JP2012115239A - Sugarcane-stalk-number-related marker and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sugarcane-stalk-number-related marker among the quantitative traits thereof, and a method for producing a sugarcane strain with increased stalk number.SOLUTION: The sugarcane-stalk-number-related marker comprises consecutive nucleic acid regions selected from regions sandwiched by specific base sequences in sugarcane chromosome. The method for producing a sugarcane strain with increased stalk number is also provided, comprising a step of extracting a chromosome of a progeny plant where at least one parent thereof is sugarcane; and a step of assaying the presence/absence of the stalk-number-related marker in the chromosome obtained by the preceding step. In this method, the progeny plant is in the form of seeds or juvenile seedlings, and the first step comprises extracting the chromosome from the seeds or juvenile seedlings.

Description

  The present invention relates to a stem number related marker capable of selecting a sugarcane line having a characteristic that the number of stems is increased, and a method of using the same.

  Sugarcane is cultivated for food, such as sugar raw materials and alcoholic beverage raw materials, and is used in various industrial fields including use as a biofuel raw material. Under such circumstances, desired properties (e.g., sugar content, growth potential, sprouting ability, disease resistance and insect resistance, cold resistance, increased leaf length and leaf area, increased stem length and number of stems) There is a need to develop a new variety of sugarcane with

  Generally, there are three methods for identifying plant varieties and lines: “characteristic comparison” comparing characteristic data, “comparative cultivation” cultivating and comparing under the same conditions, and “DNA analysis” analyzing DNA. Line identification by characteristic comparison and comparative cultivation has many problems, such as a long-term field survey that requires a reduction in accuracy and a large number of man-hours due to differences in cultivation conditions. In particular, sugarcane has a much larger plant body than other gramineous crops such as rice and corn, and it is difficult to carry out line identification by field survey. In addition, in order to identify varieties and lines having traits characteristic to traits such as leaf length, number of stems, and stem length, it is necessary to cultivate sugarcane for a long time and then collect these characteristic data. Even if sugarcane is cultivated for a long period of time, it is difficult to accurately identify these traits because they are easily affected by the environment.

  Furthermore, for the production of new sugarcane varieties, first, tens of thousands of hybrids are produced by crossing, seedlings are selected therefrom, and more excellent strains are selected step by step, and finally 2 to 2 having the desired characteristics. Three new varieties can be obtained. Thus, in order to produce a new variety of sugarcane, it is necessary to cultivate and evaluate a very large number of lines, and it is necessary to prepare a large-scale field and take a lot of time and effort.

  Therefore, development of a method for identifying a sugarcane line having a desired characteristic using a marker present in the genome is required. In particular, when a new variety in sugarcane is produced, if a marker excellent in various characteristics can be used, the above-mentioned problems unique to sugarcane can be avoided, which can be a very effective tool. However, sugarcane is highly polyploidy and has a large number of chromosomes (about 100 to 130), so the development of marker technology is delayed. In sugarcane, although USDA reports on genotyping using SSR markers (Non-Patent Document 1), the accuracy is low due to the small number of markers and the number of polymorphisms from each marker, and the scope of application is American.・ Because it is limited to Australian varieties, it cannot be used for system identification of major varieties and useful genetic resources in Japan and Taiwan / India.

  Non-Patent Document 2 suggests the possibility of creating a genetic map in sugarcane by increasing the number of markers, comparing the characteristics of each marker, and verifying them. However, Non-Patent Document 2 does not disclose a sufficient number of markers, and no marker linked to the target characteristics has been found.

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

  Therefore, an object of the present invention is to provide a marker related to the number of stems among quantitative characters of sugarcane.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have prepared a large number of markers in sugarcane, and the number of stems is increased by the linkage analysis between quantitative traits and markers in the progeny of the hybrid. A marker linked to a quantitative trait was found and the present invention was completed.

The present invention includes the following.
[1] A region sandwiched between the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 10, a region sandwiched by the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 29 in the sugarcane chromosome, SEQ ID NO: 30 A sugarcane stalk comprising a continuous nucleic acid region selected from a region sandwiched between the base sequence shown in FIG. Number related marker.
[2] The sugarcane stem number-related marker according to [1], wherein the nucleic acid region includes any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 47.
[3] The nucleic acid region comprises a region sandwiched between the base sequence shown in SEQ ID NO: 4 and the base sequence shown in SEQ ID NO: 6 in the sugarcane chromosome, the base sequence shown in SEQ ID NO: 21 and the base sequence shown in SEQ ID NO: 23 It is located in a region sandwiched between a base sequence shown in SEQ ID NO: 30 and a base sequence shown in SEQ ID NO: 32, or a region sandwiched between a base sequence shown in SEQ ID NO: 45 and a base sequence shown in SEQ ID NO: 46 [1] The number-related marker of stems.

