence shown in SEQ ID NO: 35 and a marker (N914100) consisting of the nucleotide sequence shown in SEQ ID NO: 38.
 
A continuous nucleic acid region existing in any of 2 regions containing markers shown in tables 1 to 3 can be used as a sugarcane-sugar-yield-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-sugar-yield-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-sugar-yield-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-sugar-yield-related marker falls within the above range, the nucleic acid region can be specifically detected according to a standard method.
 
In particular, among the 5 types of markers contained in the 12.4-cM region of the NiF8 sugarcane variety, a sugarcane-sugar-yield-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 5. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 5. In addition, among the 19 types of markers contained in the 32.0-cM region of the Ni9 sugarcane variety, a sugarcane-sugar-yield-related marker is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 9. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 9. Further, among the 23 types of markers contained in the 31.7-cM region of the Ni9 sugarcane variety, a sugarcane-sugar-yield-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: 38. 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: 38.
 
In addition, a nucleic acid region containing a single marker selected from among the 47 types of markers shown in tables 1 to 3 can be used as a sugarcane-sugar-yield-related marker. For example, it is preferable to use, as a sugarcane-sugar-yield-related marker, a nucleic acid region containing a marker (N823594) consisting of the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 12.4-cM region of the NiF8 sugarcane variety, a nucleic acid region containing a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 8 located closest to the peak position in the 32.0-cM region of the Ni9 sugarcane variety, or a nucleic acid region containing a marker (N911604) consisting of the nucleotide sequence shown in SEQ ID NO: 36 located closest to the peak position in the 31.7-cM region of the Ni9 sugarcane variety. 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-sugar-yield-related marker, any of the above 47 types of markers can be directly used. Specifically, one or more type(s) of markers selected from among the 47 types of such markers can be directly used as a sugarcane-sugar-yield-related marker. For example, it is preferable to use, as a sugarcane-sugar-yield-related marker, a marker (N823594) consisting of the nucleotide sequence shown in SEQ ID NO: 4 located closest to the peak position in the 12.4-cM region of the NiF8 sugarcane variety, a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 8 located closest to the peak position in the 32.0-cM region of the Ni9 sugarcane variety, or a marker (N911604) consisting of the nucleotide sequence shown in SEQ ID NO: 36 closest to the peak position in the 31.7-cM region of the Ni9 sugarcane variety.
 
     Sugarcane Marker Identification 
     As described above, sugarcane-sugar-yield-related markers were identified from among 3004 markers originally obtained from chromosomes of the NiF8 sugarcane variety and 4569 markers originally obtained from chromosomes of the Ni9 sugarcane variety in the present invention. These 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, these 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 by 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 by 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 adapter, adapter(s) corresponding to one or more restriction enzyme(s) 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 by 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 ligated 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 1e. 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 and 4569 markers, including the above 47 types of sugarcane-sugar-yield-related markers shown in SEQ ID NOS: 1 to 47, can be identified from the sugarcane varieties NiF8 and Ni9, respectively.
 
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 and 4569 markers, including the aforementioned 47 types of sugarcane-sugar-yield-related markers shown in SEQ ID NOS: 1 to 47, were identified from the sugarcane varieties NiF8 and Ni9, respectively.
 
     Use of Sugarcane-Sugar-Yield-Related Markers 
     The use of sugarcane-sugar-yield-related markers makes it possible to determine whether a sugarcane progeny line or the like, which has a phenotype exhibiting unknown sugar yield, is a line having a phenotype showing an increase in sugar yield. The expression “the use of sugarcane-sugar-yield-related markers” used herein indicates the use of a DNA microarray having probes corresponding to sugarcane-sugar-yield-related markers in one embodiment. The expression “probes corresponding to sugarcane-sugar-yield-related markers” indicates oligonucleotides that can specifically hybridize under stringent conditions to sugarcane-sugar-yield-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-sugar-yield-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 sugar yield, is a line having a phenotype showing an increase in sugar yield. 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 sugar yield, is a line having a trait characterized by an increase in sugar yield by detecting the above sugarcane-sugar-yield-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 sugar yield, and thus which can be used as a subject to be determined whether to have a trait characterized by an increase in sugar yield 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, specific DNA fragment concentration can be increased by selectively amplifying the 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-sugar-yield-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.; 0.2 SDS; and 6×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-sugar-yield-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 determination of the actual sugar yield thereof becomes possible. 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-sugar-yield-related marker(s). In particular, when a novel sugarcane variety is created, it is preferable to produce several tens of thousands of seedlings by crossing and then to identify a novel sugarcane variety using sugarcane-sugar-yield-related markers prior to or instead of seedling selection. The use of such sugarcane-sugar-yield-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.
 
