JP2008212005A - Primer set for detecting dna marker linking to maize pleiophylly-associated gene locus, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer set for detecting a DNA marker linked to a gene locus instrumental in the increase in female ear upper leaves of maize, a method for discriminating the presence of a pleiophylly-associated gene locus, and to provide a method for preparing maize pleiophylly phylesis group. <P>SOLUTION: The primer set is for detecting a DNA marker linked to a pleiophylly-associated gene locus and detects a DNA marker linked to a pleiophylly-associated gene locus (lfy1) boarded on a third chromosome of maize. The method for discriminating the presence of a pleiophylly-associated gene locus of investigation individual comprises performing PCR with respect to a maize genome DNA by using the primer set, and examining the length of amplified fragment. The method for preparing a phylesis, having a large number of female ear upper leaves in its posterity includes selecting of a seedling of individual that has a pleiophylly-associated gene locus by the method described. By using these means, multilobed phylesis in a seedling stage can be selected. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a primer set for detecting a DNA marker linked to a corn leafy leaf related gene locus, a method for selecting a seedling of a leafy individual in the process of growing a corn leafy self-breeding line, and production of a corn leafy line This method relates to the method, and more specifically, PCR is performed on maize genomic DNA using a primer set that can detect a DNA marker linked to a multilobe-related locus, and the genotype is determined by examining the length of the amplified fragment. Thus, the present invention relates to a new seedling selection method for multileafy individuals and a method for producing a maize multileafy line, which enables selection of individuals having an improved number of upper leaves of ears at the seedling stage.

  In feed corn, which uses not only the seed but also the stem and leaves, an increase in the number of leaves is considered to be an important trait that leads to an increase in yield. In general, the number of leaves above the top ear of corn (the number of upper leaves of the ear) is 5 to 7 in the inbred line. However, a mutant having an increased number of upper ear leaves was found, and by introducing the trait, a multileaf inbred line having about 10 upper ear leaves was created.

  Several reports have shown that this trait is a dominant trait governed by a locus called the multilobe associated locus (lfy1), but where the locus is located on the chromosome. It is not clarified whether to ride (Non-Patent Document 1).

  The characteristics of the leafy leaf can only be identified after the appearance of the ear, but if it becomes possible to utilize a DNA marker linked to the leafy associated gene, Selection is possible, and it is also considered effective for homology of multileaf related genes.

Shaver, DL. , 38th Annual Corn and Sorghum Research Conference: 161-180 (1983)

In such a situation, in view of the above-mentioned prior art, the present inventors set the hybrid second generation (F ₂ ) whose parents are the maize multileaf line 957L and the non-leafy line Na12 as the analysis population. As a result of creating a linkage map using various DNA markers and performing QTL analysis, it succeeded in identifying that the multilobe-related locus (lfy1) sits on the third chromosome, and the vicinity of the locus The amplified fragment length polymorphism (AFLP) marker was converted to STS, and A4M48 was found. Furthermore, by adding a simple repeat sequence (SSR) marker around the leafy loci associated locus (lfy1) and creating a high-density linkage map, the lobe associated locus (lfy1) is closely related. The present inventors have found a linked SSR marker and further researched to complete the present invention.

  An object of the present invention is to provide a primer set that makes it possible to elucidate a locus related to the number of upper leaves of maize ears and to detect a DNA marker linked to the locus. Further, the present invention provides a method for selecting young seedlings of multileaf individuals using the primer set and producing a line having a large number of upper ear spikes in the progeny, and a maize multileaf line produced by the method Is intended to provide.