[4] extracting a chromosome of a progeny plant in which at least one parent is sugarcane;
A method for producing a sugarcane line having an increased number of stems, comprising the step of measuring the presence / absence of the stem number-related marker according to any one of [1] to [3] in the chromosome obtained above.
[5] The method for producing a sugarcane line according to [4], wherein a DNA chip including a probe corresponding to the stem number related marker is used in the measuring step.
[6] The method for producing a sugarcane line according to [4], wherein the progeny plant is a seed or a seedling, and a chromosome is extracted from the seed or the seedling.

  ADVANTAGE OF THE INVENTION According to this invention, the novel sugarcane-stalk-number-related marker linked with the characteristic that the number of stems increases among the quantitative characters in sugarcane can be provided. By using the sugarcane-stalk-number-related marker according to the present invention, the number of stems in a sugarcane mating line can be identified. Thereby, the sugarcane line | wire which has the characteristic that the number of stems increased can be identified at a very low cost.

It is a schematic diagram which shows the manufacture flow of the DNA microarray used when obtaining the marker of a sugarcane chromosome. It is a schematic diagram which shows the process of the signal detection using a DNA microarray. It is a characteristic view which shows raw material stem number data about the sugarcane varieties and the line | group group used in the Example. It is a characteristic view which shows the result (2nd linkage group) of the QTL analysis regarding the number of raw material stems in sugarcane variety NiF8. It is a characteristic view which shows the result (1st linkage group) of the QTL analysis regarding the number of raw material stems in sugarcane variety Ni9. It is a characteristic view which shows the result (46th linkage group) of the QTL analysis regarding the number of raw material stems in sugarcane variety Ni9. It is a characteristic view which shows the result (73rd linkage group) of the QTL analysis regarding the number of raw material stems in sugarcane variety Ni9. It is a characteristic view which shows the signal value of N812115 (NiF8 2nd linkage group marker) in each system | strain. It is a characteristic view which shows the signal value of N918161 (the marker of Ni9 1st linkage group) for every system | strain. It is a characteristic view which shows the signal value of N916624 (Ni9 46th linkage group marker) in each line. It is a characteristic view which shows the signal value of N910631 (the marker of Ni9 73rd linkage group) for every system | strain.

  Hereinafter, a sugarcane-stalk-number-related marker and a method for using the same according to the present invention, particularly, a method for producing a sugarcane line using a sugarcane-stalk-number-related marker will be described.

Sugarcane stem number related marker The sugarcane stem number related marker according to the present invention is a specific region existing on the sugarcane chromosome, and is linked to a causative gene (group) of a trait that increases the number of sugarcane stems. It has the function of distinguishing the trait of increasing the number of sugarcane stems. That is, in a progeny line obtained using a known sugarcane line, it is possible to determine that the line has an increase in the number of stems by confirming the presence of a sugarcane stem number related marker.

  Here, the number of stems means the number of stems per sugarcane individual. In addition, in the Example in this specification, the number of raw material stems is used as the number of stems. “Number of raw material stems” means the number of raw material stems per unit area (for example, 1a (R)). Here, `` raw material stem '' is a portion of the stem that is used as a raw material when producing raw sugar, etc., and is a stem after removing low-sugar tops, leaves, roots, etc. from untreated sugarcane stems, In general, it refers to those of 1 m or more. The number of stems and the number of raw material stems have a correlation. If the number of raw material stems is increased, it can be determined that the number of stems is also increased.

  Sugarcane means a plant belonging to the genus Sugarcane. Moreover, sugarcane means to include both so-called noble species (scientific name: Saccharum officinarum) and wild species (scientific name: Saccharum spontaneum). Known sugarcane varieties / lines are not particularly limited, and include all varieties / lines usable in Japan, varieties / lines used outside Japan, and the like. For example, sugarcane domestic breeds are not particularly limited, but Ni1, NiN2, NiF3, NiF4, NiF5, Ni6, NiN7, NiF8, Ni9, NiTn10, Ni11, Ni12, Ni14, Ni15, Ni16, Ni17, NiTn19, NiTn20 , Ni22, Ni23, and the like. Further, the main sugarcane varieties in Japan are not particularly limited, and examples thereof include NiF8, Ni9, NiTn10, and Ni15. Furthermore, the main varieties introduced into sugarcane in Japan are not particularly limited, and examples include F177, Nco310, and F172.

  In addition, the progeny line may be a homozygous line in which both mother and father are sugarcane varieties / lines, either one is sugarcane varieties / lines and the other is closely related Erianthus arundinaceus Such a hybrid system may be used. The progeny varieties may be those obtained by so-called backcrossing.

  Sugarcane stem number-related markers include a gene linkage map containing 3004 markers uniquely acquired from the chromosome of sugarcane variety NiF8 and a gene linkage map containing 4569 markers uniquely acquired from the chromosome of sugarcane variety Ni9 and sugarcane stem It was newly identified by QTL (Quantitative Trait Loci) analysis using numerical data. The number of sugarcane stems is a quantitative trait that is considered to involve many genes and takes a continuous distribution. Genetic analysis software QTL Cartographer (Wang S., CJ Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) is used for QTL analysis. , Composite interval mapping (CIM) method is applied.

