# Patent

## Title
Marker associated with resistance to smut in plant belonging to genus Saccharum, and use thereof

## Abstract

The present invention relates to a marker associated with resistance to smut which is a quantitative trait of sugarcane. Specifically, a marker associated with resistance to sugarcane smut, 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: 14 or a different similar region, is provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2012/060671, filed on Apr. 20, 2012, which claims priority from Japanese Patent Application No. 2011-101050, filed on Apr. 28, 2011, and Japanese Patent Application No. 2012-094995, filed on Apr. 18, 2012, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a marker associated with resistance to smut whereby a sugarcane line resistant to smut 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, 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 resistant to a certain disease, an inoculation test is carried out using a causative microorganism of a disease after long-term cultivation of sugarcane, and then disease resistance data are collected by observing lesions and the like. However, transmission of the causative microorganism to an external environment must be securely prevented when the test is carried out, and thus it is necessary to provide, for example, facilities such as a large-scale special-purpose greenhouse, a special-purpose field or isolation facility from an external environment. Further, for creation of a novel sugarcane variety, first, tens of thousands of hybrids are created via crossing, followed by seedling selection and stepwise selection of desirable 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 the above large-scale greenhouse or field and undertake highly time-consuming efforts.
Therefore, it has been required to develop a method for identifying a sugarcane line having disease resistance 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 Literature 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 Literature 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 Literature 2, an insufficient number of markers are disclosed and markers linked to desired characteristics have not been found.
Meanwhile, as a marker associated with disease resistance, a marker associated with black root rot resistance in sugar beet disclosed in Patent Literature 1 is known. In addition, a technique of selecting a Zea mays variety using a maker linked to a desired trait is disclosed in Patent Literature 2.
The level of infectiousness of the causative microorganism of sugarcane smut is high, and therefore the onset of smut quickly results in the infection of the entire field. Crops of sugarcane affected with smut cannot be used as raw material for sugar production, and even they die. Therefore, the development of smut will cause a significant decline in yield within the following year or later. Damage due to smut has been reported in more than 28 countries, including Brazil, the U.S., Australia, China, and Indonesia. Smut can be prevented by sterilization treatment prior to planting; however, preventive effects are limited to the period of early growth. Thus, cultivation of a sugarcane variety imparted with smut resistance has been awaited.
CITATION LIST
Non-Patent Literature


    Non-Patent Literature 1: Maydica 48 (2003)319-329 “Molecular genotyping of sugarcane clones with microsatellite DNA markers”
    Non-Patent Literature 2: Nathalie Piperidis et al., Molecular Breeding, 2008, Vol. 21, 233-247


Patent Literature


    Patent Literature 1: WO 2007/125958
    Patent Literature 2: JP Patent Publication (Kokai) No. 2010-516236 A


SUMMARY OF INVENTION
Technical Problem
In view of the above, an object of the present invention is to provide a marker associated with resistance to smut, 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 plant 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 smut resistance. This has led to the completion of the present invention.
The present invention encompasses the following.
(1) A marker associated with resistance to sugarcane smut, 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: 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: 32, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 51, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 52 and the nucleotide sequence shown in SEQ ID NO: 62, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 63 and the nucleotide sequence shown in SEQ ID NO: 72, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 85 of a sugarcane chromosome.
(2) The marker associated with resistance to sugarcane smut 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 85 or a part of the nucleotide sequence.
(3) The marker associated with resistance to sugarcane smut 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: 5 and the nucleotide sequence shown in SEQ ID NO: 9, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 32, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66, or a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 72 and the nucleotide sequence shown in SEQ ID NO: 80 of a sugarcane chromosome.
(4) A method for producing a sugarcane line having improved smut resistance 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/or a chromosome of a parent sugarcane plant; and a step of determining the presence or absence of the marker associated with resistance to sugarcane smut according to any one of (1) to (3) in the obtained chromosome.
(5) The method for producing a sugarcane line according to (4), wherein a DNA chip comprising a probe corresponding to the marker associated with resistance to sugarcane smut 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.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application Nos. 2011-101050 and 2012-94995, which are priority documents of the present application.
Advantageous Effects of Invention
According to the present invention, a novel marker associated with resistance to sugarcane smut linked to a sugarcane quantitative trait such as smut resistance can be provided. With the use of the marker associated with resistance to sugarcane smut of the present invention, smut resistance of a line obtained by crossing sugarcane lines can be tested. Thus, a sugarcane line characterized by improved smut resistance 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 data on smut resistance examined on Jun. 23, 2010, for sugarcane variety/line groups used in the Examples.
FIG. 4 is a characteristic chart showing data on smut resistance examined on Jul. 21, 2010, for sugarcane variety/line groups used in the Examples.
FIG. 5 is a characteristic chart showing data on smut resistance examined on Aug. 18, 2010, for sugarcane variety/line groups used in the Examples.
FIG. 6 is a characteristic chart showing data on smut resistance examined on Sep. 2, 2010, for sugarcane variety/line groups used in the Examples.
FIG. 7 is a characteristic chart showing QTL analysis results regarding smut resistance (the 5th linkage group in NiF8).
FIG. 8 is a characteristic chart showing QTL analysis results regarding smut resistance (the 17th linkage group in NiF8).
FIG. 9 is a characteristic chart showing QTL analysis results regarding smut resistance (the 40th linkage group in NiF8).
FIG. 10 is a characteristic chart showing QTL analysis results regarding smut resistance (the 1st linkage group in Ni9).
FIG. 11 is a characteristic chart showing QTL analysis results regarding smut resistance (the 13th linkage group in Ni9).
FIG. 12 is a characteristic chart showing QTL analysis results regarding smut resistance (the 14th linkage group in Ni9).
FIG. 13 is a characteristic chart showing signal levels of N802870 for individual lines.
FIG. 14 is a characteristic chart showing signal levels of N827136 for individual lines.
FIG. 15 is a characteristic chart showing signal levels of N812680 for individual lines.
FIG. 16 is a characteristic chart showing signal levels of N916081 for individual lines.
FIG. 17 is a characteristic chart showing signal levels of N919839 for individual lines.
FIG. 18 is a characteristic chart showing signal levels of N918761 for individual lines.
FIG. 19 is a characteristic chart showing signal levels of N901160 for individual lines.