The present invention for solving the above-described problems comprises the following technical means.
(1) A primer set for detecting a DNA marker linked to a multilobe-related gene locus, and detecting a DNA marker linked to a multilobe-related gene locus (lfy1) seated on corn chromosome 3 Primer set characterized by being able to.
(2) The primer set according to (1) above, which can detect a DNA marker located within a range of 10 centmorgans from the multileaf associated gene locus (lfy1).
(3) The primer set according to (2), wherein the DNA marker is AFLP marker A4M48.
(4) In any one of the above (1) to (3), which is composed of a pair of base sequences selected from the forward primer of SEQ ID NO: 1-6 and the reverse primer of 7-12: The primer set described.
(5) Presence or absence of the leafy lobe-related gene locus of the investigated individual by performing PCR on the corn genomic DNA using the primer set according to any one of (1) to (4) above and examining the length of the amplified fragment How to identify.
(6) By using the method according to (5) above, a young corn seedling of an individual having a multilobe-related gene locus is selected, and a line having a large number of upper ear leaves in its progeny is produced. How to create sex lines.
(7) A corn for feed produced by the method according to (6) above, characterized by increasing the number of upper lobes of ears.

Next, the present invention will be described in more detail.
The present invention relates to a primer set for detecting a DNA marker linked to a multilobe-related gene locus, comprising a DNA marker linked to a multilobe-related gene locus (lfy1) seated on corn chromosome 3 It can be detected.

  In the present invention, it is possible to detect a DNA marker located within the range of 10 centmorgans from the multilobe-related gene locus (lfy1), the primer set includes forward primers of SEQ ID NOs: 1 to 6 and 7 to 12 in the Sequence Listing. It is a preferred embodiment that it is composed of a pair of base sequences selected from the reverse primers.

  In addition, the present invention provides a method for identifying the presence or absence of a leafy loci associated with a research individual by performing PCR on corn genomic DNA using the above primer set and examining the length of the amplified fragment, The method of selecting young seedlings of individuals having a leafy loci-related locus by the method and producing a line with a large number of upper ear spikes in the progeny, and further, a corn leafy line produced by the above method It is characterized by a point.

  The present invention provides a primer set that is capable of detecting a DNA marker that is linked to a gene locus (lfy1) involved in the number of corn ear upper leaves and can be used for genotype determination. Hereinafter, the primer set of this invention and its new utilization method are demonstrated.

  In the present invention, a plurality of DNA markers closely linked to the leafy loci-related gene locus are found, but the DNA marker targeted by the present invention is not limited to these, and the leaflet-related loci are not limited thereto. Any one that is closely linked to the locus (lfy1) can be used as well.

  In the present invention, a primer pair capable of detecting these DNA markers has been successfully constructed. When PCR was performed on maize genomic DNA using the primer pair, it was found that DNA fragments having different base numbers were amplified in multileafy and non-leafy lines. Therefore, by investigating the length of the amplified fragment, it is determined whether the region on which the marker is seated is derived from a multileafy line or a non-leafy line. It was found that the presence or absence of the locus (lfy1) can be identified.

  The genomic DNA extraction method used for the determination of the genotype of the above DNA marker is not particularly limited, and the CTAB method generally used for DNA extraction from plant leaf pieces is modified and implemented as necessary. do it. It is also possible to use a commercially available DNA extraction kit as appropriate. The PCR conditions for amplifying the DNA fragment of the DNA marker are exemplified in the examples described later, but are not limited thereto, and may be arbitrarily changed according to the primer pair and the equipment used. be able to. Similarly, the electrophoresis conditions can be changed, and an agarose gel can be used if there is a sufficient difference in the length of the amplified fragments.

  In the present invention, genomic DNA extraction techniques, PCR conditions for DNA fragment amplification, electrophoresis conditions, and the like can be arbitrarily set. According to the present invention, as described above, the DNA marker is detected using the specific primer pair of the present invention, and the presence or absence of the multilobe-related gene locus (lfy1) is identified using the DNA marker as an index. By selecting the young seedlings, it becomes possible to produce a multileaf line with a large number of upper ear spikes in its progeny.

  As a maize line subjected to PCR by the method of the present invention, any line can be used as long as it is a line using a multi-leaf line having a multi-leaf associated locus (lfy1) as a single parent. Regarding the DNA marker, it is preferable to use a DNA marker having a clear difference in the length of the amplified fragment between, for example, a multileaf line used as a breeding material and a non-leafy line.