  Specifically, by the above-described QTL analysis, peaks having a rod score (LOD score) equal to or higher than a predetermined threshold (for example, 3.0) were found in the four regions included in the respective gene linkage maps. Specifically, about 48.6 cM (centiorgan) region containing the peak (sugar cane variety NiF8) and about 32.0 cM region, about 33.7 cM region and about 30.4 cM region (cane variety Ni9) containing the peak were identified. . Here, “Morgan (M)” is a unit that relatively indicates the distance between genes on a chromosome, and is a value obtained by setting the cross value as a percentage. In the sugarcane chromosome, 1 cM corresponds to about 2000 kb. In addition, it is suggested that the causal gene (group) of the trait which increases the number of stems exists in this peak position or its vicinity.

  The 48.6 cM region containing the above peak of sugarcane variety NiF8 is a region in which the 10 types of markers shown in Table 1 are included in this order.

  The 32.0 cM region containing the above peak of sugarcane variety Ni9 is a region containing 19 types of markers shown in Table 2 in this order.

  The 33.7 cM region including the above-mentioned peak of sugarcane variety Ni9 is a region including the 12 types of markers shown in Table 3 in this order.

  The region of 30.4 cM including the above peak of sugarcane variety Ni9 is a region including 12 kinds of markers shown in Table 4 in this order among the above markers.

  In Table 1-4, a linkage group is a number assigned to each of a plurality of linkage groups identified in the QTL analysis. In Table 1-4, the marker name is a name given to the marker uniquely acquired in the present invention. In Table 1-4, the signal threshold is a threshold for determining the presence or absence of a marker.

  The peak contained in the above 48.6 cM region of sugarcane variety NiF8 is present in a region sandwiched between a marker (N812269) consisting of the base sequence shown in SEQ ID NO: 4 and a marker (N825528) consisting of the base sequence shown in SEQ ID NO: 6. Yes.

  Further, the peak contained in the 32.0 cM region of sugarcane variety Ni9 is present in a region sandwiched between a marker (N918536) consisting of the base sequence shown in SEQ ID NO: 21 and a marker (N919743) consisting of the base sequence shown in SEQ ID NO: 23 is doing.

  Furthermore, the peak contained in the 33.7 cM region of sugarcane variety Ni9 is present in a region sandwiched by a marker (N917999) consisting of the base sequence shown in SEQ ID NO: 30 and a marker (N917862) consisting of the base sequence shown in SEQ ID NO: 32 is doing.

  Furthermore, the peak contained in the 30.4 cM region of the sugarcane variety Ni9 is located in the region sandwiched by the marker (N912675) consisting of the base sequence shown in SEQ ID NO: 45 and the marker (N910631) consisting of the base sequence shown in SEQ ID NO: 46. Existing.

  A continuous nucleic acid region selected from the four regions containing the markers shown in Table 1-4 can be used as a sugarcane-stalk-number-related marker. Here, the nucleic acid region is a base whose identity with other regions existing in the sugarcane chromosome is 95% or less, preferably 90% or less, more preferably 80% or less, and most preferably 70% or less. An area consisting of an array. If the identity of the nucleic acid region serving as a sugarcane-stalk-number-related marker and other regions is in the above range, the nucleic acid region can be specifically detected according to a standard method. Here, the identity value can be calculated using default parameters using, for example, BLAST.

  In addition, the base length of the nucleic acid region serving as a sugarcane-stalk-number-related marker can be at least 8 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and most preferably 30 bases in length. If the base length of the nucleic acid region serving as a sugarcane-stalk-number-related marker is in the above range, the nucleic acid region can be specifically detected according to a conventional method.

  In particular, as a sugarcane-stalk-number-related marker, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence shown in SEQ ID NO: 6 among the 10 types of markers contained in the above 48.6 cM region of sugarcane variety NiF8 Is preferably selected from. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 4 and the base sequence shown in SEQ ID NO: 6. Moreover, as a sugarcane-stalk-number-related marker, among 19 types of markers included in the 32.0 cM region of sugarcane variety Ni9, a region sandwiched between the base sequence shown in SEQ ID NO: 21 and the base sequence shown in SEQ ID NO: 23 Is preferably selected from. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 21 and the base sequence shown in SEQ ID NO: 23. Furthermore, as a sugarcane-stalk-number-related marker, a region sandwiched between the nucleotide sequence shown in SEQ ID NO: 30 and the nucleotide sequence shown in SEQ ID NO: 32 among the 12 types of markers contained in the 33.7 cM region of the sugarcane variety Ni9 Is preferably selected from. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 30 and the base sequence shown in SEQ ID NO: 32. Furthermore, as the sugarcane-stalk-number-related marker, among the six types of markers included in the 30.4 cM region of sugarcane variety Ni9, the marker is sandwiched between the nucleotide sequence shown in SEQ ID NO: 45 and the nucleotide sequence shown in SEQ ID NO: 46. It is preferably selected from the region. This is because the peak exists in a region sandwiched between the base sequence shown in SEQ ID NO: 45 and the base sequence shown in SEQ ID NO: 46.

