JP2002112773A - Method for discriminating sex of papaya using dna marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for discriminating the sex of papaya (fruit tree having three sex expression types comprising male, female and bisexual and the bisexual fruit has high commercial value as the fruit). SOLUTION: The present invention relates to a sex discrimination marker for papaya containing a DNA composed of either one of two specific kinds of base sequences of papaya or its polymorphic compound, a primer for the amplification of the sex discrimination marker of papaya containing a part of the DNA and expressed by either one of the sequence numbers 4-7: ggtaagagtt tttcccaagc 4; tgcacgattt agattagatg 5; ggatagcttg cccaggtcac 6; and gttgtgctgc gctatcttgc 7, a kit for the sex discrimination of papaya containing either one of the above primers for the amplification of sex discrimination marker as a constituent element, a probe for the detection of the sex discrimination marker for papaya containing a part or total of the above DNA, a kit for the sex discrimination of papaya containing the probe as a constituent element and a method for the sex discrimination of papaya comprising the analysis of the presence of the sex discrimination marker of papaya.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA specifically present in male and amphoteric papayas, a papaya sex identification marker containing the DNA, a primer for amplifying the sex identification marker,
The present invention relates to a probe for detecting the sex identification marker, a kit for sex identification of papaya containing the primer or the probe as a component, and a method for sex identification of papaya using the presence or absence of the sex identification marker as an index.

[0002]

BACKGROUND OF THE INVENTION Papaya (Carica papaya) is male, female,
A fruit tree having three sex phenotypes of both sexes, the one with high commercial value as a fruit is the amphoteric fruit. Sunrise, a main papaya variety cultivated in Okinawa Prefecture, is an amphoteric self-breeding variety, and its seedlings have females and amphoteric in a ratio of 1: 2. Therefore, in gender identification of the sunrise seedlings, it is important to distinguish between females and amphoteric. Conventionally, gender identification of papaya has been carried out by a cultivation method in which a plurality of plants are supposedly planted at one location and a necessary strain is left by sex identification based on the flower type, since it is not possible to judge by sex until a flower blooms. However, this method increases the cost of raising seedlings (fertilizer, water, pots, labor costs, land occupation, etc.) at the seed and seedling company and the cultivation costs at the site (fertilizer, water, labor costs, and difficulties in planned cultivation). The development of a papaya sex identification method (before flowering) was urgently needed.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to DNA specifically present in male and bisexual papayas, a papaya sex identification marker containing the DNA, a primer for amplifying the gender identification marker, and a papaya sex identification marker. An object of the present invention is to provide a detection probe, a kit for sex identification of papaya containing the primer or the probe as a constituent element, and a method for sex identification of papaya using the presence or absence of the sex identification marker as an index.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, obtained a DNA marker specifically present in male and amphoteric papayas by RAPD method. And succeeded in completing the present invention. That is, the present invention is a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2, or a DNA containing a polymorph of the DNA.

Further, the present invention is a papaya sex identification marker containing a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2, or a polymorph of the DNA. Furthermore, the present invention relates to the properties of papaya containing a part of the above DNA (for example, one having a length of 17 to 30 mer, more specifically, for example, one represented by any of SEQ ID NOs: 4 to 7). It is a primer for amplifying a discriminating marker.

Further, the present invention is a papaya sex discrimination kit comprising the above-mentioned primer for amplifying a sex discriminating marker as a constituent element. Furthermore, the present invention is a probe for detecting a papaya sex identification marker containing a part (for example, one having a length of 20 to 450 mer) or all of the above DNA.
Furthermore, the present invention is a papaya sex identification kit comprising the above-described sex identification marker detection probe as a constituent element.

Further, the present invention is a method for identifying sex of papaya, which comprises analyzing the presence or absence of the papaya sex identification marker in a subject papaya. here,
Analysis of the presence or absence of the papaya sex identification marker can be performed by detecting an amplification product derived from the sex identification marker obtained by the polymerase chain reaction. In the polymerase chain reaction, a DNA derived from the subject papaya is used as a template,
It can be carried out using any of the above-mentioned primers for amplifying a sex identification marker as a primer. The analysis of the presence or absence of a papaya sex identification marker can also be performed by detecting a hybridized product obtained by a probe hybridization reaction. The probe hybridization reaction can be performed by hybridizing the probe for detecting a sex identification marker to DNA derived from a subject papaya. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The gender identification method of papaya of the present invention is different from the conventional gender identification method in that gender identification is performed based on DNA sequence differences between papayas caused by sex differences. is there. More specifically, it is a method of identifying the sex of the subject papaya using the presence or absence of a sex identification marker as an index. Here, the “sex discriminating marker” is a DNA having a base sequence specifically found in a specific sex, and refers to a DNA that can be used for discriminating sex. The papaya gender identification marker can be separated as follows.