DESCRIPTION OF EMBODIMENTS
The marker associated with resistance to sugarcane smut 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 marker associated with resistance to sugarcane smut is described.
<Markers Associated with Resistance to Sugarcane Smut>
The marker associated with resistance to sugarcane smut of the present invention corresponds to a specific region present on a sugarcane chromosome and is linked to a causative gene (or a group of causative genes) for a trait characterized by smut resistance. Thus, it can be used to identify a trait characterized by smut resistance. Specifically, it is possible to determine that a progeny line obtained using a known sugarcane line is a line having a trait characterized by the improvement of smut resistance by confirming the presence or absence of the marker associated with resistance to sugarcane smut in such progeny line. In the present invention, the term “smut” refers to a disease characterized by lesion formation due to infection with a microorganism of the genus Ustilago. One example of a microorganism of the genus Ustilago is Ustilago scitaminea. 
In addition, the term “marker associated with resistance to sugarcane smut” refers to both a marker linked to a trait characterized by the improvement of smut resistance and a marker linked to a trait characterized by the reduction of smut resistance. For example, if the presence of the former marker in a certain sugarcane variety is confirmed, it is possible to determine that the variety has improved smut resistance. Further, if the presence of the former marker and the absence of the latter marker in a certain sugarcane variety are confirmed, it is possible to determine that the variety has improved smut resistance with high accuracy. It is also possible to determine that a certain sugarcane variety has improved smut resistance by confirming only the absence of the latter marker.
The term “sugarcane” used herein refers to a plant belonging to the genus Saccharum of the family Poaceae. In addition, the term “sugarcane” includes so-called noble cane (scientific name: Saccharum officinarum) and wild cane (scientific name: Saccharum spontaneum), Saccharum barberi, Saccharum sinense, and the earlier species of Saccharum officinarum (Saccharum robustum). The term “known sugarcane variety/line” is not particularly limited. It includes any variety/line available in Japan and any variety/line available 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 marker associated with resistance to sugarcane smut of the present invention has been newly identified by QTL (Quantitative Trait Loci) analysis using a genetic linkage map containing 3004 markers and 4569 markers originally obtained from sugarcane chromosomes, and sugarcane smut resistance data. In addition, many genes are presumably associated with sugarcane smut resistance, which is a quantitative trait characterized by a continuous distribution of sugarcane smut resistance. That is, sugarcane smut resistance is evaluated based on the incidence of smut characterized by such continuous distribution. 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, seven relevant regions included in the above genetic linkage map with LOD scores equivalent to or exceeding a given threshold (e.g., 2.5) have been found by QTL analysis described above. That is, the following 7 regions have been specified: an approximately 18.7-cM (centimorgan) region including the relevant region, an approximately 39.2-cM region including the relevant region, an approximately 19.2-cM region including the relevant region, an approximately 32.0-cM region including the relevant region, an approximately 39.5-cM region including the relevant region, an approximately 53.4-cM region including the relevant region, and an approximately 38.0-cM region including the relevant region. 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 a causative gene (or a group of causative genes) for a trait that causes the improvement of smut resistance could be present at the peak positions or in the vicinity thereof.
The 18.7-cM region is a region that comprises 14 types of markers listed in table 1 below in the order shown in table 1 and is linked to a trait characterized by the reduction of smut resistance.











TABLE 1





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:







NiF8_5
N827337
CCTCGTCATGCACCCGTGCCTCTTCTTCCTCTTGCTGTT
1,000
SEQ ID NO: 1




GCTCCTCCTCC




 



N802879
GGAATTGTTGTAGATTTGTTTTGTGATGGAAAGATCATAC
1,000
SEQ ID NO: 2




CTCAGCTACAAGAAGTAAATATCCTTTTCCA




 



N804818
GGCATTAGAAGAAAGGTGGAAGAATAAGGTTTGAGCCC
1,000
SEQ ID NO: 3




TTATTTATTTGCTTTGGTGATGGAT




 



N816296
CCATTCTACTTCTACCAACCATAAAACAGGAGGAGCATG
1,000
SEQ ID NO: 4




CATGCACATGC




 



N804607
ATTGCTTGCTCGCTGCAACTTGGGCCATGTTTAGTTCCT
1,000
SEQ ID NO: 5




CGAATTTGAGT




 



N802870
AGTGAAGAGATTGGATTTCTAGGGTTACTTTATAAAGTGT
1,500
SEQ ID NO: 6




CAACACCTTAGATCTGTTTTTTAGT




 



N813249
GGCCGGCACGAGCATCAGGGTCAAGACTCAAGAGCTC
1,500
SEQ ID NO: 7




AAGTGCTTGCTTT




 



N813609
TACTTTGTCTCGTTCCAGTAGTCCATCAAGCAAGCCTCG
1,000
SEQ ID NO: 8




TACACAAGTCC




 



N815502
TGCACTGGGGATACCAGTTGAGTTGATTGCACAACTTG
1,000
SEQ ID NO: 9




CGCTACACCATG




 



N815101
GCCGCCTGATGGAAACGGTCGTCGCATCCAAAGACGC
1,000
SEQ ID NO: 10




ACATGGTTTAGCA




 



N823481
AGTACCTGTTCTGCTGCACTACATAACAGTACTTTTCAG
1,000
SEQ ID NO: 11




TGAACGAACAGTGTTTTC




 



N801028
AGCGGATAGCGCTAGCATGTCATTCTCTCCCCTCGCTA
1,500
SEQ ID NO: 12




GCACGTTATTCC




 



N810798
GTTGCGGCGTGTGTTGATGATGTAAAGAATACTCGTCC
1,500
SEQ ID NO: 13




GTGAGAAATTATCA




 



N821515
ACGTGACGACGACGACGATGCAGCTGGGGCTTGGCGT
1,000
SEQ ID NO: 14




GGAATGGTTGTCG











The 39.2-cM region is a region that comprises 8 types of markers listed in table 2 below in the order shown in table 2 and is linked to a trait characterized by the improvement of smut resistance.











TABLE 2





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:







NiF8_17
N826561
GGCCTTGTTTAAATGTCACCTAAATTCTAAATTTTACACT
1,000
SEQ ID NO: 15




CTTTTCATAACATCGAATCTTAAAA




 



N827136
AAACTGAGGGATTACTTTCCAATTGAAATGTCATCCACC
1,500
SEQ ID NO: 16




ACAAACACAAAAGGCATACTCA




 



N826325
ACACTACACTGTGTAGGCAATGAGCAGCTCTGTTGCAC
1,000
SEQ ID NO: 17




AGCAAAGCCAAA




 



N803928
GGATGTGAAGTATGTATGTGTTTTCAGATGGACCAAGG
1,000
SEQ ID NO: 18




AAGCTGCATGGG




 



N822568
TACGGTGGTACAAAGCTTAGATCAATGATCAAGCTACAA
1,000
SEQ ID NO: 19




AACACACAAAGATAGTCAGTAGAAAAAGT




 



N829026
GACGACGAGGTGGGCAGCGCCAGTGCGCTACTACCTT
1,000
SEQ ID NO: 20




CTTTCTTGCAACT




 



N815300
GTATGGTTATGTTGGTACTAAAGGTTTCTGACTATTGTAT
1,000
SEQ ID NO: 21




TGTATTGTTGTGTTATAATGGGTTCAATG




 



N826906
GGCTGCAATACCTGTTCCTCATCTCATCTATTCGTGCAA
1,000
SEQ ID NO: 22




AGTTGCTGGTC









The 19.2-cM region is a region that comprises 10 types of markers listed in table 3 below in the order shown in table 3 and is linked to a trait characterized by the reduction of smut resistance.











