TW202242141A - Test kit for identifying all-hermaphrodite papaya and testing method for identifying all-hermaphrodite papaya derived from taiwan seed station no. 7 and its progeny - Google Patents

Abstract

The present disclosure provides a test kit for identifying all-hermaphrodite papaya, which is used for identifying all-hermaphrodite papaya derived from Taiwan Seed Station No. 7 and its progeny and detecting whether a papaya sample has a nucleotide sequence of SEQ ID NO: 1. The test kit for identifying all-hermaphrodite papaya includes a first primer set and a second primer set. The first primer set includes a forward primer of SEQ ID NO: 2 and a reverse primer of SEQ ID NO: 3. The second primer set includes a forward primer of SEQ ID NO: 4 and a reverse primer of SEQ ID NO: 5. The present disclosure also provides a testing method for identifying all-hermaphrodite papaya derived from Taiwan Seed Station no. 7 and its progeny using the aforementioned test kit.

Description

Molecular markers for identifying papaya seedling No. 7 and its descendants of hermaphrodite papaya, test kits for identifying papaya seedling 7 and its descendants of hermaphrodite papaya

The present invention relates to a molecular marker for identifying the sex of papaya, a test set for identifying the sex of papaya, and a method for identifying the sex of papaya, in particular to a molecular marker for identifying papaya seedling No. 7 and its descendants of all hermaphrodite papaya, A test set for differentiating hermaphrodite papaya and a method for distinguishing hermaphrodite papaya seedling No. 7 and its descendants.

Papaya ( Carica papaya ), also known as papaya, stone melon, pomegranate fruit, etc., is a softwood fruit tree native to tropical America. Papaya is rich in carotene, vitamin C, a high amount of enzymes and a variety of ingredients beneficial to human health, making its planting volume grow steadily all over the world.

The gender form of papaya is one of the most important characteristics in cultivation. It has three basic tree types: male plant, female plant, and hermaphrodite plant. Among them, female plants only open female flowers, and male plants or hermaphrodite plants need to provide pollen to make the fruit develop normally; male plants generally only open male flowers but not fruit, and are mostly used as pollination trees; hermaphrodite plants open hermaphrodite flowers, and their fruits are more It is pear-shaped or long-shaped, and has the characteristics of a small fruit cavity, which makes the hermaphrodite papaya have better economic value and is widely welcomed by papaya cultivators.

In order to plant and cultivate hermaphrodite papaya, during the papaya cultivation process, human efforts are often used to identify and remove female plants in order to obtain a higher proportion of hermaphrodite plants. However, from germination, growth to the formation of flower organs of papaya, plants of different sexes are roughly the same in various external forms such as leaf shape, stem color, and plant shape. After the plant blooms, the gender can be identified according to its flower shape and inflorescence. In this way, not only must all the papaya seedlings be planted until they mature and bloom, so that the cost of the seedlings remains high, and a large amount of labor expenditure is also required. Benefits are not ideal. Furthermore, the traits of papaya seedlings will be affected by cultivation in different environments, and degeneration and deformity may even occur when the environmental temperature is too high or low, so that the accuracy and benefits of human identification and removal of female plants are still to be determined. Discuss.

In addition, among the offspring produced by selfing or intercrossing of hermaphrodite papaya plants, the number of hermaphrodite plants accounts for about 67%. Papaya cultivators must also plant 2 to 3 papaya seedlings at a time, and they will form after 3 to 4 months after planting. Flower vessels can only confirm their gender by the structure of the flower vessel. Therefore, it is impossible to obtain 100% offspring of hermaphrodites through selfing or mutual hybridization of hermaphrodites.

In order to solve the aforementioned problems, molecular markers related to papaya sex have been vigorously developed to identify the sex of papaya seedlings when they cannot be visually identified. However, the current molecular markers related to the sex of papaya are still difficult to identify the sex of papaya one by one in a large number, and are rarely used in the production of papaya seedlings.

In order to meet the demands of planting and cultivating 100% hermaphrodite papaya, the Seedling Improvement and Breeding Farm of the Agricultural Committee of the Executive Yuan further selected and bred a papaya variety that can obtain 100% hermaphrodite offspring - Papaya Seedling No. 7. However, after introducing the hermaphroditism of Papaya Seedling No. 7 into other papaya varieties, it is still impossible to effectively distinguish the hermaphrodite papaya and the fully hermaphrodite papaya in the descendants of Papaya Seedling No. 7, which still limits its subsequent application. .

In view of this, how to develop a method for distinguishing papaya seedling No. 7 and its descendants of papaya papaya fully hermaphrodite, so as to effectively select papaya papaya papaya plants with better economic value in the early stage of selection of papaya varieties, is a technology in the art The goal of the people's joint efforts.

One aspect of the present invention is to provide a molecular marker for distinguishing papaya seedling No. 7 and its descendants of fully hermaphrodite papaya, comprising a nucleotide sequence as shown in SEQ ID NO: 1, wherein such as SEQ ID NO: 1 The nucleotide sequence shown contains a single nucleotide polymorphism (SNP) locus of monodehydroascorbate reductase 4 (MDAR4).