1. Searching for sex discrimination markers Searching for sex discrimination markers is performed by a method for detecting a genetic polymorphism between living organisms, for example, by using amplified fragment polymorphism (random amplified p.
olymorphic DNA; RAPD) [William et al .: Nucleic Acids Re
s. 18: 6531-6535 (1990)].
That is, first, a conventional method such as the CTAB method [Mur
DNA is extracted using ray et al .: Nucleic Acids Res. 8: 4321-4325 (1980)] or the like. At this time, the plant tissue used for extraction is not limited, but leaves are preferably used. Further, these leaves are preferably crushed so that DNA extraction can be performed efficiently. For example, the leaves can be frozen with liquid nitrogen and crushed in a mortar.

[0010] Next, using the papaya DNA as a template, a polymerase chain reaction (PCR) is performed using one or two kinds of random sequence primers (also called random primers). As a random primer that can be used here, 10 to
A 12-mer base length is exemplified. In the present invention, as a random primer, independently designed and synthesized
10 consisting of the nucleotide sequence of 5'-ttggcacggg-3 '(SEQ ID NO: 3)
Mer primers were used. The PCR reaction conditions are not particularly limited, but are preferably 92 to 96 ° C for 1 to 2 minutes (dissociation of double-stranded DNA), 37 to 42 ° C for 1 to 2 minutes (primer annealing), 70 to 74 ° C. C. for 1 to 2 minutes (synthesis of complementary strand), preferably 30 to 40 cycles. PCR can be performed using a heat block type program temperature controller (for example, PCR Thermal Cycler (Takara Shuzo)).

After the PCR, the PCR amplification product is detected. As a detection method, gel electrophoresis (eg, agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc.) and subsequent gel staining (eg, ethidium bromide staining, etc.)
, But is not limited thereto. For example, after agarose gel electrophoresis, when the gel is stained with ethidium bromide and observed under ultraviolet light, a barcode-like band pattern is obtained. This band pattern
When PCR was performed using female papaya-derived DNA as a template, PCR was performed using male papaya-derived DNA as a template, and PCR was performed using amphoteric papaya-derived DNA as a template. A PCR amplification product specifically found can be used as a papaya sex identification marker.

By the way, the sexual phenotypes that appear in the offspring due to the cross between papayas and their appearance ratios are as shown in Table 1. Sunrise, which is the main variety of papaya produced in Okinawa Prefecture, is an amphoteric self-breeding variety, and its seedlings (also referred to as young plants) have females and amphoteric in a ratio of 1: 2 as shown in Table 1. Therefore, in the sex identification of the sunrise seedlings, a method for identifying a female and hermaphrodite is desired. From female papaya
It is not seen when performing PCR using DNA as a template, only when performing PCR using papaya-derived DNA as a template,
Alternatively, PCR using amphoteric and male papaya-derived DNA as template
Can be used as a sex identification marker for identifying amphoteric or male papaya from female papaya.

[0013]

[Table 1]

2. Determination of Nucleotide Sequence of Sex Identification Marker The nucleotide sequence of the sex identification marker of papaya obtained in the above 1 can be determined by a nucleotide sequencing method well known in the art. That is, for example, the band of the sex identification marker is extracted from the gel after the agarose gel electrophoresis of the PCR amplification product in the above item 1, and ligated to a base sequence determination plasmid (for example, pT7Blue (manufactured by NOVAGEN)). Then, after transforming the ligation product into E. coli, colony PC
Escherichia coli containing the plasmid into which the sex identification marker has been inserted is screened by the R method. A plasmid is prepared from the obtained Escherichia coli, and the base sequence of the inserted DNA fragment is determined by a known method such as the dideoxy method. In the present invention, the base sequence of the DNA fragment obtained as a sex identification marker is set to SEQ ID NO: 1, and the sequence of its complementary strand is set to SEQ ID NO: 2.
Shown in Consists of the nucleotide sequences represented by SEQ ID NOs: 1 and 2
DNA is present in the form of double strands with each other in amphoteric and male papayas, and both are useful as papaya sex identification markers.

The sex identification marker of the present invention is not limited to those shown in SEQ ID NOs: 1 and 2, as long as it can identify amphoteric or male paia from female papaya. “Polymorphs” in which one or several bases are substituted, deleted, added or inserted in Nos. 1 and 2 are also included in the sex identification marker of the present invention. Here, "polymorphism" refers more specifically to a DNA polymorphism in which papaya individuals of the same sex have a difference in the corresponding nucleotide sequence at one or several levels.