TABLE 3





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:



















NiF8_40
N816552
TCGGGTTGGAGGCAAGGAAGAAAGGAGCTAGATTGCT
1,500
SEQ ID NO: 23




CGGCTGCTGGTGC




 



N827448
ACAGTAGTGCAACTGCGACGACGATGTGTGGGTATATG
1,000
SEQ ID NO: 24




TTCCATAGCTTG




 



N829378
TTTTGATTGGCCTTGCAGATGTTGCAGCGATGGCACTC
900
SEQ ID NO: 25




GTGGCAAACAGA




 



N829404
AACCATGCTGAAAACGTCTTCCGTTTACAGTTTATGGTA
1,000
SEQ ID NO: 26




TATCCGCTTAAAACTAACTCGATC




 



N828725
AATCTAAATGACTAATGAGACCGTGAGAGCTGCTTAGC
1,000
SEQ ID NO: 27




TTAATGGTGCATCCCTTTTTAAACT




 



N812680
AAGAACACTGCTAAGGATGGTCACAATTTGGAAACTGA
1,500
SEQ ID NO: 28




AGTTTTATCTCTGGTTCGGT




 



N811688
AAGCTGCATCTGATTCTCATCCAAACCTGCTCTGCTCA
1,000
SEQ ID NO: 29




TTATCATTACTTCGT




 



N819703
CCAACCAACAGCAAGAACACCAAGACGCACATAATGA
1,500
SEQ ID NO: 30




GGCCCATGAAGTA




 



N815648
TTTACACCAGTGAACTGACAAAAAATCGAAGTGGTGCG
1,000
SEQ ID NO: 31




GTACATAAGAACATTTACATCCAACT




 



N821999
GACCAATCTAGGAAAAACAATTGCACAAATGACTACATT
1,000
SEQ ID NO: 32




TATTATGGCAAATCAATTTTCTTCAGTCATTGTA









The 32.0-cM region is a region that comprises 19 types of markers listed in table 4 below in the order shown in table 4 and is linked to a trait characterized by the improvement of smut resistance.











TABLE 4





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:



















Ni9_1
N915070
ATAGTCTACCTATACTGGTGCCACAAGTCAACAAGTGAT
1,000
SEQ ID NO: 33




GGCAATACCCATTCAAATT




 



N915209
TGGCAATACCCATTCAAATTGCGTCAAATGTGAATAAAT
1,000
SEQ ID NO: 34




GGAGGTAGATGACTAACACCTTTGTTTCAAAA




 



N916186
CTGCAATACAATGCGGTGGAAGCGGATTGGTGGAAGG
1,000
SEQ ID NO: 35




CATGCATGCATCA




 



N902342
CCAAATACCTAAGTGCACTTTTTTCTGAGGCCAAATACC
900
SEQ ID NO: 36




TAGGTTCGAAAGATTCGT




 



N919949
CCGCCTCAAAAGGAAGTAACACAGGAACATGATCATAC
1,000
SEQ ID NO: 37




GGAGTAGTACTAT




 



N920597
CTTGCCGGCCGGGACCCTGCTGGCACGATCAAGCGA
1,500
SEQ ID NO: 38




CTACAGTACAATGC




 



N916081
CAAAGAAAGCACATTACCGCGTATGTTACCAACTTCCTA
4,000
SEQ ID NO: 39




TGTTGACTATCCAAATACTG




 



N902047
GGATTGGTCTAGTACAATCTTTATTGAAGACGAAAGATT
1,500
SEQ ID NO: 40




TATGCATGGTGATTAGTTGAGCCTGT




 



N916874
CAAATATGACGATGGAAATATATAGTACTATTAATAAGAC
1,000
SEQ ID NO: 41




ATAACTTGCAGCATATATTAATTTCATAGGATAAG




 



N918161
CTAGTTAGAGCATCTCCAAGCGTACTCAGAAGAGTCGC
1,000
SEQ ID NO: 42




CCAATCTAGCAA




 



N918536
CAGAGAAACTGGGAACGAAACAGGACAATACATCTGTA
900
SEQ ID NO: 43




CGTTTGGCTTGT




 



N901676
TCCCTGTACTGTATGGTCGCCACAAATGCATATTGATAG
1,000
SEQ ID NO: 44




ACATGTTTATGATGTAGAATTTGATGTTTACA




 



N919743
AAATCAATAAAGAAAGGCACGCTGAAAATAAGATGGTC
1,000
SEQ ID NO: 45




TGATCGAGCTCCTGTGTTTAGTACAA




 



N901176
ATTCCAATGAACTAAGGGTAAGTAGAGATTATTATATATA
1,500
SEQ ID NO: 46




AATCAATGATACACAAACTGATCAATCAACTAA




 



N916035
GCCTTCTTGATCTCTCAGACTAAGAACATAGGCCCAGA
1,500
SEQ ID NO: 47




GTGAGGGGAAAC




 



N921010
CGTTCGCTTGAGCTTATTAGATAAAATCAATCAGCAATA
1,500
SEQ ID NO: 48




AAATAATATTTTTTTCTAATAAAAATCAGCA




 



N915635
TTTATCAGCTTCGGAAATCAGCTTGAGCTGACGAAGAC
4,000
SEQ ID NO: 49




ATCAATCTTCTACATCAGAT




 



N901348
ACATGTATGTGCAAAATATCTTGAGACCCTCTGCTTTAA
1,500
SEQ ID NO: 50




CATGCATGTCCTTCACATGT




 



N920207
CAGCTCTGTCATTGCCGCCAAACACATATGCGCCTTCA
1,500
SEQ ID NO: 51




TGCCCTTCTCCC









The 39.5-cM region is a region that comprises 11 types of markers listed in table 5 below in the order shown in table 5 and is linked to a trait characterized by the reduction of smut resistance.











TABLE 5





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:



















Ni9_13
N914284
AGCCATCCCGCAGAGGCTCTTGATGTCCTTTGAGCTG
1,000
SEQ ID NO: 52




TCCTAAAACCACT




 



N901453
CTATGTGTTGGGCTTATATGTGATGCATCTTTCCTTTTG
1,000
SEQ ID NO: 53




AATTCAGGGTAGTGCTGATA




 



N900044
GTGCTGATACGCCACCAGCCGAAACAAATGGTGATAG
1,000
SEQ ID NO: 54




CTCTAGCGCACAG




 



N919839
AAATCCTGAAGGCCGAAGCCCGTAGACATGTTCACCCT
1,500
SEQ ID NO: 55




AGCAAACAAAGG




 



N901567
GCATCGGCTGGTGCTGGTAGGGATAAACCTCTGCTCC
1,500
SEQ ID NO: 56




GCTTGATATTTTT




 



N911103
TTCGCTTGAGTTTTATCAGCAGAATTAACAGTTATATAGC
800
SEQ ID NO: 57




GGTGTTTTTTCTCTCACACTAAATCAGTAAA




 



N918508
CTTGCCTACTTCTTGCATAGATGCTTAGTTTACATTTTAC
1,500
SEQ ID NO: 58




CTGAAATTTATTAATATCGATCACTACAAAT




 



N918344
GAACAAGGAGCATCCATATATGTATGGCACTTTGACATT
1,000
SEQ ID NO: 59




GTTGGCTATGTCTAGCTT




 



N919696
GGAAAAGCAAGCAGCTCGTGTAGCAATAGTTGGCATTG
1,000
SEQ ID NO: 60




GCAACAGACGCC




 



N916172
GGTAAAATTATGCAAGTTCCCACGAAATTTGGCATATGA
1,500
SEQ ID NO: 61




AAGTGCCCTTAAAAATTAAGGTTT




 



N916129
GAGCTTTTATTTATGCTAACCTGTAACAATAAATTGTCTT
1,500
SEQ ID NO: 62




TGAGCATGGTTTGTTTGATGATCTCAATGACCG









The 53.4-cM region is a region that comprises 10 types of markers listed in table 6 below in the order shown in table 6 and is linked to a trait characterized by the reduction of smut resistance.