In the present invention, examples of the analysis population used for estimating the leafy loci-related loci include, for example, population individuals of the hybrid second generation (F ₂ ) of the leafy line 957L and the nonleafy line Na12. However, the present invention is not limited thereto, and a suitable second-generation hybrid individual of a multileafy line and a non-multileafy line is used. Observe the number of upper lobes of each individual and extract DNA for later analysis. The number of upper leaves of the ear is measured as the number of leaves higher than the uppermost ear.

  Next, for example, a restriction enzyme fragment length polymorphism (RFLP) marker, an amplified fragment length polymorphism (AFLP) marker, and a simple repeat sequence (SSR) marker are preferably used for the creation of a linkage map. The RFLP analysis can be performed, for example, by using a corn genome-derived and cDNA-derived probe and performing polymorphism analysis using, for example, an ECL Southern hybrid system manufactured by GE Healthcare Bioscience.

  AFLP analysis can be performed according to the method of Vos et al. And the method of Myburg et al. The restriction enzyme digestion of genomic DNA is preferably performed with, for example, EcoRI and MseI or a combination of PstI and MseI, and the EcoRI primer and the PstI primer are used after being labeled with a fluorescent dye.

  The polymorphism can be detected by, for example, DNA analysis system LIC-4200L-2 (manufactured by Aloka). In the SSR polymorphism analysis, SSR markers registered and published in Maize Genetics and Genomics Database (MaizeGDB, http://www.maizeggdb.org/) can be used. Among the primer pairs of each SSR marker, the forward primer can be labeled with, for example, an IRD800 fluorescent dye. However, the present invention is not limited to these, and any device and fluorescent dye can be used for polymorphism detection by AFLP or SSR.

  Polymerase chain reaction (PCR) for SSR amplification is performed under conditions as described in, for example, MaizeGDB. PCR conditions include, for example, 1) 94 ° C. for 5 minutes, 2) 94 ° C. for 1 minute, 65 ° C. for 1 minute, 72 ° C. for 1.5 minutes, 2 cycles, 3) 94 ° C. for 1 minute, 65 ° C. (every cycle) 1) 72 ° C 1.5 minutes 10 cycles 4) 94 ° C 1 minute 55 ° C 1 minute 72 ° C 1.5 minutes 30 cycles 5) 72 ° C 7 minutes However, the present invention is not limited to this, and suitable conditions can be set arbitrarily. The detection of the polymorphism is performed by, for example, the DNA analysis system LIC-4200L-2.

  Based on the results of the polymorphism analysis of the above RFLP, AFLP and SSR, QTL analysis software MapmanagerQTX is used to perform linkage analysis and QTL analysis on multileaf nature. In the present invention, it was found that there is a multilobe associated locus (lfy1) on chromosome 3 and that one AFLP marker, A4M48, is linked to lfy1. Therefore, the base sequence of the amplified fragment of the A4M48 marker was determined, and a primer set for efficiently performing polymorphism analysis of the marker was newly designed (STS conversion).

  When PCR is performed using this primer set, for example, a 282 base pair fragment is amplified when genomic DNA of non-leafy strain Na12 is used as a template, but when genomic DNA of leafy strain 957L is used as a template. A 280 base pair fragment is amplified, and the genotype of the marker can be determined from the difference in amplified fragment length. In the case of other non-multileafy lines and multileafy lines, the genotype of the same marker can be similarly determined from the difference in the length of the amplified fragments.

  In addition, in order to find a marker that is more closely linked to the multilobe-related gene locus (lfy1), for example, among the SSR markers published in MaizeGDB, sit on Bin numbers 3.08 to 3.09, Those having a difference in polymorphism between Na12 and 957L, which are the parent lines of this analysis group, are selected. Polymorphism analysis of these markers is performed on the above-mentioned analysis population, and a high-density linkage map is created. This linkage map is again subjected to QTL analysis using QTL analysis software MapQTL.