The DNA of the present invention containing the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2 or a polymorph of the DNA is prepared by the above-described method 1, but the DNA of the present invention is prepared in addition to the determined nucleotides. It can also be prepared by designing and synthesizing a primer based on the sequence, and performing PCR using the primer as a template with DNA extracted from male and amphoteric papaya strains.

3. Sex Discrimination of Papaya by Analysis of the Sex Discrimination Marker of the Present Invention By analyzing the presence or absence of the sex discrimination marker of the present invention in a test papaya, the sex of the test papaya can be discriminated. Analysis of the presence or absence of a sex identification marker can be performed by a PCR method, a probe method, or the like.

(1) Analysis of Sex Identification Marker by PCR Method Sex identification of papaya based on analysis of the sex identification marker by PCR method can be performed as follows. First, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2
Alternatively, a primer for amplifying a sex identification marker containing a part of the nucleotide sequence is designed and synthesized based on the nucleotide sequence of the polymorphic product of the DNA. Here, the “part of the base sequence” refers to a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 or the DNA
It refers to a continuous nucleotide chain having a base length of 17 to 30 mer, preferably 20 to 30 mer, most preferably 20 to 25 mer in the polymorphism of NA. More specifically, it is a nucleotide chain which specifically hybridizes to a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2 or a polymorph of the DNA and functions as a primer. Here, “function as a primer” refers to providing a DNA polymerase with a 3′-terminal OH group in a complementary strand synthesis reaction of PCR and functioning to initiate a nucleotide chain synthesis reaction by the enzyme. For the synthesis of the primer, a known method known as a method for synthesizing an oligonucleotide can be used, but more simply, a DNA synthesizer (for example, Applied Biosystem
(Manufactured by Sharp Corporation).

More specifically, in the analysis of the gender identification marker by the PCR method, a gender identification marker comprising a 5 'primer and a 3' primer capable of amplifying a part or the whole region of the gender identification marker is used. A pair of amplification primers is required. For example, primers for amplifying gender identification markers
As the 5 'primer, 5'-ggtaagagtttttcccaagc-
3 ′ (SEQ ID NO: 4) and 5′-tgcacgatttagattagatg-3 ′ (SEQ ID NO: 5). As the 3′-side primer, 5′-ggatagcttgcccaggtcac-3 ′ (SEQ ID NO: 6), 5′-g
ttgtgctgcgctatcttgc-3 ′ (SEQ ID NO: 7). Those skilled in the art can easily design and synthesize other appropriate sequences based on the nucleotide sequence of SEQ ID NO: 1 or 2, and use these primers. It is not limited to. In addition, the corresponding position of each of the above primers in SEQ ID NO: 1 is shown in FIG. In FIG. 1, the direction of the arrow indicates the direction of the array.
These primers can be used as a primer pair for amplifying the sex identification marker region by appropriately combining the 5′-side primer and the 3′-side primer.
An appropriate combination can be appropriately selected by those skilled in the art. That is, it is an essential condition to select a primer pair so that the 5 'primer is present upstream (in the direction from 5' to 3 ') of the 3' primer. Such a combination of primers is preferably SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 4
And SEQ ID NO: 7, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 5 and SEQ ID NO: 7, but the combination of primers is optional.

Sex identification of papaya seedlings can be performed using the primers of SEQ ID NOs: 4 to 7. More specifically, among female, amphoteric and male papaya seedlings obtained from a mating combination of "female x male", "female x bisexual", "male x male" and "amphoter x bisexual", Can be identified.

The specific procedure is as follows. That is, first, DNA is extracted from the tissue of a papaya seedling according to a conventional method. The tissue of the plant used for DNA extraction is not particularly limited, but leaves are preferably used. further,
These leaves are preferably pulverized so that nucleic acid extraction can be performed efficiently. For example, the leaves can be pulverized using liquid nitrogen. DNA extraction can be performed using a known method, and preferably, CTAB method [Murra
y et al., Nucleic Acids Res. 8, 4321-4325 (1980)].

Next, using the extracted DNA as a template, the above-mentioned primer pair for amplifying the sex identification marker
Perform R to amplify the target DNA fragment. The temperature conditions for PCR can be appropriately determined by those skilled in the art based on experience, and are not particularly limited.
The reaction is successively carried out for 1 to 2 minutes (dissociation of double-stranded DNA), 1 to 2 minutes at 50 to 55 ° C (annealing of primer), and 1 to 2 minutes at 70 to 74 ° C (synthesis of complementary strand). 25
Perform ~ 30 cycles. A heat block type programmed temperature controller can be used to set the temperature of the above reaction. Examples of such devices include the PCR Thermal Cycler
(Manufactured by Takara Shuzo).