Another aspect of the present invention is to provide a test kit for identifying hermaphrodite papaya, which is used to identify papaya seedling No. 7 and its descendants, including a first primer set and a second primer set. The first primer set includes a forward primer as shown in SEQ ID NO: 2 and a reverse primer as shown in SEQ ID NO: 3. The second primer set includes a forward primer as shown in SEQ ID NO: 4 and a reverse primer as shown in SEQ ID NO: 5. Wherein, the test kit for identifying fully hermaphrodite papaya is used to detect whether a papaya sample has the nucleotide sequence shown in SEQ ID NO: 1.

According to the above-mentioned test kit for distinguishing all hermaphrodite papaya, it may further include a reagent required for reverse transcription polymerase chain reaction.

Yet another aspect of the present invention is to provide a method for identifying papaya seedling No. 7 and its descendants of hermaphrodite papaya, which includes the following steps. A test nucleic acid sample of a papaya sample is provided. A test kit for differentiating hermaphrodite papaya as described in the preceding paragraph is provided. Carrying out a first nucleic acid amplification step, which uses the nucleic acid sample to be tested as a template and uses the aforementioned first primer set to carry out a polymerase chain reaction to obtain a first reaction product. A second nucleic acid amplification step is carried out, which uses the nucleic acid sample to be detected as a template and uses the aforementioned second primer set to perform a polymerase chain reaction to obtain a second reaction product. A detection step is performed to detect whether the first reaction product has a first amplicon product, and detect whether the second reaction product has a second amplicon product. Wherein, when the first reaction product does not have the first amplicon product and the second reaction product has the second amplicon product, the papaya sample will be judged as a fully amphoteric papaya sample.

According to the above-mentioned method for identifying papaya seedling No. 7 and its offspring's all-amptersexual papaya, the molecular weight of the first amplicon product can be equal to 403 bp, and the molecular weight of the second amplicon product can be equal to 282 bp .

According to the aforementioned method for identifying papaya seedling No. 7 and hermaphrodite papaya of its descendants, the aforementioned nucleic acid sample to be tested can be DNA or RNA.

According to the aforementioned method for identifying the hermaphrodite papaya of papaya seedling No. 7 and its descendants, when the aforementioned nucleic acid sample to be tested is RNA, the method for identifying the hermaphrodite papaya of papaya seedling No. 7 and its descendants may further include performing a reverse reaction The transcription step is to perform a reverse transcription reaction on the aforementioned nucleic acid sample to be tested.

According to the above-mentioned method for identifying papaya seedling No. 7 and its offspring's all-hermaphrodite papaya, the above-mentioned detection step can be performed by a gel electrophoresis system.

According to the aforementioned method for identifying papaya seedling No. 7 and hermaphrodite papaya of its descendants, the aforementioned papaya samples can be papaya seeds, papaya leaves or papaya flowers.

In this way, the test kit for identifying all-hermaphrodite papaya and the method for identifying all-hermaphrodite papaya seedling No. 7 and its descendants of the present invention detect whether the papaya sample has the method for identifying papaya seedling No. 7 and its descendants of the present invention. Molecular markers of hermaphrodite papaya can quickly and accurately select papaya species with better economic value in the early stage of papaya variety selection, which has the hermaphrodite characteristics of papaya seedling No. 7, thereby reducing the manpower for seed selection and planting in the future cost, and has application potential in relevant markets.

The present invention provides a test set for identifying all hermaphrodite papaya and a method for identifying papaya seedling No. 7 and its descendants of hermaphrodite papaya, which is used to identify papaya seedling No. 7 and its descendants by detecting whether there are papaya samples of the present invention The molecular markers of hermaphrodite papaya can quickly and accurately select papaya plants with better economic value in the early stage of papaya variety selection and have the full hermaphrodite characteristics of papaya seedling No. 7, and can further ensure that the identified All hermaphrodite papaya plants can obtain 100% hermaphrodite offspring through main biological methods.

The papaya of the present invention may comprise the Florida species, Risheng species, seedling No. 7 species of papaya ( Carica papaya L.) or the offspring obtained by hybridizing with seedling No. 7 species, or the aforementioned descendants are further mixed with Florida species, The descendants of Rishengzhong, Zhongmiao No.7 and other papaya varieties through cross breeding. Preferably, the hermaphrodite papaya described in the present invention is the papaya of seedling No. 7 species or hermaphrodite papaya of its descendants.