Causative genes (i.e., gene group) for a trait that causes an increase in sugarcane sugar yield can be isolated using the above sugarcane-sugar-yield-related markers. A conventionally known method can be used as an isolation method (see “Illustrated bio-experiment practice 4 (Bio-Jikken Illustrated 4): Effortless Cloning,” Kazuhiro Makabe (1997), Shujunsha Co., Ltd.). For example, causative genes (i.e., gene group) for a trait that causes an increase in the sugar yield of a non-sugarcane graminaceous plant can be isolated by screening a different graminaceous-plant-derived genomic DNA or cDNA instead of the sugarcane genomic DNA or cDNA using primers or probes corresponding to the sugarcane-sugar-yield-related markers.
 
     In addition, a transformed plant characterized by an increase in sugar yield can be produced by transformation of plant cells using a recombinant vector including a causative gene for a trait that causes an increase in sugarcane sugar yield obtained above. 
     EXAMPLES 
     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: 48) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 49)) 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 4 hours or longer. 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: 50)) 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, 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 FLX454 (Roche) or 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 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: 48) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 49)) 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 4 hours or longer. 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: 50)) 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 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 9mer wobble (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 isopropanol 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&#39;s Guide. Signals from the label were detected. 
     3. Identification of QTL for Sugarcane Sugar Yield and Development of Markers 
     (1) Creation of Genetic Map Datasheet 
     Genotype data of possible NiF8-derived 3004 markers and Ni9-derived 4569 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 Sugar Yield Data 
     The tested sugarcane varieties (NiF8 and Ni9) and the progeny line (line 191) were planted (13 individuals in each plot (2.2 m 2 )) in April 2009. In March 2010, stalks of 5 individuals were harvested from each plot. The harvested stalks were prepared as millable stalks. The juice extracted therefrom was used for calculation of the recoverable sugar percent in the sugarcane by the following calculation method. 
     Method for Calculating the Recoverable Sugar Percent 
     CCS Method (Australia Method) 
       Recoverable sugar percent (%)=(3 ×P ×(95 −F )− B (97 −F ))/200
 
     P: Polarization of sugarcane juice (%); B: Brix of sugarcane juice (%); F: fiber content (%) 
     Based on the recoverable sugar percent, the available sugar yield was calculated by the following calculation method for each line. The obtained available sugar yields were used as sugar yield data. 
     Available Sugar Yield Calculation Method 
       Available sugar yield (kg/a)=Millable stalk weight (kg/a)×Recoverable sugar percent (%)/100
 
       FIG. 3  is a chart summarizing sugar yields determined for each line. In addition, NiF8 and Ni9 are included in the “120 kg/a” data zone. 
     (3) Quantitative Trait (Quantitative Trait Loci: QTL) Analysis 
     Based on the genetic map datasheet obtained in (1) above and the sugar-yield 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, N.C.; http://statgen.ncsu.edu/qticart/cartographer.html). Upon analysis, the LOD threshold was determined to be 3.0. As a result, as shown in  FIGS. 4 to 6 , peaks exceeding the LOD threshold were observed in the following ranges: the range between markers N812648 and N820026 present in the 12th linkage group of the NiF8 sugarcane variety; the range between markers N915070 and N920207 present in the 1st linkage group of the Ni9 sugarcane variety; and the range between markers N902029 and N918557 present in the 25th linkage group of the Ni9 sugarcane variety. It was possible to specify the obtained peaks as shown in table 4, suggesting the presence of causative genes (i.e., gene group) each having the function of causing an increase in sugar yield at the peak positions. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Linkage 
                 Position 
                 Range 
                   