Finally, the amplified nucleic acid is detected from the reaction mixture obtained as described above. As a detection method, a method usually used in the art can be used, and preferably, the above-described method of performing agarose gel electrophoresis and subsequent gel staining with ethidium bromide can be used. As a result, the PCR-amplified fragment is detected only in the male and amphoteric subject papaya, but not in the female. If the PCR-amplified fragment is detected, the sex identification marker of the present invention is present in the subject papaya, and it can be evaluated that the subject papaya is not a female. If no PCR-amplified fragment is detected, the subject papaya can be evaluated as a female.

Further, when the sex of the parents of the test papaya seedling is specified, the sex of the test papaya seedling can be specified by the presence or absence of the PCR-amplified fragment.
That is, if a PCR-amplified fragment is detected in a papaya seedling obtained by a `` female x male '' mating combination, the subject papaya can be evaluated as a male,
If not detected, the subject papaya can be assessed as a female. In addition, in the papaya seedlings obtained by the mating combination of "female x amphoteric", if the PCR-amplified fragment is detected, the subject papaya can be evaluated as being amphoteric. It can be evaluated as a female. Furthermore, in the papaya seedlings obtained by the cross combination of "male x male", if the PCR-amplified fragment is detected, the subject papaya can be evaluated as a male. It can be evaluated as a female. Similarly, in the papaya seedlings obtained by the hybridization combination of `` amphoter x amphibian '', if the PCR amplified fragment is detected, the subject papaya can be evaluated as amphoteric, and if not detected, the subject papaya is It can be evaluated as a female.

(2) Analysis of Sex Identification Marker by Probe Method Sex identification of papaya based on analysis of the sex identification marker by the probe method can be performed as follows.
First, a DN consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2
Based on the nucleotide sequence of A or the polymorphic product of the DNA, a probe for detecting a sex identification marker containing a part or all of the nucleotide sequence is prepared. Here, the “part of the base sequence” refers to a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 or the DNA
It refers to a continuous nucleotide chain having a base length of 20 to 450 mer, preferably 50 to 450 mer, most preferably 100 to 450 mer in the NA polymorphism. The nucleotide sequence of the nucleotide chain is a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2.
Alternatively, it may be a nucleotide sequence in which one or several bases are substituted, deleted, added or inserted, as long as the DNA specifically hybridizes to the polymorphism of the DNA. For example, SEQ ID NO: 1
DNA having a base sequence in which one or more bases are substituted, deleted, added or inserted with respect to the base sequence shown in
Is a site-directed mutagenesis method [Zoller et al .: Nucleic Acids R
es. 10 (20): 6487-6500 (1982)], and can be easily prepared by those skilled in the art.

A probe for detecting a sex-identifying marker is prepared from a cloned DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2 or a plasmid containing a polymorph of the DNA, using a suitable DNA fragment with an appropriate restriction enzyme. Can be cut out, or can be prepared by PCR using the plasmid or DNA prepared from amphoteric or male papaya as a template. SEQ ID NO: 1
Or having a base sequence in which one or several bases are substituted, deleted, added or inserted, as long as it specifically hybridizes to DNA consisting of the base sequence represented by 2 or a polymorphism of the DNA. DNA was obtained by site-directed mutagenesis [Zoller et al .:
Nucleic Acids Res. 10 (20): 6487-6500 (1982)], and can be easily prepared by those skilled in the art. The label of the probe DNA fragment is a radioactive label (for example,
^{32 P, 35 S, 3 H} ), non-radioactive labels (e.g. biotin, it can be done by digoxigenin) or the like, specifically, after removal of the phosphate of the 5 'end of the probe DNA with phosphatase, polynucleotide kinase [γ- ³² P] ATP γ
-A method of transferring a phosphate group to the 5 'end of the DNA,
A method of incorporating a nucleotide substrate labeled with [ ³² P], [ ³⁵ S], [ ³ H], biotin, or the like, together with the synthesis of the probe DNA by a nick translation method or the like. More simply, labeling of the probe DNA with ³² P can be performed using a commercially available kit such as rediprime ^™ II (manufactured by Amersham Pharmacia Biotech).

As a specific procedure, a method utilizing Southern hybridization is exemplified as follows. That is, first, DNA is extracted from papaya by the same method as described above, and digested with an appropriate restriction enzyme. The restriction enzyme to be used is not particularly limited, but EcoRI is preferably used.