  In the present invention, it has been found that five SSR markers (umc1578, umc1361, bnlg1496, mmc0001, umc1641) are linked to lfy1 as much as or more closely than the A4M48 marker. In the present invention, primer pairs were constructed to enable detection of these DNA markers. These DNA markers are all SSR markers published in MaizeGDB, and more detailed information on these markers and information on other markers can be obtained on the same website.

In order to verify the selection effect by the above-mentioned A4M48 marker and five SSR markers, for example, three populations are used. In this case, for example, the backcrossing in which F ₁ of the multileafy line 914L and three different non-leafy inbred lines (eg, TD16, KK8, KK11) are further crossed with each non-leafy line An example is a method of training 90 individuals in each group using 1 generation (BC ₁ generation), measuring the number of upper lobes of the ear, and genotyping using lfy1 neighborhood markers.

  In the above method, the lfy1 vicinity marker to be investigated in each population is the one that has been confirmed in advance that there is a difference in the amplified fragment length between 914L and each non-leafy line, and that genotype can be determined. It is done. In the case of backcrossing in which a non-leafy line is a recurrent parent, genotypes of multi-leaf type and non-leafy type hetero and non-leafy type homozygos may appear.

  Comparing the average number of upper ear spikes between the group that showed heterogeneity in the A4M48 marker genotype and the group that showed non-leafy homozygosity, the three populations showed a heterogeneity between the leafy type and the non-leafy type. It was shown that the number of upper leaves in the ear was significantly higher than that of the non-multileaf homozygote, and the selection effect by the A4M48 marker was found.

  Similar results were also observed for the four SSR markers examined, and it was found that selection of multileaf individuals using DNA markers was effective. Further, when the investigated markers were linked, it was found that umc1578, bnlg1496, and mmc0001 were seated within 1 cM from lfy1, A4M48 was seated at 7 cM, and umc1361 was seated at 7-8 cM. .

  The present invention discriminates the presence or absence of a multilobe-related gene locus in a research individual by detecting a DNA marker linked to the multilobe-related gene locus, whereby a seedling of an individual having a multileafy gene locus The most characteristic feature is that a method for selecting a line and producing a line with a large number of upper ear leaves in its progeny has been established. In the present invention, methods and means relating to selection of seedlings and production of progeny can be appropriately selected and used according to conventional methods.

  In the present invention, a specific primer set capable of detecting the above DNA marker is provided. Similarly, any primer set capable of detecting a DNA marker located within the range of 10 centmorgans from the multileaf associated locus (lfy1) is used. Can be used. In the present invention, a known DNA marker can be used as a DNA marker. However, a primer set capable of detecting a DNA marker closely linked to the multilobe-related gene locus (lfy1) seated on corn chromosome 3 is used. Using corn genomic DNA, PCR is performed, and the length of the amplified fragment is examined to identify the presence or absence of the leafy loci associated with the individual being investigated, thereby selecting the seedlings of individuals having the leafy loci However, a method for producing a line having a large number of upper ear leaves in the progeny is a novel method that cannot be expected from the prior art.

The present invention has the following effects.
(1) According to the present invention, it is possible to provide a primer set capable of detecting a DNA marker linked to a multileafy loci (lfy1) locused on corn chromosome 3.
(2) It is possible to identify the presence or absence of the multilobe associated gene locus (lfy1) of the individual to be investigated by performing PCR on corn genomic DNA using the primer set provided by the present invention and examining the length of the amplified fragment it can.
(3) Thereby, it becomes possible to select a young seedling of an individual having a multileafy-related gene locus, and to efficiently and reliably produce a maize multileafy line in its progeny.

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

1) Analytical population For the estimation of multilobe-related loci, 80 hybrid second-generation (F ₂ ) populations of multileaf line 957L and non-leafy line Na12 were trained, and the upper ear leaves of each individual Number observation and DNA extraction were performed and used for later analysis. The number of upper leaves of the ear was measured as the number of leaves higher than the uppermost ear.