  Further, the sugarcane-stalk-number-related marker can be a nucleic acid region containing one type of marker selected from the 47 types of markers shown in Table 1-4 above. For example, as a sugarcane stem number-related marker, a nucleic acid region comprising a marker (N812269) consisting of the base sequence shown in SEQ ID NO: 4 closest to the peak position contained in the above 48.6 cM region of sugarcane variety NiF8, sugarcane variety Ni9 The nucleic acid region containing the marker (N901676) consisting of the base sequence shown in SEQ ID NO: 22 closest to the peak position contained in the above 32.0 cM region, and the peak position contained in the above 33.7 cM region of sugarcane variety Ni9 A marker (N912675) consisting of a nucleic acid region comprising a marker (N919703) consisting of a base sequence shown in the closest SEQ ID NO: 31 and a nucleotide sequence shown in SEQ ID NO: 45 closest to the position of the peak contained in the 30.4 cM region of the sugarcane variety Ni9 It is preferred to use a nucleic acid region comprising At this time, the base sequence of the nucleic acid region containing the marker can be identified by inverse PCR using a primer designed based on the base sequence of the marker.

  Furthermore, as the sugarcane-stalk-number-related markers, the above 47 types of markers themselves can be used. That is, one or more of these 47 types of markers can be used as sugarcane-stalk-number-related markers. For example, as a sugarcane stem number related marker, a marker (N812269) consisting of the base sequence shown in SEQ ID NO: 4 closest to the peak position contained in the above 48.6 cM region of the sugarcane variety NiF8, the above 32.0 cM of the sugarcane variety Ni9 A marker (N901676) consisting of the base sequence shown in SEQ ID NO: 22 closest to the position of the peak contained in the region of NO, and the base sequence shown in SEQ ID NO: 31 closest to the position of the peak contained in the 33.7 cM region of sugarcane variety Ni9 It is preferable to use the marker (N919703) consisting of the nucleotide sequence shown in SEQ ID NO: 45 closest to the position of the peak contained in the 30.4 cM region of the sugarcane variety Ni9.

Identification of markers in sugarcane In the present invention, as described above, sugarcane stem number related markers from 3004 markers uniquely acquired from the chromosome of sugarcane variety NiF8 and 4569 markers uniquely acquired from the chromosome of sugarcane variety Ni9 Identified. Here, these markers will be described. In identifying this marker, a DNA microarray to which the method disclosed in Japanese Patent Application No. 2009-283430 is applied can be used.

  Specifically, a DNA microarray having a probe designed by the method disclosed in Japanese Patent Application No. 2009-283430 is used for these markers uniquely obtained from the sugarcane chromosome. As shown in FIG. 1, the probe design method first extracts genomic DNA from sugarcane (step 1a). Next, the extracted genomic DNA is digested with one or more restriction enzymes (step 1b). In the example shown in FIG. 1, genomic DNA is digested using two types of restriction enzymes, restriction enzyme A and restriction enzyme B, in this order. Here, the restriction enzyme is not particularly limited, and for example, PstI, EcoRI, HindIII, BstNI, HpaII, HaeIII and the like can be used. In particular, the restriction enzyme can be appropriately selected in consideration of the appearance frequency of the recognition sequence so that a genomic DNA fragment having a length of 20 to 10,000 bases is obtained when the genomic DNA is completely digested. Moreover, when using a some restriction enzyme, it is preferable that the genomic DNA fragment after using all the restriction enzymes is 200-6000 base length. Furthermore, when using a plurality of restriction enzymes, the order of restriction enzymes to be used for the treatment is not particularly limited, and when the processing conditions (solution composition, temperature, etc.) are common, a plurality of restriction enzymes are used in the same reaction system. May be used in That is, in the example shown in FIG. 1, restriction enzyme A and restriction enzyme B are used in this order to digest genomic DNA, but restriction enzyme A and restriction enzyme B are simultaneously used in the same reaction system. Genomic DNA may be digested, or genomic DNA may be digested using restriction enzyme B and restriction enzyme A in this order. Further, the number of restriction enzymes used may be 3 or more.

  Next, an adapter is bound to the genomic DNA fragment after the restriction enzyme treatment (step 1c). Here, the adapter is not particularly limited as long as it can bind to both ends of the genomic DNA fragment obtained by the restriction enzyme treatment described above. As an adapter, for example, a primer that has a single strand complementary to the protruding ends (sticky ends) formed at both ends of genomic DNA by restriction enzyme treatment, and is used in the amplification process described in detail later Having a primer binding sequence capable of hybridizing can be used. As the adapter, one having a single strand complementary to the protruding end (adhesive end) and having a restriction enzyme recognition site for incorporation into a vector for cloning can also be used.

  When genomic DNA is digested using a plurality of restriction enzymes, a plurality of adapters corresponding to each restriction enzyme can be prepared and used. That is, a plurality of adapters having a single strand complementary to each of a plurality of types of protruding ends generated when genomic DNA is digested with a plurality of restriction enzymes can be used. At this time, the plurality of adapters corresponding to the plurality of restriction enzymes may have a common primer binding sequence so that a common primer can hybridize, or different primers can hybridize with each other. May have different primer binding sequences.