Next, the DNA digested with the restriction enzyme is subjected to agarose gel electrophoresis as described above, and the DNA is blotted on a membrane according to a conventional method. The membrane is not particularly limited, but is preferably a nylon membrane such as Hybond N + (manufactured by Amersham Pharmacia Biotech).

Further, the probe for detecting a sex identification marker is hybridized to the membrane after blotting according to a conventional method. Prehybridization and hybridization are performed at 65 ° C for 4 hours and 16 hours, respectively. These conditions are not particularly limited, since those skilled in the art can appropriately set them based on experience. After the hybridization, the nylon membrane is washed with a 65 ° C. SSC solution (containing 0.1% SDS). Washing conditions are not limited since those skilled in the art can appropriately set them based on experience, but 2 × SSC solution (containing 0.1% SDS) is added twice, 0.1 times.
× SSC solution (containing 0.1% SDS) can be performed twice.
In addition, each cleaning time can be performed in about 20 minutes.

Finally, the washed blot can be analyzed by autoradiography or BAS2000 (manufactured by Fuji Photo Film Co., Ltd.). This allows
A band (positive band) is detected only in the male and amphoteric test plants, but not in the female.

As in the case of the analysis of the sex identification marker by the PCR method, when the sex of the parents of the test papaya seedling is specified, the sex of the test papaya seedling is determined by the presence or absence of a positive band. It is also possible. That is,
In the papaya seedling obtained by the "female x male" mating combination, if a positive band is detected, the subject papaya can be evaluated as a male; if not, the subject papaya is a female. Can be evaluated. If a positive band is detected in the papaya seedlings obtained by the mating combination of "female x amphoteric", the subject papaya can be evaluated as amphoteric. Can be evaluated. Furthermore, if a positive band is detected in the papaya seedlings obtained by the “male × male” mating combination, the subject papaya can be evaluated as a male. Can be evaluated. Similarly, "bisexual x bisexual"
If the positive band is detected in the papaya seedlings obtained by the mating combination, the subject papaya can be evaluated as amphoteric, and if not detected, the subject papaya can be evaluated as a female. it can.

4. Papaya Sex Identification Kit Based on the papaya sex identification method of the present invention, it is possible to package and supply a kit necessary for carrying out the identification method. More specifically, for example, a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2
Alternatively, a part of the DNA of the DNA containing the polymorphism of the DNA (for example, 17
A primer having a length of 識別 30 mer, more specifically, for example, a primer for amplifying a sex identification marker of papaya, including those represented by any of SEQ ID NOS: 4 to 7, and / or SEQ ID NO: 1 or 2. DNA consisting of the base sequence represented or the D
Examples of such a probe include a probe for detecting a papaya sex identification marker, which contains a part or all of a part (for example, one having a length of 20 to 450 mer) of a DNA containing a polymorphism of NA, as an essential component. The components of the kit can vary as needed. For example, a buffer that provides suitable conditions for a PCR reaction or a hybridization reaction, and reagents necessary for detection of a synthesis reaction product can be added. Using the kit, it is possible to identify the sex of papaya according to the procedure of the above 3.

[0033]

EXAMPLES The present invention will now be described specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. [Example 1] Search for sex identification marker (1) Extraction of DNA from papaya leaves DNA was extracted from 5 g of papaya leaves using the CTAB method. As the papaya used for the extraction, a total of 33 known sexes (13 males, 16 females, 4 amphoteric individuals) such as sunrise, Waimanalo, and the Okinawa Prefectural Agricultural Experiment Station Breeding Line were used.

(2) Search for sex identification marker by RAPD method Using 100 ng of the DNA extracted as described above as a template, IBRC-RP07 5 'of a random primer designed and synthesized by the inventors.
PCR was performed using -ttggcacggg-3 ′ (SEQ ID NO: 3).
That is, DNA solution 10μl (10ng / μl), sterile distilled water 4μ
l, 10 × PCR buffer 2μl, dNTP mixture 2μl (2.5m each
M), 1 μl (0.5 U / μl) of TaKaRa Ex Taq, and 1 μl (20 μM) of a 10-Mer primer designed and synthesized by the inventors were mixed, and a PCR reaction was performed. The reaction was sequentially performed at 94 ° C. for 1 minute (DNA denaturation), at 37 ° C. for 1 minute (association of primers), and at 72 ° C. for 2 minutes (DNA chain elongation), and the cycle was repeated 30 times. The temperature was set by using a heat block type program temperature controller PCR Thermal Cycler (Takara Shuzo). Next, after 10 μl of the PCR reaction solution was subjected to 2% agarose gel electrophoresis, the gel was stained with ethidium bromide, and observed under ultraviolet light to detect amplified fragments. The results are shown in FIGS.