2) Creation of a linkage map For the creation of a linkage map, a restriction enzyme fragment length polymorphism (RFLP) marker, an amplified fragment length polymorphism (AFLP) marker and a simple repeat sequence (SSR) marker were used. For RFLP analysis, polymorphism analysis was performed by the ECL Southern Hybrid System (manufactured by GE Healthcare Bioscience) using corn genome-derived and cDNA-derived probes sold by the University of Missouri, USA.

  The AFLP analysis was performed according to the method of Vos et al. (Nucleic Acids Res. 23: 4407-4414 (1995)) and the method of Myburg et al. (BioTechniques 30: 348-357 (2001)). Restriction enzyme digestion of genomic DNA was performed with a combination of EcoRI and MseI, or PstI and MseI, and EcoRI and PstI primers were labeled with IRD700 or IRD800 fluorescent dye (Aloka).

  The polymorphism was detected with a DNA analysis system LIC-4200L-2 (Aloka). For SSR polymorphism analysis, SSR markers registered and published in Maize Genetics and Genomics Database (MaizeGDB, http://www.maizeggdb.org/) were used. Of each SSR marker primer pair, the forward primer was labeled with an IRD800 fluorescent dye.

  Polymerase chain reaction (PCR) for SSR amplification was performed under the following conditions as described in Maize GDB. 1) 94 ° C. for 5 minutes, 2) 94 ° C. for 1 minute, 65 ° C. for 1 minute, 72 ° C. for 1.5 minutes, 2 cycles, 3) 94 ° C. for 1 minute, 65 ° C. (down 1 ° C. for each cycle) for 1 minute, 72 ° C 1.5 minutes 10 cycles, 4) 94 ° C 1 minute, 55 ° C 1 minute, 72 ° C 1.5 minutes 30 cycles, 5) 72 ° C 7 minutes. The polymorphism was detected with the DNA analysis system LIC-4200L-2.

  Based on the results of polymorphism analysis of a total of 247 markers of RFLP, AFLP, and SSR, QTL analysis software MapmanagerQTX (Manly, KF, Cudmore, Jr, RH, Meer, JM (2001) Mammalian Genome 12: 930-932) Was used to perform linkage analysis and QTL analysis for multilobes.

  As a result, it was shown that a multilobe associated locus (lfy1) is present on the third chromosome, and that one AFLP marker, A4M48, is linked to lfy1. Therefore, the base sequence of the amplified fragment of the A4M48 marker was determined, and a primer set for efficiently performing polymorphism analysis of the marker was newly designed (STS conversion). This primer set consists of a forward primer LEF: 5'-GGATGAGAACTATAGACA-3 '(SEQ ID NO: 1) and a reverse primer LER: 5'-TATGCCATGCCCCTTTTT-3' (SEQ ID NO: 7).

  When PCR is performed using this primer set, a 282 base pair fragment is amplified when the genomic DNA of the non-leafy line Na12 is used as a template. A base pair fragment is amplified, and the genotype of the marker can be determined from the difference in length of the amplified fragment (FIG. 1).

  In addition, in order to find a marker linked more closely to lfy1, among the SSR markers published in MaizeGDB, they sit on Bin numbers 3.08 to 3.09, and Na12 and 957L, which are the parent lines of this analysis group The ones with differences in polymorphism were selected. Polymorphism analysis of these markers was performed on the above analysis group to create a high-density linkage map.

  QTL analysis software MapQTL (Van Ooijen, JW, MP Boer, RC Jansen, C. Marypaard, 2002. MapQTL4.0, Software for the Calfontation of QT QTL analysis was performed again at Plant Research International, Wageningen, the Netherlands.

  As a result, it was shown that five SSR markers (umc1578, umc1361, bnlg1496, mmc0001, umc1641) were linked to lfy1 to the same extent or more closely than the A4M48 marker (FIG. 2). Therefore, in the present invention, primer pairs were constructed to enable detection of these DNA markers (Table 1). These DNA markers are all SSR markers published in MaizeGDB, and more detailed information on these markers and information on other markers can be obtained on the same website.