                 LOD 
                 Effect 
               
               
                 group 
                 (cM) 
                 (cM) 
                 Adjacent marker 
                 value 
                 (kg/a) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 NiF8_12 
                 6.2 
                 12.4 
                 N812648-N820026 
                 3.2 
                 15.6 
               
               
                 Ni9_1 
                 5.5 
                 32.0 
                 N915070-N920207 
                 6.3 
                 21.8 
               
               
                 Ni9_25 
                 56.0 
                 31.7 
                 N902029-N918557 
                 3.4 
                 28.5 
               
               
                   
               
            
           
         
       
     
     As shown in  FIGS. 4 to 6 , markers located in the vicinity of the peaks are inherited in linkage with causative genes (i.e., gene group) each having the function of causing an increase in sugar yield. This shows that the markers can be used as sugarcane-sugar-yield-related markers. Specifically, it has been revealed that the 47 types of markers shown in  FIGS. 4 to 6  can be used as sugarcane-sugar-yield-related markers. In addition, as examples of signals detected in 2 (6) above, table 5 shows signal levels of 47 types of markers among markers N812648 to N820026 present in the 12th linkage group of the NiF8 sugarcane variety, markers N915070 to N920207 present in the 1st linkage group of the Ni9 sugarcane variety, and markers N902029 to N918557 present in the 25th linkage group of the Ni9 sugarcane variety for NiF8 and Ni9 and their 12 progeny lines (F1 — 1 to F1 — 12). In particular, the signal levels of N812648, N916035, and N913752 are shown in  FIGS. 7-9 , respectively. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
             
            
               
                 Linkage 
                 Marker 
                 Line name 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 group 
                 name 
                 NiF8 
                 Ni9 
                 F1_1 
                 F1_2 
                 F1_3 
                 F1_4 
                 F1_5 
               
               
                   
               
               
                 NiF8_12 
                 N812648 
                 2,992 
                 572 
                 641 
                 2,980 
                 599 
                 3,219 
                 678 
               
               
                   
                 N817248 
                 1,578 
                 368 
                 441 
                 1,505 
                 434 
                 1,244 
                 396 
               
               
                   
                 N827148 
                 1,564 
                 462 
                 481 
                 2,272 
                 595 
                 1,975 
                 655 
               
               
                   
                 N823594 
                 8,706 
                 926 
                 541 
                 5,820 
                 824 
                 5,827 
                 506 
               
               
                   
                 N820026 
                 8,510 
                 622 
                 672 
                 5,656 
                 507 
                 5,863 
                 445 
               
               
                 Ni9_1 
                 N915070 
                 424 
                 1,195 
                 1,122 
                 465 
                 1,422 
                 1,197 
                 370 
               
               
                   
                 N915209 
                 560 
                 1,796 
                 1,776 
                 385 
                 2,713 
                 2,291 
                 485 
               
               
                   
                 N916186 
                 496 
                 2,002 
                 1,808 
                 448 
                 1,660 
                 1,538 
                 457 
               
               
                   
                 N902342 
                 372 
                 1,245 
                 1,003 
                 362 
                 1,209 
                 1,323 
                 605 
               
               
                   
                 N919949 
                 625 
                 1,459 
                 1,942 
                 542 
                 2,289 
                 2,715 
                 859 
               
               
                   
                 N920597 
                 450 
                 4,702 
                 3,819 
                 997 
                 5,411 
                 5,062 
                 409 
               
               
                   
                 N916081 
                 518 
                 13,678 
                 14,893 
                 441 
                 11,095 
                 9,844 
                 754 
               
               
                   
                 N902047 
                 955 
                 5,233 
                 3,853 
                 467 
                 4,584 
                 5,235 
                 775 
               
               
                   
                 N916874 
                 491 
                 3,320 
                 2,869 
                 790 
                 3,170 
                 2,894 
                 658 
               
               
                   
                 N918161 
                 438 
                 2,109 
                 1,892 
                 397 
                 2,246 
                 1,973 
                 520 
               
               
                   