  Furthermore, when digesting genomic DNA using a plurality of restriction enzymes, the adapter may be one restriction enzyme selected from the plurality of restriction enzymes used or a part of the restriction enzymes used. A corresponding adapter can also be prepared and used.

  Next, a genomic DNA fragment with adapters added to both ends is amplified (step 1d). When an adapter having a primer binding sequence is used, the genomic DNA fragment can be amplified by using a primer that can hybridize to the primer binding sequence. Alternatively, a genomic DNA fragment to which an adapter has been added can be cloned into a vector using the adapter sequence, and the genomic DNA fragment can be amplified using a primer that can hybridize to a predetermined region in the vector. In addition, PCR can be used as an example of the amplification reaction of a genomic DNA fragment using a primer.

  In addition, when digesting genomic DNA using multiple restriction enzymes and ligating multiple adapters corresponding to each restriction enzyme to genomic DNA fragments, genomic DNA fragments obtained by treatment with multiple restriction enzymes An adapter will be connected to all of these. In this case, all the genomic DNA fragments obtained can be amplified by performing a nucleic acid amplification reaction using the primer binding sequence contained in the adapter.

  Alternatively, genomic DNA is digested using a plurality of restriction enzymes, and one restriction enzyme selected from the plurality of restriction enzymes used or an adapter corresponding to a part of the restriction enzymes used is used. When ligated to a genomic DNA fragment, only the genomic DNA fragment having a recognition sequence of a selected restriction enzyme at both ends can be amplified from the obtained genomic DNA fragment.

  Next, the base sequence of the amplified genomic DNA fragment is determined (step 1e), and one or more regions having a base length shorter than the genomic DNA fragment and covering at least a part of the genomic DNA fragment are identified. Then, the specified region or regions are designed as a sugar cane probe (step 1f). The method for determining the base sequence of the genomic DNA fragment is not particularly limited, and a conventionally known method using a DNA sequencer to which the Sanger method or the like is applied can be used. Here, the region to be designed is, for example, 20 to 100 bases long, preferably 30 to 90 bases long, more preferably 50 to 75 bases long as described above.

  As described above, a large number of probes are designed using genomic DNA extracted from sugarcane, and an oligonucleotide having a target base sequence is synthesized on a carrier based on the base sequence of the designed probe. Microarrays can be made. By using the DNA microarray thus prepared, 3004 and 4569 markers including 47 types of sugarcane stem number-related markers shown in SEQ ID NOs: 1 to 47 described above were obtained from sugarcane varieties NiF8 and Ni9, respectively. Can be identified.

  More specifically, the present inventors obtained signal data for the known sugarcane varieties NiF8 and Ni9 and their progeny lines (191 lines) using the DNA microarray described above. Then, genotype data is obtained from the obtained signal data, and based on this genotype data, genetic map creation software AntMap (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm Breed Sci 56: 371-378), the position information of the marker on the chromosome was calculated by the genetic distance calculation formula Kosambi. Furthermore, based on the acquired marker position information, a genetic map data sheet was prepared by 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 47 sugarcane stem number related markers shown in SEQ ID NOs: 1-47 were identified from sugarcane varieties NiF8 and Ni9, respectively.

Use of sugarcane stalk number-related markers By using sugarcane stalk number-related markers, it is judged whether the phenotype of sugarcane lines whose phenotype is unknown in progeny strains, etc. be able to. Here, the use of a sugarcane-stalk-number-related marker means that a DNA microarray having a probe corresponding to the marker is used. The probe corresponding to the sugarcane-stalk-number-related marker means an oligonucleotide that can specifically hybridize under stringent conditions to the sugarcane-stalk-number-related marker defined above. Such oligonucleotides are, for example, at least 10 bases, 15 bases, 20 bases, 25 bases, 30 bases, 35 bases of the base sequence of the sugarcane stem number-related marker defined as described above or the complementary strand thereof. , 40 bases, 45 bases, 50 bases or more, and can be designed as a partial region or the entire region. The DNA microarray having the probe may be any microarray using a flat substrate such as glass or silicone as a carrier, a bead array using a microbead as a carrier, or a three-dimensional microarray in which a probe is fixed to the inner wall of a hollow fiber. It may be a type of microarray.

  By using the DNA microarray prepared as described above, to determine whether the phenotype of the number of stems typified by progeny strains etc. is a strain that exhibits a phenotype of increased stem number Can do. In addition to the method using the above-described DNA microarray, the sugarcane-stalk-number-related marker described above is detected using a conventionally known method, and the number of stems is determined for a sugarcane line whose phenotype is unknown. It may be determined whether the strain has a trait of increase.