FIG. 2 shows 17 papayas of known sex (male 6
The results are obtained by performing PCR using DNA extracted from leaves of an individual, 7 females and 4 amphoteric) as primers using IBRC-RP07 primers, and analyzing the amplification product by 2% agarose gel electrophoresis. M is a molecular weight marker 100 bp DNA Ladder (Bio Labs),
C is a control group in which sterilized distilled water was added instead of DNA to perform PCR. Males are, in order from the left, Okinawa wild type A, Mexican introduced species M1, Okinawa prefecture wild type IK2, Okinawa wild type IK7, Okinawa wild type D, and Mexican introduced species M5. Females are from left to right.
Okinawa wild type A, Mexico introduced species M1, Okinawa wild type IK2,
Waimanalo, Orange Queen, Okinawa Wild 2 and Sunrise. In order from the left, the genders are Waimanalo, Sunrise, Mexican-introduced M1 and Waimanalo x Mexican-introduced M1 F1.

FIG. 3 shows that DNA extracted from the leaves of 16 papayas of known sex (mating parent and its F1 group, 7 males and 9 females) was used as a template, and the IBRC-RP07 primer was used to obtain a PC.
R shows the results of analysis of the amplification products by 2% agarose gel electrophoresis. M is the molecular weight marker 100bp DNA Ladder
(Bio Labs), C added PC with sterile distilled water instead of DNA
Control group where R was performed. The male used for mating was the Okinawa Agricultural Experiment Station Breeding Line ON-7, and the female was the Okinawa Agricultural Experiment Station ON-36. As is clear from the results of FIGS. 2 and 3, 400 bp and 500 bp
Male and amphoteric-specific amplified fragments (sex discriminating markers) were detected between bp.

[Example 2] Determination of base sequence of sex identification marker (1) Cloning of sex identification marker The sex identification marker detected in Example 1 was replaced with QIAquick.
It was extracted from agarose gel using Gel Extraction Kit (QIAGEN), cloned into plasmid pT7Blue (NOVAGEN), and transformed into E. coli.

(2) Determination of base sequence of sex identification marker Next, a clone having the sex identification marker was cultured and QIAp
Recombinant plasmid was extracted using rep Spin Miniprep Kit. Using the extracted recombinant plasmid as a template, BigDye
Perform the reaction using Terminator Cycle Sequencing, FS kit (PE Biosystems Japan), and perform ABI PRISMT
The nucleotide sequence was determined using M377 DNA Sequencer (PE Biosystems Japan). The determined nucleotide sequence is shown in SEQ ID NO: 1.

[Example 3] Sex identification of papaya by PCR method Using the obtained sex identification marker, sex identification of papaya was performed by PCR method. First, a primer for amplifying a sex-identifying marker was designed and synthesized based on the base sequence determined in Example 2. That is, 5′-ggtaagagtttttttcccaagc-3 ′ (SEQ ID NO: 4) and 5 ′ primer
5'-tgcacgatttagattagatg-3 '(SEQ ID NO: 5) was used as a 3' primer, 5'-ggatagcttgcccaggtcac-3 '(SEQ ID NO: 6) and 5'-gttgtgctgcgctatcttgc-3' (SEQ ID NO: 7).
Was synthesized. On the other hand, 5 g of leaves of the papaya to be tested were transferred to a mortar, crushed by adding a small amount of liquid nitrogen, and then subjected to CTAB method.
DNA was extracted. As the papaya used for the extraction, the above 33 individuals (13 males, 16 females, 4 amphoteric individuals), mating parents (female, amphoteric) and their F1 population (14 young plants) were used. Extracted
The target DNA fragment was amplified by PCR using the DNA as a template and a 5 ′ homologous primer and a 3 ′ complementary primer. That is, DNA solution 1μl (10ng / μl), 10 × PCR buffer 2μ
l, dNTP mixture 2 μl (2.5 mM each), TaKaRa Ex Taq 1 μl
(0.5 U / μl), sterile distilled water 12 μl, sex identification marker 5 ′ homologous primer 1 μl (20 μM) and 3 ′ side complementary primer
1 μl (20 μM) was mixed and a PCR reaction was performed. As the primer, a combination of SEQ ID NO: 5 and SEQ ID NO: 6 was used.
The reaction was performed at 94 ° C. for 1 minute (dissociation of double-stranded DNA), at 55 ° C. for 1 minute (annealing of primer), and at 72 ° C. for 1 minute (synthesis of complementary strand), followed by 30 cycles. .
In addition, a heat block type program temperature controller PCR Thermal Cycler (Takara Shuzo) was used for temperature setting.
Next, after 10 μl of the PCR reaction solution was subjected to 2% agarose gel electrophoresis, the gel was stained with ethidium bromide, and observed under ultraviolet light to detect amplified fragments.