Three populations were used to verify the selection effect with the A4M48 marker and the five SSR markers described above. These are multilobed line 914L and three systems of different non multilobed inbred (TD16, KK8, KK11) and _{F 1} and further backcrossing were crossed with non multilobed line of each first generation (BC ₁ generation), 90 individuals were trained in each group, and the number of upper lobes of the ear was measured and the genotype was investigated using the lfy1 neighborhood marker.

  The lfy1 neighborhood marker to be investigated in each population is A4M48, umc1578, umc1361, which has been confirmed in advance that there is a difference in amplified fragment length between 914L and each non-leafy line, and genotype determination is possible. Five markers were used: bnlg 1496 and mmc0001. In the case of backcrossing in which a non-leafy line is a recurrent parent, genotypes of multi-leaf type and non-leafy type hetero and non-leafy type homozygos may appear.

  The average number of upper ear leaves was compared between the group in which the genotype of the A4M48 marker showed heterogeneity and the group in which non-multileaf homology was shown. As a result, it was shown that the number of upper leaves in the ear was significantly higher in the three populations than in the non-leafy type homozygous in the multileaf type and non-leafy type hetero, and the selection effect by the A4M48 marker Was shown (Table 2).

  Similar results were also observed with the four SSR markers tested, indicating that the selection of multileaf individuals using DNA markers is effective. As a result of linkage analysis of the investigated markers, it was shown that umc1578, bnlg1496, and mmc0001 are seated within 1 cM from lfy1, A4M48 is seated at 7 cM, and umc1361 is seated at 7 to 8 cM. It was.

  As described above in detail, the present invention relates to a primer set for detecting a DNA marker linked to a corn multileaf leaf-related gene locus and the use thereof. A primer set that enables detection of a DNA marker linked to the relevant gene locus (lfy1) can be provided. PCR is performed on maize genomic DNA using the primer set provided by the present invention, and the presence or absence of the leafy loci associated locus (lfy1) of the investigated individual is examined by examining the length of the amplified fragment, thereby A seedling of an individual having a leafy leaf related locus can be selected, and a maize leafy line can be efficiently and reliably produced in its progeny.

FIG. 1 shows an example of genotyping using the A4M48 marker in the first generation of backcross (BC ₁ ). FIG. 3 is an electrophoretogram of a fragment amplified by PCR using SEQ ID NOS: 1 and 2 as primers. In the right side of the figure, “← multi-leafy” indicates an amplified fragment derived from a multi-leafy parent line, and “← non-leafy” indicates an amplified fragment derived from a non-leafy parent line. In the upper part of the figure, “M” indicates a size marker, and the number of bases of the size marker is indicated on the left in the figure. 24 individuals are genotyped between size markers in the lanes at both ends. FIG. 2 shows the results of QTL analysis regarding multileaf nature. In the figure, the LOD score of each marker is indicated by “▲”, the marker confirmed to be usable for multileaf selection in the present invention is indicated by Δ, and the marker name is indicated at the bottom of the figure.

Claims (7)

  A primer set for detecting a DNA marker linked to a multilobe-related gene locus, characterized in that it can detect a DNA marker linked to a multilobe-related gene locus (lfy1) that sits on corn chromosome 3 Primer set.   The primer set according to claim 1, wherein a DNA marker located within the range of 10 centmorgans from the multilobe associated gene locus (lfy1) can be detected.   The primer set according to claim 2, wherein the DNA marker is AFLP marker A4M48.   The primer set in any one of Claim 1 to 3 comprised from a pair of base sequence selected from the forward primer of sequence number 1-6 of a sequence table, and the reverse primer of 7-12.   A method for discriminating the presence or absence of a multilobe-related gene locus in a research individual by performing PCR on maize genomic DNA using the primer set according to any one of claims 1 to 4 and examining the length of the amplified fragment.   A method for producing a maize multileaf line, comprising selecting a seedling of an individual having a leafy loci associated locus by the method according to claim 5 and producing a line having a large number of upper ear spikes in a progeny thereof. Method. A feed corn produced by the method according to claim 6, characterized by increasing the number of upper ear spike leaves.