                 N918536 
                 372 
                 1,059 
                 1,293 
                 386 
                 1,430 
                 1,384 
                 426 
               
               
                   
                 N901676 
                 648 
                 1,534 
                 1,395 
                 587 
                 1,369 
                 1,309 
                 460 
               
               
                   
                 N919743 
                 635 
                 2,361 
                 1,731 
                 388 
                 2,121 
                 2,091 
                 384 
               
               
                   
                 N901176 
                 697 
                 5,017 
                 5,027 
                 901 
                 5,193 
                 3,970 
                 773 
               
               
                   
                 N916035 
                 757 
                 4,444 
                 3,803 
                 503 
                 3,489 
                 4,026 
                 834 
               
               
                   
                 N921010 
                 521 
                 5,630 
                 5,012 
                 565 
                 4,702 
                 5,636 
                 968 
               
               
                   
                 N915635 
                 424 
                 7,875 
                 10,900 
                 548 
                 12,886 
                 11,099 
                 993 
               
               
                   
                 N901348 
                 493 
                 3,188 
                 7,451 
                 549 
                 7,426 
                 7,614 
                 756 
               
               
                   
                 N920207 
                 421 
                 5,291 
                 4,857 
                 467 
                 5,756 
                 4,121 
                 384 
               
               
                 Ni9_25 
                 N902029 
                 382 
                 2,007 
                 2,028 
                 378 
                 2,085 
                 345 
                 2,597 
               
               
                   
                 N917675 
                 389 
                 2,017 
                 2,364 
                 555 
                 2,627 
                 474 
                 1,930 
               
               
                   
                 N915680 
                 542 
                 2,862 
                 2,334 
                 478 
                 2,719 
                 455 
                 2,750 
               
               
                   
                 N917310 
                 341 
                 2,192 
                 2,595 
                 347 
                 3,199 
                 377 
                 2,465 
               
               
                   
                 N900440 
                 411 
                 1,207 
                 1,467 
                 375 
                 1,605 
                 400 
                 1,522 
               
               
                   
                 N901219 
                 627 
                 13,040 
                 11,643 
                 595 
                 12,654 
                 619 
                 10,692 
               
               
                   
                 N920418 
                 783 
                 2,150 
                 1,949 
                 839 
                 1,891 
                 683 
                 2,378 
               
               
                   
                 N919541 
                 327 
                 1,728 
                 1,568 
                 395 
                 1,500 
                 402 
                 2,311 
               
               
                   
                 N900579 
                 452 
                 5,169 
                 3,520 
                 799 
                 3,472 
                 430 
                 3,868 
               
               
                   
                 N900152 
                 633 
                 6,141 
                 6,158 
                 447 
                 5,355 
                 488 
                 5,383 
               
               
                   
                 N919576 
                 398 
                 4,932 
                 6,146 
                 473 
                 7,284 
                 450 
                 4,660 
               
               
                   
                 N911604 
                 397 
                 2,665 
                 3,072 
                 417 
                 3,093 
                 441 
                 4,105 
               
               
                   
                 N911151 
                 421 
                 9,258 
                 7,569 
                 518 
                 10,087 
                 412 
                 5,643 
               
               
                   
                 N914100 
                 433 
                 2,564 
                 2,136 
                 455 
                 2,668 
                 537 
                 1,818 
               
               
                   
                 N914316 
                 536 
                 1,466 
                 1,024 
                 322 
                 1,150 
                 574 
                 1,331 
               
               
                   
                 N912566 
                 552 
                 1,036 
                 1,164 
                 345 
                 1,201 
                 373 
                 1,362 
               
               
                   
                 N913492 
                 534 
                 1,754 
                 1,798 
                 531 
                 1,910 
                 342 
                 2,262 
               
               
                   
                 N913359 
                 347 
                 3,959 
                 3,349 
                 327 
                 3,616 
                 361 
                 3,765 
               
               
                   
                 N920944 
                 439 
                 9,424 
                 8,025 
                 489 
                 7,773 
                 393 
                 8,158 
               
               
                   
                 N918183 
                 408 
                 2,178 
                 2,404 
                 373 
                 1,861 
                 418 
                 2,959 
               