  The method of using the DNA microarray will be described in more detail. As shown in FIG. 2, first, genomic DNA is extracted from the test sugarcane. This test sugarcane is a sugarcane line whose phenotype of the number of stems is unknown, such as a progeny line, and is a sugarcane line for which it is determined whether or not it has a trait that increases the number of stems. Next, the extracted genomic DNA is digested with the restriction enzymes used in preparing the DNA microarray to prepare a plurality of genomic DNA fragments. Next, the obtained genomic DNA fragment is ligated to the adapter used in preparing the DNA microarray. Next, the genomic DNA fragment with adapters added to both ends is amplified using the primers used in preparing the DNA microarray. As a result, the test sugarcane-derived genomic DNA fragment corresponding to the genomic DNA fragment amplified in step 1d when preparing the DNA microarray can be amplified.

  In this step, a predetermined genomic DNA fragment may be selectively amplified among the genomic DNA fragments to which the adapter is added. For example, when a plurality of adapters corresponding to a plurality of restriction enzymes are used, a genomic DNA fragment to which a specific adapter is added can be selectively amplified. In addition, when genomic DNA is digested with multiple restriction enzymes, the adapter is added by adding the adapter only to the genomic DNA fragment having a protruding end corresponding to the predetermined restriction enzyme. Genomic DNA fragments can be selectively amplified. Thus, it can concentrate by selectively amplifying a predetermined genomic DNA fragment.

  Next, a label is added to the amplified genomic DNA fragment. Any conventionally known substance may be used as the label. As the label, for example, fluorescent molecules, dye molecules, radioactive molecules and the like can be used. This step can be omitted by using a nucleotide having a label in the step of amplifying the genomic DNA fragment. This is because the amplified DNA fragment is labeled by amplifying the genomic DNA fragment using a nucleotide having a label in the above step.

  Next, the genomic DNA fragment having the label is brought into contact with the DNA microarray under predetermined conditions, and the probe fixed to the DNA microarray and the genomic DNA fragment having the label are hybridized. At this time, it is preferable to use high stringency conditions for hybridization. By setting it as such a high stringency condition, it can be determined more accurately whether the sugarcane stem number related marker exists in the test sugarcane. Stringency conditions can be adjusted by reaction temperature and salt concentration. That is, higher stringency conditions are achieved at higher temperatures, and higher stringency conditions are achieved at lower salt concentrations. For example, when a probe having a length of 50 to 75 bases is used, higher stringency conditions can be obtained by setting the hybridization conditions to 40 to 44 ° C., 0.2 SDS, and 6 × SSC.

  Further, hybridization between the probe and a genomic DNA fragment having a label can be detected based on the label. That is, after the hybridization reaction of the genomic DNA fragment having the above-described label and the probe, the unreacted genomic DNA fragment is washed, and then the label of the genomic DNA fragment specifically hybridized to the probe is observed. . For example, when the label is a fluorescent substance, the fluorescence wavelength is detected, and when the label is a dye molecule, the dye wavelength is detected. More specifically, an apparatus such as a fluorescence detection apparatus or an image analyzer that is used for normal DNA microarray analysis can be used.

  As described above, by using the above-described DNA microarray, it is possible to determine whether or not the test sugarcane has the above-mentioned sugarcane stem number related marker. In particular, in the method described above, it is not necessary to grow the test sugarcane to such an extent that the actual number of stems can be measured. For example, seeds of progeny lines or seedlings germinated from the seeds can be used. Therefore, by using the sugarcane-stalk-number-related marker, the field for growing the test sugarcane and other costs for growth can be greatly reduced.

  In particular, when producing a new variety of sugarcane, first, after producing tens of thousands of hybrids by crossing, it is possible to make a determination using a sugarcane stem number related marker prior to seedling selection or instead of seedling selection. preferable. Thereby, in an actual field, the number which grows a good system | strain can be reduced significantly, and the effort and cost which relate to sugarcane new kind production can be suppressed significantly.

  By using the sugarcane-stalk-number-related marker, a causative gene or group of genes that increase the number of stems can be isolated. As an isolation method, a conventionally known method can be used ("Bio-Experiment Illustrated 4 Cloning without Difficulties" by Kazuhiro Makabe (1997), Shujunsha). For example, by producing a primer or probe corresponding to the sugarcane stem number-related marker defined above and screening the sugarcane genomic DNA or cDNA, the gene responsible for the trait that increases the number of stems can be isolated. Further, in place of the sugarcane genomic DNA or cDNA, by screening a genomic DNA or cDNA derived from other gramineous plants using a primer or probe corresponding to the sugarcane stem number related marker, It is also possible to isolate a causative gene or group of causative genes that increase the number of stems of the gramineous plant.

  In addition, a transformed plant body having a trait having an increased number of stems is produced by transforming a plant cell with a recombinant vector in which a gene causing a trait that increases the number of stems obtained above is incorporated. You can also.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

1. Creating DNA microarray probes
(1) Materials Sugarcane varieties: NiF8, Ni9, US56-15-8, POJ2878, Q165, R570, Co290 and B3439, sugarcane-related wild species: Glagah Kloet, Chunee, Natal Uba and Robustum9, and Eliansus: IJ76-349 And JW630 was used.