TABLE 6





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:







Ni9_14_1
N901178
ATCTACACAACAAATCCACTGTATTAGACGATTGTTATC
1,000
SEQ ID NO: 63




AAATGATCTTCCAGCAAATTGACATAATATGACATT




 



N918761
AGAACAGGGCCATCGTTGTTAGCGTGCGTGCTGTAA
1,500
SEQ ID NO: 64




GTTTGATTTAATTTAAAAAAAATACGTATA




 



N913735
ACGTACAAATGTTTGGGATGGCAGAGGACATGTAGTA
1,000
SEQ ID NO: 65




CAGGGTTGATTCTTTTCAATA




 



N900663
GCACCTCGCTCCTCCTTATCAAGTTTCGATTTCTGGAT
1,000
SEQ ID NO: 66




TTGCTGCTCTTG




 



N918363
AAGGCGAACAAATGATTCCCCTCAGTGACCTGAACGT
1,000
SEQ ID NO: 67




AATAGTAAAATGATACACACT




 



N918213
TCGCATGTCAGGGCTGACAAATGGCTAAAACCAGAC
1,500
SEQ ID NO: 68




GGAAGATAGACGGA




 



N900568
AACATCAGCTTAGTCTTTAGAGGTTATACCTGCTGTGC
1,000
SEQ ID NO: 69




TATTTTTTTTACTTAGTGTACACCATTCCTGA




 



N912523
CCTTAATCACGCTTGTGAAATATCACTCAAACCAACAA
1,500
SEQ ID NO: 70




TATCAATACCACCATTAATTATGCTTGTGAAATATGC




 



N900344
TTAAAGACTGAAAGAAACAATTATTGAATTAAAGAACA
1,500
SEQ ID NO: 71




ACTAGATAGAGAGCACTGGACTGAATGGTTGCAGA




 



N900802
ATCCCATCACAAAGGAAAGAATTGCACAAACAATGAC
1,500
SEQ ID NO: 72




GTGGTACCTTTAAAAGATAGAGAATGGAATAGA









The 38.0-cM region is a region that comprises 13 types of markers listed in table 7 below in the order shown in table 7 and is linked to a trait characterized by the improvement of smut resistance.











TABLE 7





Linkage
Marker

Signal



group
name
Nucleotide sequence information
threshold
SEQ ID NO:







Ni9_14_2
N901524
AAGCAACAGATGACTAGAAGTACAGTGCAGGAGACT
1,000
SEQ ID NO: 73




CCAACACTTTACTATATTAGTAGAAGA




 



N901163
TCTTCAGTTCATATCTATCATCTATCCGTCGCTCGTTTC
1,000
SEQ ID NO: 74




ATGAGACAGATCAAATAAGCAGAT




 



N911063
TTCGAGAATGAGCGCATTAGCACAAGGTTTAATTTCAT
1,000
SEQ ID NO: 75




TAATCACTTTAGGTATCTAGTTAGGTGTGTGT




 



N914692
CGCCCACCAATGCATTACCCAATGGGGTACCCGATGC
1,500
SEQ ID NO: 76




CGCCCCATTCGCA




 



N911405
GTGCAGGGTACCCGTCAATGGGCTACGGCTATGGCC
1,000
SEQ ID NO: 77




GCCCACCAATGCAT




 



N913383
AAGATAAATTTACAAGCAAAATTAGAATGTCAAATACCA
1,000
SEQ ID NO: 78




CAAATATTGAGAGCTGTGCCTGACAATTGAGGAGA




 



N914112
AGCTGTGCCTGACAATTGAGAGTGAACAGAGTACATT
1,000
SEQ ID NO: 79




TCATACTGCCCAG




 



N915180
TCCGGAGATTACAACGTCTTCAGTGACGAGAACCCG
1,000
SEQ ID NO: 80




AACAGCTGCTCGGT




 



N901160
CCCCTGACACGATATTTATTTGCCAGAATTTATGAATTA
1,500
SEQ ID NO: 81




CAGCCGCATTTCGTTGTGT




 



N916293
TTGGCAATCATCGACTAATTAGGTGTAAAAGATTCGTC
1,000
SEQ ID NO: 82




TTGTTATTTTCTACCAAATTATGAAATTTA




 



N916263
TATAGGGCCAGATAAACCATGATAATCATAGGATATTTG
1,000
SEQ ID NO: 83




CAGAAATCTTAAATTTCTGAGATTGCCAACAGAAGA




 



N917579
TATGGATCTTCCAGTTGATTACTGTTCTTTCGCTCCGC
1,000
SEQ ID NO: 84




TTTTTGCTTTTTTACTCGTGA




 