               
                   
                 N919525 
                 403 
                 3,752 
                 3,540 
                 345 
                 3,531 
                 544 
                 3,053 
               
               
                   
                 N913752 
                 520 
                 4,024 
                 4,449 
                 565 
                 4,846 
                 405 
                 4,584 
               
               
                   
                 N918557 
                 404 
                 1,789 
                 1,149 
                 366 
                 1,688 
                 391 
                 1,290 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Linkage 
                 Marker 
                 Line name 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 group 
                 name 
                 F1_6 
                 F1_7 
                 F1_8 
                 F1_9 
                 F1_10 
                 F1_11 
                 F1_12 
               
               
                   
               
               
                 NiF8_12 
                 N812648 
                 976 
                 735 
                 3,977 
                 772 
                 4,188 
                 596 
                 3,143 
               
               
                   
                 N817248 
                 386 
                 450 
                 1,404 
                 432 
                 1,325 
                 549 
                 1,996 
               
               
                   
                 N827148 
                 608 
                 649 
                 2,451 
                 589 
                 2,076 
                 460 
                 2,234 
               
               
                   
                 N823594 
                 658 
                 604 
                 6,138 
                 460 
                 7,187 
                 577 
                 8,060 
               
               
                   
                 N820026 
                 823 
                 478 
                 5,147 
                 529 
                 5,847 
                 777 
                 4,062 
               
               
                 Ni9_1 
                 N915070 
                 1,851 
                 1,612 
                 1,659 
                 1,467 
                 1,227 
                 1,359 
                 384 
               
               
                   
                 N915209 
                 2,819 
                 3,527 
                 1,918 
                 2,571 
                 1,920 
                 2,361 
                 523 
               
               
                   
                 N916186 
                 2,064 
                 2,655 
                 2,224 
                 1,966 
                 2,125 
                 1,723 
                 435 
               
               
                   
                 N902342 
                 1,566 
                 2,119 
                 1,120 
                 1,030 
                 1,178 
                 1,346 
                 394 
               
               
                   
                 N919949 
                 2,469 
                 4,064 
                 2,230 
                 2,207 
                 2,278 
                 2,360 
                 478 
               
               
                   
                 N920597 
                 4,330 
                 5,664 
                 5,734 
                 4,578 
                 4,636 
                 4,669 
                 545 
               
               
                   
                 N916081 
                 11,988 
                 11,648 
                 11,307 
                 11,559 
                 13,129 
                 10,441 
                 519 
               
               
                   
                 N902047 
                 4,827 
                 7,474 
                 4,719 
                 4,968 
                 3,724 
                 5,336 
                 656 
               
               
                   
                 N916874 
                 2,867 
                 4,067 
                 3,028 
                 2,428 
                 3,092 
                 3,046 
                 679 
               
               
                   
                 N918161 
                 1,915 
                 3,286 
                 2,575 
                 1,854 
                 2,262 
                 2,193 
                 370 
               
               
                   
                 N918536 
                 1,663 
                 1,846 
                 1,383 
                 1,306 
                 1,310 
                 1,704 
                 356 
               
               
                   
                 N901676 
                 1,466 
                 2,033 
                 1,650 
                 1,683 
                 1,879 
                 1,820 
                 944 
               
               
                   
                 N919743 
                 1,698 
                 3,369 
                 2,309 
                 2,367 
                 2,232 
                 2,076 
                 498 
               
               
                   
                 N901176 
                 4,074 
                 4,017 
                 2,870 
                 2,874 
                 2,375 
                 3,362 
                 502 
               
               
                   
                 N916035 
                 3,965 
                 4,035 
                 4,949 
                 3,940 
                 4,429 
                 4,270 
                 463 
               
               
                   
                 N921010 
                 5,107 
                 6,405 
                 5,791 
                 4,931 
                 4,664 
                 4,902 
                 510 
               
               
                   
                 N915635 
                 10,634 
                 13,276 
                 12,215 
                 9,824 
                 12,973 
                 10,501 
                 407 
               
               
                   