(2) Restriction enzyme treatment
Genomic DNA was extracted from each of these sugarcane varieties, wild relatives of sugarcane, and Eliansus using DNeasy Plant Mini Kit (QIAGEN). Genomic DNA (750 ng) was treated with restriction enzyme PstI (NEB, 25 units) at 37 ° C. for 2 hours, restriction enzyme BstNI (NEB, 25 unit) was added, and treated at 60 ° C. for 2 hours.

(3) Adapter ligation
The PstI sequence adapter (5'-CACGATGGATCCAGTGCA-3 '(SEQ ID NO: 48), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 49))) and T4 DNA Ligase (NEB) were added to the genomic DNA fragment (120ng) treated in (2). , 800 unit) was added, and the mixture was treated at 16 ° C. for 4 hours or longer. As a result, among the genomic DNA fragments treated in (2), adapters were selectively added to the genomic DNA fragments having PstI recognition sequences at both ends.

(4) PCR amplification
Add PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3 '(SEQ ID NO: 50)) and Taq polymerase (Takara Bio PrimeSTAR, 1.25 unit) to the genomic DNA fragment (15 ng) having the adapter obtained in (3) The genomic DNA fragment was amplified by PCR (98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1 minute, 30 cycles, treated at 72 ° C. for 3 minutes, and stored at 4 ° C.).

(5) Get genome sequence
The base sequence of the genomic DNA fragment PCR-amplified in (4) was determined by FLX454 (Roche) or the Sanger method. Moreover, the base sequence information inserted | pinched by the PstI recognition sequence was acquired from the sorghum whole genome sequence information stored in the genome database (Gramene: http: //www.gramene.org/).

(6) Probe design and creation of DNA microarray
Based on the genomic sequence information of (5), a 50-75 bp probe was designed. Based on the nucleotide sequence information of the designed probes, a DNA microarray having these probes was prepared.

2. Acquisition of signal data using DNA microarray
(1) Materials Sugarcane varieties and lines: NiF8 and Ni9 and 191 progenies of these crosses were used.

(2) Restriction enzyme treatment Genomic DNAs were extracted from these NiF8 and Ni9 and 191 progeny lines using DNeasy Plant Mini Kit (QIAGEN). Genomic DNA (750 ng) was treated with restriction enzyme PstI (NEB, 25 units) at 37 ° C. for 2 hours, restriction enzyme BstNI (NEB, 25 unit) was added, and treated at 60 ° C. for 2 hours.

(3) Adapter ligation
The PstI sequence adapter (5'-CACGATGGATCCAGTGCA-3 '(SEQ ID NO: 48), 5'-CTGGATCCATCGTGCA-3' (SEQ ID NO: 49))) and T4 DNA Ligase (NEB) were added to the genomic DNA fragment (120ng) treated in (2). , 800 unit) was added, and the mixture was treated at 16 ° C. for 4 hours or longer. As a result, among the genomic DNA fragments treated in (2), adapters were selectively added to the genomic DNA fragments having PstI recognition sequences at both ends.

(4) PCR amplification
Add PstI sequence adapter recognition primer (5'-GATGGATCCAGTGCAG-3 '(SEQ ID NO: 50)) and Taq polymerase (Takara Bio PrimeSTAR, 1.25 unit) to the genomic DNA fragment (15 ng) having the adapter obtained in (3) The genomic DNA fragment was amplified by PCR (98 ° C. for 10 seconds, 55 ° C. for 15 seconds, 72 ° C. for 1 minute, 30 cycles, treated at 72 ° C. for 3 minutes, and stored at 4 ° C.).

(5) Labeling After the PCR amplified fragment obtained in (4) above was purified with a column (QIAGEN), Cy3 9mer wobble (TriLink, 1O.D.) was added, treated at 98 ° C. for 10 minutes, Allowed to stand on ice for 10 minutes. Thereafter, Klenow (NEB, 100 unit) was added and the mixture was treated at 37 ° C. for 2 hours. And the labeled sample was prepared by isopropanol precipitation.

(6) Hybrid signal detection
Using the labeled sample of (5), according to NimbleGen Arrays User's Guide, 1. Hybridization was carried out using the DNA microarray prepared in step 1, and a signal based on the label was detected.

3. QTL identification and marker development of sugarcane raw material stem number
(1) Creation of genetic map data sheet 2. From the signal data of the sugarcane varieties NiF8 and Ni9 detected in step 1 and their progeny lines (191 lines), genotype data that can serve as 3004 markers derived from NiF8 and 4569 markers derived from Ni9 were obtained. Based on this genotype data, genetic map creation software AntMap (Iwata H, Ninomiya S (2006) AntMap: constructing genetic linkage maps using an ant colony optimization algorithm. Breed Sci 56: 371-378) The position information of the marker on the chromosome was calculated by the calculation formula Kosambi. Furthermore, based on the acquired marker position information, a genetic map data sheet was prepared by Mapmaker / EXP ver.3.0 (A Whitehead Institute for Biomedical Research Technical Report, Third Edition, January, 1993).