FIG. 4 shows the results. (1) shows the results of analyzing 17 papayas of known sex (6 males, 7 females, 4 amphoteric individuals). M is the molecular weight marker 100bp DNA Ladder
(BioLabs), C: PCR by adding sterile distilled water instead of DNA
This is a control plot where the test was performed. Males are Okinawan wild species in order from the left
A, Mexican introduced species M1, Okinawa wild type IK2, Okinawa wild type
It is IK7, Okinawa Pref. Wild Species D, and Mexico Introduced Species M5. Females are, in order from the left, Okinawa wild type A, Mexican introduced species M1, Okinawa wild type IK2, Waimanalo, Orange Queen, Okinawa wild type 2, and sunrise. In order from the left, the genders are Waimanalo, Sunrise, Mexican-introduced M1 and Waimanalo x Mexican-introduced M1 F1. (2) is the result of analyzing 16 papayas of known sex (mating parent and its F1 group, 7 males and 9 females). M is the molecular weight marker 100 bp D
NA Ladder (Bio Labs), C is a control group in which sterilized distilled water was added instead of DNA and PCR was performed. Male used for mating is Okinawa Prefectural Agricultural Experiment Station Breeding Line ON-7, female is Okinawa Prefectural Agricultural Experimental Station
ON-36. (3) shows the result of analyzing the mating parents (female and bisexual) and their F1 population (14 young plants). M is a molecular weight marker of 100 bp DNA Ladder (Bio Labs), and C is a control group in which sterilized distilled water was added instead of DNA to perform PCR. Both sexes used for mating were sunrise and females were Okinawa Prefectural Agricultural Experiment Station ON-3
4 As is clear from FIG. 4, the target amplified fragment was detected only in the male and amphoteric samples (FIG. 4). In addition,
The F1 population (14 young plants) was collated with the above results after flowering. In addition, even when other sets of primers were used, similarly good identification results were obtained. From the above, it was found that sex identification of papaya can be performed based on the analysis of the sex identification marker of the present invention by the PCR method.

Example 4 Sex Identification of Papaya by Probe Method Sex identification of papaya was performed by the probe method using the obtained sex identification marker. First, the restriction enzyme EcoRI
After 10 μg of male, female and amphoteric DNAs digested with the above method were subjected to 0.8% agarose gel electrophoresis, the DNAs were subjected to alkali blotting on Hybond N + (Amersham Pharmacia Biotech) according to a conventional method. On the other hand, the amplified fragment obtained in Example 3 was labeled with ³² P. That is, 20 ng of the amplified fragment
After labeling with ³² P using rediprimeTMII (manufactured by Amersham Pharmacia Biotech), ProbeQuantTM G-50 Mic
ro Column (Amersham Pharmacia Biotech)
The purified product was used as a probe. Next, after pre-hybridizing the blot prepared above at 65 ° C. for 4 hours, the heat-denatured probe was added, and
Hybridization was carried out at ℃ for 16 hours. Next, the blot was blotted for 20 minutes at 65 ° C. in 2 × SSC solution (containing 0.1% SDS)
Twice, followed by 0.1 x SSC solution (containing 0.1% SDS) at 65 ° C for 20
Washed twice for 2 minutes. Finally, wash the washed blot with BAS20
00 (manufactured by Fuji Photo Film Co., Ltd.).

FIG. 5 shows the results. Male is Okinawa Pref.IK
7. The female is Weinamaro and both genders are sunrise. FIG.
As is clear from the above, the band was detected only in the male and bisexual test plants, but not in the female. From the above, it was found that sex identification of papaya can be performed based on the analysis of the sex identification marker of the present invention by the probe method.

[0043]

According to the present invention, sex identification of papaya can be performed using a DNA marker. Therefore, the use of the method of the present invention enables rapid and highly sensitive sex identification of papaya at the seedling raising stage.