                 N901348 
                 8,703 
                 3,731 
                 8,233 
                 4,969 
                 3,510 
                 7,406 
                 507 
               
               
                   
                 N920207 
                 3,913 
                 3,124 
                 4,904 
                 3,466 
                 3,101 
                 3,567 
                 480 
               
               
                 Ni9_25 
                 N902029 
                 2,420 
                 414 
                 472 
                 1,889 
                 358 
                 1,992 
                 349 
               
               
                   
                 N917675 
                 2,579 
                 494 
                 374 
                 2,283 
                 582 
                 2,492 
                 339 
               
               
                   
                 N915680 
                 2,075 
                 630 
                 491 
                 1,968 
                 525 
                 2,773 
                 531 
               
               
                   
                 N917310 
                 2,258 
                 497 
                 331 
                 2,204 
                 355 
                 2,430 
                 432 
               
               
                   
                 N900440 
                 1,635 
                 423 
                 453 
                 1,486 
                 388 
                 1,664 
                 475 
               
               
                   
                 N901219 
                 10,352 
                 697 
                 476 
                 12,366 
                 743 
                 9,943 
                 566 
               
               
                   
                 N920418 
                 2,382 
                 705 
                 810 
                 2,295 
                 759 
                 2,235 
                 728 
               
               
                   
                 N919541 
                 2,366 
                 424 
                 430 
                 2,118 
                 401 
                 2,435 
                 366 
               
               
                   
                 N900579 
                 3,450 
                 448 
                 437 
                 3,666 
                 511 
                 3,853 
                 482 
               
               
                   
                 N900152 
                 4,878 
                 490 
                 469 
                 7,271 
                 877 
                 5,543 
                 534 
               
               
                   
                 N919576 
                 7,137 
                 484 
                 380 
                 5,258 
                 434 
                 7,056 
                 412 
               
               
                   
                 N911604 
                 1,301 
                 443 
                 427 
                 2,361 
                 758 
                 3,369 
                 446 
               
               
                   
                 N911151 
                 6,548 
                 439 
                 456 
                 8,372 
                 487 
                 6,867 
                 475 
               
               
                   
                 N914100 
                 1,814 
                 392 
                 467 
                 2,442 
                 636 
                 2,411 
                 503 
               
               
                   
                 N914316 
                 1,869 
                 409 
                 519 
                 1,189 
                 394 
                 1,268 
                 384 
               
               
                   
                 N912566 
                 1,425 
                 414 
                 354 
                 1,009 
                 435 
                 1,611 
                 368 
               
               
                   
                 N913492 
                 1,937 
                 419 
                 326 
                 1,536 
                 332 
                 3,012 
                 381 
               
               
                   
                 N913359 
                 3,854 
                 369 
                 334 
                 3,421 
                 354 
                 3,424 
                 363 
               
               
                   
                 N920944 
                 6,319 
                 404 
                 352 
                 6,413 
                 330 
                 9,191 
                 341 
               
               
                   
                 N918183 
                 2,418 
                 355 
                 442 
                 2,109 
                 660 
                 2,329 
                 403 
               
               
                   
                 N919525 
                 2,216 
                 375 
                 365 
                 3,103 
                 496 
                 3,623 
                 358 
               
               
                   
                 N913752 
                 4,659 
                 479 
                 411 
                 4,805 
                 371 
                 4,457 
                 397 
               
               
                   
                 N918557 
                 1,395 
                 376 
                 385 
                 1,337 
                 428 
                 1,405 
                 395 
               
               
                   
               
            
           
         
       
     
     Signal levels of 47 types of markers were found to be very high for the progeny lines such as F1 — 1, F1 — 3, F1 — 4, F1 — 6, F1 — 8, F1 — 9, F1 — 10, and F1 — 11 with relatively high sugar yields. These results also revealed that 47 types of markers among markers N812648 to N820026 present in the 12th linkage group of the NiF8 sugarcane variety, markers N915070 to N920207 present in the 1st linkage group of the Ni9 sugarcane variety, and markers N902029 to N918557 present in the 25th linkage group of the Ni9 sugarcane variety can be used as sugarcane-sugar-yield-related markers. 
     All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.