N918080
TACTCGTGAGGGTCCATCTATGACCTATCCTGTGTTCT
1,000
SEQ ID NO: 85




TTACTAGCGAAA









In addition, in tables 1 to 7, “Linkage group” represents the number given to each group among a plurality of linkage groups specified by QTL analysis. In tables 1 to 7, “Marker name” represents the name given to each marker originally obtained in the present invention. In tables 1 to 7, “Signal threshold” represents a threshold used for determination of the presence or absence of a marker.
In addition, the peak contained in the 18.7-cM region is present in a region sandwiched between a marker (N804607) consisting of the nucleotide sequence shown in SEQ ID NO: 5 and a marker (N815502) consisting of the nucleotide sequence shown in SEQ ID NO: 9. The peak contained in the 39.2-cM region is present in a region sandwiched between a marker (N803928) consisting of the nucleotide sequence shown in SEQ ID NO: 18 and a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22. The peak contained in the 19.2-cM region is present in a region sandwiched between a marker (N829378) consisting of the nucleotide sequence shown in SEQ ID NO: 25 and a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32. The peak contained in the 32.0-cM region is present in a region sandwiched between a marker (N915070) consisting of the nucleotide sequence shown in SEQ ID NO: 33 and a marker (N918161) consisting of the nucleotide sequence shown in SEQ ID NO: 42. The peak contained in the 39.5-cM region is present in a region sandwiched between a marker (N911103) consisting of the nucleotide sequence shown in SEQ ID NO: 57 and a marker (N918344) consisting of the nucleotide sequence shown in SEQ ID NO: 59. The peak contained in the 53.4-cM region is present in a region sandwiched between a marker (N918761) consisting of the nucleotide sequence shown in SEQ ID NO: 64 and a marker (N900663) consisting of the nucleotide sequence shown in SEQ ID NO: 66. The peak contained in the 38.0-cM region is present in a region sandwiched between a marker (N901524) consisting of the nucleotide sequence shown in SEQ ID NO: 73 and a marker (N915180) consisting of the nucleotide sequence shown in SEQ ID NO: 80.
A continuous nucleic acid region existing in any of 7 regions containing markers shown in tables 1 to 7 can be used as a marker associated with resistance to sugarcane smut. 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 marker associated with resistance to sugarcane smut to a different region falls within the above range, the nucleic acid region can be specifically detected according to a standard method. The identity value 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 marker associated with resistance to sugarcane smut 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 marker associated with resistance to sugarcane smut 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 18.7-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 5 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: 5 and the nucleotide sequence shown in SEQ ID NO: 9. In addition, among the 8 types of markers contained in the 39.2-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 18 and the nucleotide sequence shown in SEQ ID NO: 22. Further, among the 10 types of markers contained in the 19.2-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 32. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 25 and the nucleotide sequence shown in SEQ ID NO: 30. Furthermore, among the 19 types of markers contained in the 32.0-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 33 and the nucleotide sequence shown in SEQ ID NO: 42. Moreover, among the 11 types of markers contained in the 39.5-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 57 and the nucleotide sequence shown in SEQ ID NO: 59. Among the 10 types of markers contained in the 53.4-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 64 and the nucleotide sequence shown in SEQ ID NO: 66. Among the 13 types of markers contained in the 38.0-cM region, a marker associated with resistance to sugarcane smut is preferably designated as existing in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 80. This is because the above peak is present in the region sandwiched between the nucleotide sequence shown in SEQ ID NO: 73 and the nucleotide sequence shown in SEQ ID NO: 80.
In addition, a nucleic acid region containing a single marker selected from among the 85 types of markers shown in tables 1 to 7 can be used as a marker associated with resistance to sugarcane smut. For example, it is preferable to use, as a marker associated with resistance to sugarcane smut, a nucleic acid region containing a marker (N802870) consisting of the nucleotide sequence shown in SEQ ID NO: 6 located closest to the peak position in the 18.7-cM region, a nucleic acid region containing a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22 located closest to the peak position in the 39.2-cM region, a nucleic acid region containing a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32 located closest to the peak position in the 19.2-cM region, a nucleic acid region containing a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 35 located closest to the peak position in the 32.0-cM region, a nucleic acid region containing a marker (N918508) consisting of the nucleotide sequence shown in SEQ ID NO: 58 located closest to the peak position in the 39.5-cM region, a nucleic acid region containing a marker (N913735) consisting of the nucleotide sequence shown in SEQ ID NO: 65 located closest to the peak position in the 53.4-cM region, or a nucleic acid region containing a marker (N901163) consisting of the nucleotide sequence shown in SEQ ID NO: 74 located closest to the peak position in the 38.0-cM region. In such case, the nucleotide sequence of a nucleic acid region containing the marker can be specified by flanking sequence analysis such as inverse PCR analysis using primers designed based on the nucleotide sequence of such marker.
Further, as a marker associated with resistance to sugarcane smut, any of the above 85 types of markers can be directly used. Specifically, one or more type(s) of markers selected from among the 85 types of such markers can be directly used as a marker associated with resistance to sugarcane smut. For example, it is preferable to use, as a marker associated with resistance to sugarcane smut, a marker (N802870) consisting of the nucleotide sequence shown in SEQ ID NO: 6 located closest to the peak position in the 18.7-cM region, a marker (N826906) consisting of the nucleotide sequence shown in SEQ ID NO: 22 located closest to the peak position in the 39.2-cM region, a marker (N821999) consisting of the nucleotide sequence shown in SEQ ID NO: 32 located closest to the peak position in the 19.2-cM region, a marker (N916186) consisting of the nucleotide sequence shown in SEQ ID NO: 35 located closest to the peak position in the 32.0-cM region, a marker (N918508) consisting of the nucleotide sequence shown in SEQ ID NO: 58 located closest to the peak position in the 39.5-cM region, a marker (N913735) consisting of the nucleotide sequence shown in SEQ ID NO: 65 located closest to the peak position in the 53.4-cM region, or a marker (N901163) consisting of the nucleotide sequence shown in SEQ ID NO: 74 located closest to the peak position in the 38.0-cM region.
<Sugarcane Marker Identification>
As described above, markers associated with resistance to sugarcane smut were identified from among 3004 markers and 4569 markers originally obtained from sugarcane chromosomes in the present invention. The 3004 markers and the 4569 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, the 3004 markers and the 4569 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 prepare and use, as an adaptor, adapter(s) corresponding to one or a part of 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.
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, when genomic DNA is digested using a plurality of restriction enzymes, followed by ligation of adapter(s) corresponding to one or a part of restriction enzyme(s) selected from among a plurality of the used restriction enzymes to genomic DNA fragments, 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, one or more 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.
A DNA microarray can be produced by designing many probes using genomic DNA extracted from sugarcane as described above 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, the 3004 markers and the 4569 markers, including the above 85 types of markers associated with resistance to sugarcane smut shown in SEQ ID NOS: 1 to 85, 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, the 3004 markers and the 4569 markers, including the aforementioned 85 types of markers associated with resistance to sugarcane smut shown in SEQ ID NOS: 1 to 85, were identified.
<Use of Markers Associated with Resistance to Sugarcane Smut>
The use of markers associated with resistance to sugarcane smut makes it possible to determine whether a sugarcane progeny line or the like, which has a phenotype exhibiting unknown smut resistance, is a line having a phenotype showing the improvement of smut resistance. The expression “the use of markers associated with resistance to sugarcane smut” used herein indicates the use of a DNA microarray having probes corresponding to markers associated with resistance to sugarcane smut in one embodiment. The expression “probes corresponding to markers associated with resistance to sugarcane smut” indicates oligonucleotides that can specifically hybridize under stringent conditions to markers associated with resistance to sugarcane smut 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 markers associated with resistance to sugarcane smut 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 as a carrier, 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 smut resistance, is a line having a phenotype showing the improvement of smut resistance. 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 smut resistance, is a line having a trait characterized by the improvement of smut resistance by detecting the above markers associated with resistance to sugarcane smut 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 smut resistance, and/or a sugarcane line used as a parent for producing a progeny line, and thus which can be used as a subject to be determined whether to have a trait characterized by the improvement of smut resistance or not. In addition, it is also possible to evaluate smut resistance in a sample plant which is a non-sugarcane plant such as a graminaceous plant (e.g., Sorghum or Erianthus).
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 markers associated with resistance to sugarcane smut 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.; 0.21 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 markers associated with resistance to sugarcane smut with the use of the DNA microarray. Here, as described above, as the marker associated with resistance to sugarcane smut, a marker linked to a trait characterized by the improvement of smut resistance and a marker linked to a trait characterized by the reduction of smut resistance are provided. Markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 are linked to a trait characterized by the improvement of smut resistance. Meanwhile, markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 are linked to a trait characterized by the reduction of smut resistance.
Therefore, if any one of the markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 is present in a sugarcane sample, it is possible to determine that the sample is of a variety with improved smut resistance. Further, if any one of the markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 is absent in a sugarcane sample, it is possible to determine that the sample is of a variety with improved smut resistance. Preferably, if any one of the markers associated with resistance to sugarcane smut designed based on the three aforementioned regions identified in tables 2, 4, and 7 is present in a sugarcane sample, and if any one of the markers associated with resistance to sugarcane smut designed based on the four aforementioned regions identified in tables 1, 3, 5, and 6 is absent in the sugarcane sample, it is possible to determine with high accuracy that the sample is of a variety with improved smut resistance.
In particular, according to the method described above, it is not necessary to cultivate sugarcane samples to such an extent that determination using an actual smut resistance test 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 sugarcane samples and other factors such as cost of cultivation can be significantly reduced with the use of the markers associated with resistance to sugarcane smut. In addition, the use of markers associated with resistance to sugarcane smut makes it possible to reduce the cost of facilities such as a large-scale special-purpose greenhouse, a special-purpose field, or isolation facility from an external environment, without the need to actually cause infection with a causative microorganism of smut (Ustilago scitaminea).
In particular, when a novel sugarcane variety is created, it is preferable to produce several tens of thousands of seedlings via crossing and then to identify a novel sugarcane variety using markers associated with resistance to sugarcane smut prior to or instead of seedling selection. The use of such markers associated with resistance to sugarcane smut 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.
Alternatively, upon creation of a new sugarcane variety, firstly, it may be determined whether or not a marker associated with resistance to sugarcane smut is present in a parent variety used for crossing, thereby allowing selection of a parent variety with excellent smut resistance. It can be expected that a progeny line with excellent smut resistance will be obtained with high frequency by creating a parent variety with excellent smut resistance on a priority basis. The use of such marker(s) associated with resistance to sugarcane smut makes it possible to significantly reduce the number of excellent lines that need to be cultivated. This allows drastic reduction of time-consuming efforts and the cost required to create a novel sugarcane variety.
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: 86) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 87)) 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. overnight. 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: 88)) 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 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: 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: 86) and 5′-CTGGATCCATCGTGCA-3′ (SEQ ID NO: 87)) 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. overnight. 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: 88)) 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-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 Smut Resistance and Development of Markers
(1) Creation of Genetic Map Datasheet
Genotype data of possible 3004 markers and 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 Smut Resistance Data
From Oct. 26 to 28, 2009, stalks of NiF8, Ni9, and the 191 hybrid progeny line were harvested. They were subjected to treatment for stimulating germination at room temperature and high humidity for 2 to 3 days, followed by wound inoculation with smut spores. For wound inoculation, wounds were made on both sides of buds (6 wounds in total; approximately 4.0 mm in depth), and then a spore suspension (107 to 108 spores/ml) was applied to the wounds using a brush. Smut spores in the spore suspension were collected from smut whips of Ni9 stocks naturally infected with smut, which were cultivated in Okinawa in 2009. Seedlings subjected to wound inoculation were cultivated for 2 to 3 days at room temperature and high humidity and planted in nursery boxes from Oct. 30 to Nov. 1, 2009 (40 buds/box, 2 boxes/line). The planted seedlings were cultivated at high humidity in a greenhouse until Sep. 2, 2010. The degree of the development of smut was investigated by counting, as the number of affected seedlings, the number of seedlings showing a symptom of smut, which is the outgrowth of a smut whip from the apex of a stalk. After the count of the affected seedlings, the plant bodies of affected seedlings were harvested at the ground level so that they could be removed. The number of seedlings affected with smut was investigated on Jun. 23, Jul. 21, Aug. 18, and Sep. 2, 2010 for a total of four instances. The incidence of smut was calculated as a percentage of the number of germinating stocks (excluding stocks killed by non-smut causes) accounted for by the number of affected stocks. FIGS. 3, 4, 5, and 6 show the study results of Jun. 23, 2010, the study results of Jul. 21, 2010, the study results of Aug. 18, 2010, and the study results of Sep. 2, 2010, respectively.
(3) Quantitative Trait (Quantitative Trait Loci: QTL) Analysis
Based on the genetic map datasheet obtained in (1) above and the smut resistance 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.; statgen.ncsu.edu/qticart/cartographer.html). Upon analysis, the LOD threshold was determined to be 2.5. As a result, as shown in FIGS. 7 to 12, the presence of QTL regarding sugarcane smut resistance was confirmed in the following seven ranges: the range between markers N827337 and N821515 present in the 5th linkage group (August 18), the range between markers N826561 and N826906 present in the 17th linkage group (June 23, July 21, and August 18), and the range between markers N816552 and N821999 present in the 40th linkage group (July 21, August 18, and September 2) of the NiF8 sugarcane variety; the range between markers N915070 and N920207 present in the 1st linkage group (July 21, August 18, and September 2), the range between markers N914284 and N916129 present in the 13th linkage group (July 21, August 18, and September 2), and the range between markers N901177 and N900802 (June 23) and the range between markers N901524 and N918080 (June 23 and July 21) present in the 14th linkage group of the Ni9 sugarcane variety. Specifically, peaks exceeding the LOD threshold were observed in the above seven ranges. It was possible to specify the obtained peaks as shown in table 8, suggesting the presence of a causative gene (or a group of causative genes) having the function of causing the improvement of smut resistance at the each peak positions. In addition, the “Effect (%)” column in table 8 indicates an increase or a decrease in the incidence of smut. Therefore, if the value of “Effect (%)” is negative, it means that the QTL (quantitative trait locus) is linked to a trait characterized by the improvement of smut resistance. If the value of “Effect (%)” is positive, it means that the QTL is linked to a trait characterized by the reduction of smut resistance.