[0044]

[Sequence List] SEQUENCE LISTING <110> Okinawa prefecture <120> A method of identifying of papaya sex using a DNA marker <130> P00-0757 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 450 <212> DNA <213> Carica papaya <400> 1 ttggcacggg ctctgagcca gggtcgtggt aagagttttt cccaagccat ttttttcatt 60 gcttcttctc ttgaaatttt agtaaactca ataaaacttt ggaaaaacta attaaaataa 120 aagggctaat aattctacat gaaaaatttt gcacgattta gattagatgt attgtaattt 180 taaattcgtt aagtaccaaa aagttagagt attacaacac taaactgggc cgaacctagt 240 tggggtccag agaattaggc aagatctcgc gggggtcaat taaactggat tagaccatca 300 acctctatac agacgtgcta agtagctgca cagcgcgaca cgccatggga gcaatgtgac 360 ctgggcaagc tatcccgcac gtgcacatcg cgcgtatata aacctgcgca ctcagcaaga 420 tagcgcagca caacatgctg cccgtgccaa 450 <210> 2 <211> 450 <212> DNA <213> Carica papaya <400> 2 ttggcacggg cagcatgttg tgctgcgcta tcttgctgag tgcgcaggtt tatatacgcg 60 cgatgtgcac gtgcgggata gcttgcccag gtcacattgc tcccatggcg tgtcgcgctg 120 tgcagctact tagcacgtct gtatagaggt tgatggtcta atccagttta attgaccccc 180 gcgagatctt gcctaattct ctggacccca actaggttcg gcccagttta gtgttgtaat 240 actctaactt tttggtactt aacgaattta aaattacaat acatctaatc taaatcgtgc 300 aaaatttttc atgtagaatt attagccctt ttattttaat tagtttttcc aaagttttat 360 tgagtttact aaaatttcaa gagaagaagc aatgaaaaaa atggcttggg aaaaactctt 420 accacgaccc tggctcagag cccgtgccaa 450 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 3 ttggcacggg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 4 ggtaagagtt tttcccaagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 5 tgcacgattt agattagatg 20 <210> 6 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 6 ggatagcttg cccaggtcac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: synthetic DNA <400> 7 gttgtgctgc gctatcttgc 20

[0045]

[Sequence List Free Text] SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram showing the positions of the sequences of SEQ ID NOS: 4 to 7 in the entire base sequence of a papaya identification marker.

FIG. 2: DNs extracted from leaves of 17 known papaya individuals
4 is an electrophoresis photograph showing the results of PCR when A was used as a template.

FIG. 3. DNs extracted from leaves of 16 papayas of known sex
4 is an electrophoresis photograph showing the results of PCR when A was used as a template.

FIG. 4 is an electrophoretic photograph showing the results of sex identification of papaya based on analysis of sex identification markers by PCR.

FIG. 5 is an electrophoretic photograph showing the results of sex identification of papaya based on the analysis of sex identification markers by the probe method.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA08 AA11 BA80 CA09 HA12 4B063 QA01 QA13 QA18 QQ04 QQ42 QQ52 QR08 QR55 QR62 QS02 QS25 QS34

Claims (14)

[Claims] 1. A DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 or 2, or a DNA containing a polymorph of the DNA. 2. A papaya sex identification marker comprising a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2 or a polymorph of the DNA. 3. A primer for amplifying a sex identification marker of papaya, comprising a part of the DNA according to claim 1. 4. The primer for amplifying a sex identification marker of papaya according to claim 3, wherein a part of the DNA has a length of 17 to 30 mer. 5. The primer for amplifying a sex identification marker of papaya according to claim 3, wherein a part of the DNA is represented by any of SEQ ID NOs: 4 to 7. 6. A papaya sex discrimination kit comprising the primer for amplifying a sex discrimination marker according to any one of claims 3 to 5 as a component. 7. A probe for detecting a papaya sex identification marker containing a part or all of the DNA according to claim 1. 8. The probe for detecting a sex identification marker of papaya according to claim 7, wherein a part of the DNA has a length of 20 to 450 mer. 9. A papaya gender identification kit comprising the gender identification marker detection probe according to claim 7 or 8 as a constituent element. 10. A papaya gender identification method, characterized by analyzing the presence or absence of the papaya gender identification marker according to claim 2 in a subject papaya. 11. The papaya sex according to claim 10, wherein the analysis of the presence or absence of the sex identification marker of papaya is performed by detecting an amplification product derived from the sex identification marker obtained by polymerase chain reaction. Identification method. 12. The polymerase chain reaction is carried out using a papaya-derived DNA as a template as a template and the primer for amplifying a gender identification marker according to any one of claims 3 to 5 as a primer. 12. The method for identifying sex of papaya according to 11. 13. The papaya sex identification method according to claim 10, wherein the analysis of the presence or absence of the papaya sex identification marker is performed by detecting a hybridized product obtained by a probe hybridization reaction. 14. The probe hybridization reaction according to claim 13, wherein the probe hybridization reaction is carried out by hybridizing the probe for detecting a sex identification marker according to claim 7 to DNA derived from a subject papaya. Sex identification method of papaya.