(2) Acquisition of raw material stem number data In April 2009, 13 sugarcane varieties: NiF8 and Ni9 and 191 progenies of the mating were planted in one section (2.2 m ² ). In March 2010, all stems were harvested for each plot. The harvested stalks were measured, and the number of stalks having a stem length of 1 m or more when adjusted to the raw material stalk was used as the raw material stalk number and used as the raw material stalk number data of each line. The measured number of raw material stems of each line is summarized in FIG. NiF8 is included in the data section of 1000 (lines / a) and Ni9 is included in the data section of 1100 (lines / a).

(3) Quantitative trait loci (QTL) analysis Based on the genetic map data sheet obtained in (1) above and the raw stem number data obtained in (2) above, genetic analysis software QTL Cartographer (Wang S., CJ Basten, and Z.-B. Zeng (2010). Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC) and QTL using the Composite interval mapping (CIM) method. Analysis was performed. At this time, the LOD threshold was set to 3.0. As a result, as shown in FIG. 4-7, in the section of markers N813145 to N812115 present in the second linkage group of sugarcane variety NiF8, in the section of markers N915070 to N920207 present in the first linkage group of sugarcane variety Ni9, Peaks exceeding the LOD threshold were obtained in the section of markers N917999 to N916624 in the 46th linkage group of sugarcane variety Ni9 and in the section of markers N919527 to N900970 in the 73rd linkage group of sugarcane variety Ni9. . The obtained peak can be identified as shown in Table 5, and it was suggested that a causal gene (group) having a function of increasing the number of raw material stems exists at the position of the peak.

  And as shown in FIGS. 4-7, since the marker located in the vicinity of the peak is linked to a causative gene (group) having a function of increasing the number of raw material stems, It was shown that it can be used. That is, it was revealed that the 47 types of markers shown in FIGS. 4-7 can be used as sugarcane raw material stem number related markers. The above 2. (6) As an example of signal detection in NiF8 and Ni9, and the 12th progeny lines (F1_1 to F1_12), markers N813145 to N812115 existing in the second linkage group of sugarcane variety NiF8, the first linkage group of sugarcane variety Ni9 47 markers included in markers N915070 to N920207, markers N917999 to N916624 present in the 46th linkage group of sugarcane variety Ni9, and markers N919527 to N900970 present in the 73rd linkage group of sugarcane variety Ni9 Are shown in Table 6-9, among which marker N812115 present in the second linkage group of sugarcane variety NiF8, marker N918161 present in the first linkage group of sugarcane variety Ni9, marker present in the 46th linkage group of sugarcane variety Ni9 The signal values of N916624 and marker N910631 in the 73rd linkage group of sugarcane variety Ni9 are shown in FIGS. 8-11, respectively.

  For the progeny lines F1_2, F1_5, F1_10, etc., which have a relatively large number of raw material stems, the signal values of these 49 kinds of markers were shown to be very high. From these results, markers N813145 to N812115 present in the second linkage group of sugarcane variety NiF8, markers N915070 to N920207 present in the first linkage group of sugarcane variety Ni9, and markers present in the 46th linkage group of sugarcane variety Ni9 It has been clarified that 47 types of markers included in markers N919527 to N900970 present in the 73rd linkage group of N917999 to N916624 and sugarcane cultivar Ni9 can be used as sugarcane raw material stem number related markers.

Claims (6)

  A region sandwiched by the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 10, a region sandwiched by the nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 29, and the base shown in SEQ ID NO: 30 in the sugarcane chromosome A sugarcane-stalk-number-related marker comprising a continuous nucleic acid region selected from a region sandwiched between a sequence and a base sequence shown in SEQ ID NO: 41, or a region sandwiched between a base sequence shown in SEQ ID NO: 42 and a base sequence shown in SEQ ID NO: 47 .   2. The sugarcane stem number-related marker according to claim 1, wherein the nucleic acid region comprises any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 47.   The nucleic acid region is a region sandwiched between the base sequence shown in SEQ ID NO: 4 and the base sequence shown in SEQ ID NO: 6 in the sugarcane chromosome, and the region sandwiched between the base sequence shown in SEQ ID NO: 21 and the base sequence shown in SEQ ID NO: 23 Characterized by being located in a region sandwiched between the base sequence shown in SEQ ID NO: 30 and the base sequence shown in SEQ ID NO: 32, or in a region sandwiched between the base sequence shown in SEQ ID NO: 45 and the base sequence shown in SEQ ID NO: 46 The stem number related marker according to claim 1. Extracting a chromosome of a progeny plant in which at least one parent is sugarcane;
A method for producing a sugarcane line having an increased number of stems, comprising the step of measuring the presence / absence of the stem number-related marker according to any one of claims 1 to 3 in the chromosome obtained above.   5. The method for producing a sugarcane line according to claim 4, wherein a DNA chip having a probe corresponding to the stem number related marker is used in the measuring step.   The method for producing a sugarcane line according to claim 4, wherein the progeny plant is a seed or a seedling, and a chromosome is extracted from the seed or the seedling.

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