TABLE 8







Investigation
Position
Range


Effect


Variety
Linkage group
date
(cM)
(cM)
Close marker
LOD score
(%)






















NiF8
5
8/18
3.8
18.7
N827337 - N821515
2.6
8.2


NiF8
17
6/23
94.3
39.2
N826561 - N826906
6.5
−16.5


NiF8
17
7/21
94.3
39.2
N826561 - N826906
3.6
−10.1


NiF8
17
8/18
94.3
39.2
N826561 - N826906
3.5
−9.7


NiF8
40
7/21
34.1
19.2
N816552 - N821999
3.3
9.6


NiF8
40
8/18
34.1
19.2
N816552 - N821999
3.0
8.9


NiF8
40
9/2
34.1
19.2
N816552 - N821999
3.4
8.4


Ni9
1
7/21
5.5
32.0
N915070 - N920207
2.8
−8.7


Ni9
1
8/18
5.5
32.0
N915070 - N920207
2.6
−8.3


Ni9
1
9/2
5.5
32.0
N915070 - N920207
3.3
−8.5


Ni9
13
7/21
12.9
39.5
N914284 - N916129
2.6
8.3


Ni9
13
8/18
12.9
39.5
N914284 - N916129
2.6
8.3


Ni9
13
9/2
12.9
39.5
N914284 - N916129
2.6
7.4


Ni9
14_1
6/23
97.3
53.4
N901178 - N900802
2.6
15.2


Ni9
14_2
6/23
137.7
38.0
N901524 - N918080
5.2
−21.3


Ni9
14_2
7/21
137.7
38.0
N901524 - N918080
4.7
−16.2









As shown in FIGS. 7 to 12, a marker located in the vicinity of the peak is inherited in linkage with a causative gene (or a group of causative genes) having the function of causing the improvement or reduction of smut resistance. This shows that the markers can be used as markers associated with resistance to sugarcane smut. Specifically, it has been revealed that the 85 types of markers shown in FIGS. 7 to 12 can be used as markers associated with resistance to sugarcane smut.
In addition, as examples of signals detected in 2 (6) above, table 9 shows signal levels of 14 types of markers among markers N827337 to N821515 present in the 5th linkage group of NiF8 for NiF8 and Ni9 and their progeny lines. In particular, the signal levels of N802870 are shown in FIG. 13.











TABLE 9





Linkage group
Marker name
NiF8
Ni9
F1






























NiF8_5
N827337
1,629
529
1,354
344
439
403
1,330
1,593
1,823
1,495
1,717
512
495
739



N802879
4,193
393
2,706
370
347
372
1,484
2,319
1,707
1,897
1,803
365
518
389



N804818
3,093
591
2,173
531
494
408
2,480
3,233
3,589
4,092
4,075
533
635
613



N816296
1,489
379
1,440
510
358
342
1,445
1,822
1,671
1,664
1,691
355
396
336



N804607
2,125
375
1,454
393
361
394
1,258
1,266
1,422
1,416
1,311
660
382
495



N802870
5,498
828
4,275
377
412
444
5,198
4,496
4,195
4,631
4,207
446
655
361



N813249
6,034
778
4,329
553
498
764
4,208
3,754
3,864
3,749
3,330
627
711
414



N813609
2,821
701
2,178
750
901
869
2,820
3,222
3,729
2,888
3,552
566
945
840



N815502
2,044
481
2,452
806
493
436
2,390
2,587
2,088
2,211
2,088
493
640
425



N815101
2,055
446
2,660
549
419
344
3,184
2,673
3,153
3,105
3,116
504
347
346



N823481
2,096
509
1,200
457
487
393
1,629
1,460
1,870
1,925
1,920
528
585
402



N801028
6,877
907
5,694
886
799
651
5,083
3,359
3,578
4,019
4,197
792
377
930



N810798
5,506
633
5,171
823
608
513
5,720
4,545
5,463
6,279
5,907
561
847
775



N821515
3,768
819
3,190
790
489
418
4,899
3,485
3,282
3,331
3,603
553
921
515









Signal levels of 14 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in NiF8. These results also revealed that 14 types of markers among markers N827337 to N821515 present in the 5th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 10 lists signal levels of 8 types of markers among markers N826561 to N826906 present in the 17th linkage group of NiF8 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N827136 are shown in FIG. 14.











TABLE 10





Linkage group
Marker name
NiF8
Ni9
F1






























NiF8_17
N826561
1,977
525
462
1,514
574
1,629
744
864
1,136
1,415
528
1,752
453
1,350



N827136
5,717
514
390
3,279
405
2,898
423
433
4,050
3,633
445
3,857
547
3,723



N826325
1,620
404
421
1,103
358
1,102
381
408
1,081
1,409
408
1,458
381
1,317



N803928
2,082
403
390
1,517
427
1,875
412
426
1,696
1,520
393
1,743
322
1,620



N822568
3,592
501
753
2,556
466
2,502
360
506
2,159
2,941
425
2,733
571
2,580



N829026
1,766
540
432
1,656
452
1,759
396
656
2,159
2,325
456
1,906
558
2,041



N815300
3,128
669
708
1,951
974
2,189
460
439
2,271
1,981
687
2,039
372
2,028



N826906
2,339
447
407
1,704
754
2,139
679
485
2,122
2,554
361
1,915
480
2,281









Signal levels of 8 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in NiF8. These results also revealed that 8 types of markers among markers N826561 to N826906 present in the 17th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 11 lists signal levels of 10 types of markers among markers N816552 to N821999 present in the 40th linkage group of NiF8 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N812680 are shown in FIG. 15.











TABLE 11





Linkage group
Marker name
NiF8
Ni9
F1






























NiF8_40
N816552
2,731
770
630
622
845
3,929
4,570
4,215
3,666
4,651
4,394
900
719
747



N827448
2,470
450
357
391
340
2,664
3,031
2,325
2,650
2,035
2,473
376
564
467



N829378
3,344
609
443
505
453
1,508
2,045
2,319
1,428
1,093
1,506
752
603
497



N829404
2,700
752
723
758
771
2,641
2,295
2,645
2,128
2,792
3,259
523
748
794



N828725
2,053
461
548
542
433
1,860
2,282
2,130
1,645
2,066
1,435
496
368
453



N812680
7,958
377
309
322
343
6,921
7,267
5,468
5,905
9,015
8,278
317
378
490



N811688
4,885
410
680
954
496
3,324
4,237
3,073
2,636
2,919
3,384
520
471
649



N819703
4,612
736
617
820
633
4,054
5,138
3,636
5,267
5,107
3,622
761
648
907



N815648
3,391
471
472
550
363
2,902
3,116
2,694
2,747
3,836
3,554
393
680
483



N821999
3,255
678
427
422
427
2,959
2,237
3,538
2,036
2,680
3,002
904
413
401









Signal levels of 10 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in NiF8. These results also revealed that 10 types of markers among markers N816552 to N821999 present in the 40th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 12 lists signal levels of 19 types of markers among markers N915070 to N920207 present in the 1st linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N916081 are shown in FIG. 16.











TABLE 12





Linkage
Marker





group
name
NiF8
Ni9
F1






























Ni9_1
N915070
424
1,195
418
1,393
1,122
1,717
480
1,356
1,359
1,707
370
424
424
403



N915209
560
1,796
435
2,840
1,776
3,016
601
2,376
2,361
3,451
446
541
462
484



N916186
496
2,002
403
2,447
1,808
1,571
415
2,608
1,723
2,687
406
671
466
432



N902342
372
1,245
349
1,049
1,003
1,045
420
1,062
1,346
2,206
333
352
401
398



N919949
625
1,459
406
2,169
1,942
2,723
738
1,679
2,360
3,490
650
680
444
572



N920597
450
4,702
399
5,028
3,819
5,583
348
6,733
4,669
7,196
436
431
393
537



N916081
516
13,678
954
16,011
14,893
10,082
634
10,528
10,441
11,232
504
785
435
604



N902047
955
5,233
658
4,400
3,853
4,711
825
3,378
5,336
6,194
581
992
555
854



N916874
491
3,320
486
2,511
2,869
3,276
708
2,304
3,046
4,073
416
791
409
430



N918161
438
2,109
411
1,989
1,892
2,109
397
1,690
2,193
2,643
406
610
398
335



N918536
372
1,059
508
1,229
1,293
1,368
487
1,253
1,704
1,967
511
423
395
381



N901676
648
1,534
702
2,407
1,395
1,389
705
1,590
1,820
1,918
521
577
483
582



N919743
635
2,361
437
1,703
1,731
1,990
471
2,385
2,076
3,665
568
417
399
398



N901176
697
5,017
408
3,009
5,027
5,059
820
5,316
3,362
3,347
764
454
715
420



N916035
757
4,444
684
3,088
3,803
3,576
580
4,310
4,270
4,272
448
585
485
581



N921010
521
5,630
448
6,214
5,012
7,792
909
5,074
4,902
5,904
557
658
559
611



N915635
424
7,875
538
12,542
10,900
15,388
568
9,698
10,501
14,732
391
505
400
469



N901348
493
3,188
558
6,692
7,451
6,466
605
3,553
7,406
2,655
659
584
638
438



N920207
421
5,291
349
4,550
4,857
6,695
385
1,962
3,567
11,697
449
478
416
450









Signal levels of 19 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in Ni9. These results also revealed that 19 types of markers among markers N915070 to N920207 present in the 1st linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 13 lists signal levels of 11 types of markers among markers N914284 to N916129 present in the 13th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N919839 are shown in FIG. 17.











TABLE 13





Linkage group
Marker name
NiF8
Ni9
F1






























Ni9_13
N914284
439
1,102
380
551
1,358
562
1,234
1,463
1,247
877
1,235
492
467
1,342



N901453
788
1,511
661
638
1,120
560
1,380
2,056
2,671
807
1,415
512
533
1,821



N900044
688
1,693
646
652
1,600
554
1,632
2,841
2,410
618
1,619
603
577
1,639



N919839
389
4,719
364
329
4,261
332
4,331
6,387
5,540
360
4,307
352
334
7,400



N901567
426
2,890
374
399
3,213
526
3,981
5,253
5,212
434
3,364
449
383
4,346



N911103
500
902
388
424
1,069
414
1,246
2,352
1,465
408
1,036
417
630
1,500



N918508
686
5,836
513
667
4,963
778
4,317
7,106
5,911
599
4,037
587
547
7,786



N918344
385
1,970
352
578
1,765
470
2,000
3,298
2,428
409
2,079
426
483
2,488



N919696
497
2,696
496
427
2,327
685
1,928
2,433
2,370
414
2,043
680
657
2,763



N916172
471
1,960
448
378
3,025
445
2,990
4,282
3,314
433
2,666
403
445
3,518



N916129
526
5,026
641
506
3,393
858
3,465
5,094
5,261
478
4,030
383
558
6,621









Signal levels of 11 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in Ni9. These results also revealed that 11 types of markers among markers N914284 to N916129 present in the 13th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 14 lists signal levels of 10 types of markers among markers N901178 to N900802 present in the 14th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N918761 are shown in FIG. 18.











TABLE 14





Linkage group
Marker name
NiF8
Ni9
F1






























Ni9_14_1
N901178
617
1,779
1,250
508
414
1,929
1,698
1,986
470
584
1,104
498
1,454
539



N918761
607
5,766
6,048
453
631
5,410
4,505
6,440
519
484
2,747
453
4,233
473



N913735
850
2,996
2,251
557
519
2,576
2,759
3,188
496
555
1,903
461
2,490
651



N900663
686
3,173
2,014
412
466
2,351
3,156
4,168
475
423
1,810
559
2,534
662



N918363
477
1,964
1,961
573
481
1,895
2,092
2,809
516
486
2,012
496
2,223
583



N918213
760
2,319
3,224
882
798
3,485
3,433
4,402
579
507
3,767
678
2,874
509



N900568
1,040
3,437
3,017
581
368
2,479
3,246
3,387
571
476
2,098
525
1,821
833



N912523
626
6,398
6,371
476
565
4,799
5,756
7,064
526
813
4,739
424
4,431
541



N900344
692
5,788
6,366
838
759
3,590
5,674
6,474
588
729
6,622
640
5,622
542



N900802
717
6,090
6,668
453
537
4,639
6,414
8,043
618
905
5,322
430
6,032
619









Signal levels of 10 types of markers were found to be remarkably high for progeny lines exhibiting reduction of smut resistance among the linkage groups present in Ni9. These results also revealed that 10 types of markers among markers N901178 to N900802 present in the 14th linkage group can be used as markers associated with resistance to sugarcane smut.
Similarly, table 15 lists signal levels of 13 types of markers among markers N901524 to N918080 present in the 14th linkage group of Ni9 in NiF8 and Ni9 and the progeny lines. In particular, the signal levels of N901160 are shown in FIG. 19.











TABLE 15





Linkage group
Marker name
NiF8
Ni9
F1






























Ni9_14_2
N901524
428
2,677
2,763
425
457
343
2,534
1,954
617
445
3,083
1,291
2,532
674



N901163
379
3,462
2,099
909
861
632
3,268
3,426
600
380
3,196
3,202
3,260
392



N911063
575
4,326
5,570
385
466
384
2,738
7,453
398
409
7,625
8,794
2,220
381



N914692
625
3,592
4,016
811
577
419
3,574
3,955
742
959
3,573
4,756
4,094
714



N911405
386
1,893
1,692
470
404
411
1,715
2,402
411
437
1,762
2,052
1,660
396



N913383
580
2,923
2,202
710
421
596
2,256
2,986
821
798
2,768
3,336
2,062
708



N914112
564
3,387
3,390
825
417
730
1,913
3,528
1,000
987
2,934
3,753
2,600
966



N915180
537
2,482
2,950
566
485
396
2,590
2,968
438
497
2,242
3,021
3,110
729



N901160
452
4,333
6,165
375
394
333
4,340
6,432
424
417
7,227
8,545
4,069
359



N916293
560
2,069
1,908
725
868
520
1,420
3,179
463
517
1,720
2,129
1,681
464



N916263
414
2,358
1,775
379
348
359
2,136
2,174
522
404
2,041
1,850
2,502
491



N917579
485
2,335
1,873
501
459
395
2,209
3,210
440
390
2,959
1,816
3,724
378



N918080
469
1,238
1,171
432
532
361
1,621
1,567
380
402
1,269
1,015
1,940
442









Signal levels of 13 types of markers were found to be remarkably high for progeny lines exhibiting excellent smut resistance among the linkage groups present in Ni9. These results also revealed that 13 types of markers among markers N901524 to N918080 present in the 14th linkage group can be used as markers associated with resistance to sugarcane smut.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A method for producing a sugarcane line having improved smut resistance, said method comprising:
extracting genomic DNA from a progeny plant obtained from parent plants, at least one of which is a sugarcane plant, and/or a genomic DNA of a parent sugarcane plant;
determining by nucleic acid assay the presence of a marker associated with resistance to sugarcane smut in the obtained genomic DNA, and selecting the progeny plant as a plant having improved smut resistance based on the presence of said marker; and
using the selected plant as a parent plant for crossing, to thereby produce progeny plant(s),
wherein said marker consists of a continuous nucleic acid region in the obtained genomic DNA, and wherein said marker comprises at least 20 continuous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs:5 to 9.

2. The method for producing a sugarcane line according to claim 1, wherein a DNA chip that comprises a probe corresponding to the marker associated with resistance to sugarcane smut is used in the determination step.

3. The method for producing a sugarcane line according to claim 1, wherein the progeny plant used in said genomic DNA extracting step is in the form of seeds or a young seedling and the genomic DNA is extracted from the seeds or the young seedling.

4. The method for producing a sugarcane line according to claim 1, wherein the progeny plant(s) produced using the selected plant as a parent plant for crossing is in the form of seeds or a young seedling.