The sex of papaya in the present invention refers to male, female, hermaphrodite and all hermaphrodites. In detail, according to the theory proposed by Storey in 1941 and 1953, the sex of papaya plants (male, female and hermaphrodite) is controlled by three alleles: M ₁ (male, dominant gene), M ₂ (bisexual, dominant gene) and m (female, recessive gene), where the genotypes of mm, mM ₁ and mM ₂ are female, while the dominant alleles (M ₁ M ₁ , M ₁ M ₂ and M ₂ M ₂ ) genotypes are lethal. Usually the ratio of hermaphrodites to females is 2:1 in the offspring produced by selfing hermaphrodites, and the ratio of hermaphrodites to females in the offspring produced by hybridization of hermaphrodites and females is 1:1. Furthermore, the all-hermaphroditic papaya in the present invention means that the progeny plants produced by selfing and capable of normal growth are all hermaphrodites.

Various embodiments of the invention are discussed in more detail below. However, this embodiment may be an application of various inventive concepts, and may be embodied in various specific ranges. The specific embodiments are for illustrative purposes only and do not limit the scope of the disclosure.

[The present invention is used to distinguish the molecular marker of papaya seedling No. 7 and its offspring's all-hermaphrodite papaya]

The molecular marker of the present invention for distinguishing papaya seedling No. 7 and its descendants of fully hermaphrodite papaya comprises a nucleotide sequence as shown in SEQ ID NO: 1, wherein the nucleotide sequence as shown in SEQ ID NO: 1 Contains a single nucleotide polymorphism (single nucleotide polymorphism, SNP) locus of monodehydroascorbate reductase 4 (monodehydroascorbate reductase 4, which will be referred to as "MDAR4" for simplicity in the following). MDAR4 is one of the enzymes that plants participate in the ascorbic acid-dependent hydrogen peroxide (H ₂ O ₂ ) scavenging pathway, and is related to seed germination, and it is deduced that it also plays a similar function in papaya, and is involved in papaya seed germination and grow.

In detail, the molecular marker used to identify papaya seedling No. 7 and its progeny's all-hermaphrodite papaya of the present invention is the gene coding sequence (Coding sequence, CSD) of MDAR4 comprising the SNP locus, as shown in Table 1, compared In the papaya Bacterial Artificial Chromosome (BAC) gene bank number AC238599.1, the molecular marker of the present invention used to identify papaya seedling No. 7 and its descendants' fully hermaphrodite papaya compared with the normal MDAR4 gene coding sequence contains Three insertion-deletion SNP sites, the insertion-deletion SNP sites will lead to the loss of Val362 in the translated amino acid sequence, and affect the germination and growth of papaya seeds. Table I Papaya BAC Number BAC sequence variation position reference nucleotide SEQ ID NO: 1 sequence variation type AC238599.1 22,431 bp to 22,433 bp GTA missing

Therefore, when the X chromosome of the papaya sample comprises the nucleotide sequence shown in SEQ ID NO: 1, if the papaya sample is not a hermaphrodite and does not have a normal MDAR4 gene coding sequence, the papaya sample will be in its seed stage. Unable to germinate and grow normally. Furthermore, when the papaya sample is a fully hermaphroditic plant with normal growth, the offspring of the female plant obtained by selfing will have seed lethality due to the defective expression of the MDAR4 gene. The offspring after growth will all be all hermaphrodite offspring with the all hermaphroditism of No. 7 papaya seedlings (being the all hermaphrodite plant of the present invention). Thus it can be seen that the molecular markers of the present invention used to identify papaya seedling No. 7 and its descendants' fully hermaphrodite papaya plants can be effectively and accurately identified to obtain papaya seedling No. 7 fully hermaphrodite papaya plants, and can be further applied to selection of papaya seedlings All hermaphrodite papaya plants in the descendants of No. 7, so as to effectively select papaya papaya plants with better economic value at the early stage of papaya variety selection, which have the full hermaphrodite characteristics of papaya seedling No. 7, and have application potential in related markets .

Furthermore, the molecular markers of the present invention used to identify papaya seedling No. 7 and its descendants of the all-hermaphroditic papaya include the nucleotide sequence itself as shown in SEQ ID NO: 1, and may also include derivatives thereof and Those having the nucleotide sequence shown in SEQ ID NO: 1, such as PCR products, HRM products, RFLP products, RNA transcribed from the above sequence or polypeptides translated from the above sequence, etc., and the present invention does not refer to this limit.

In addition, in this specification, the gene coding sequence of normal MDAR4 is further referred to as "Gs allele", and the gene coding sequence of the present invention expressing defective MDAR4 (ie, the nucleus shown in SEQ ID NO: 1 Nucleotide sequence) is referred to as "gs allele of the present invention" to simplify the relevant description.

[The test set for differentiating all hermaphrodite papaya of the present invention]

The test set for differentiating hermaphrodite papaya of the present invention is used to identify hermaphrodite papaya seedling No. 7 and its descendants, and includes a first primer set and a second primer set. The first primer set includes a forward primer as shown in SEQ ID NO: 2 and a reverse primer as shown in SEQ ID NO: 3. The second primer set includes a forward primer as shown in SEQ ID NO: 4 and a reverse primer as shown in SEQ ID NO: 5.

In detail, in the test set for identifying all-hermaphrodite papaya of the present invention, the first primer set and the second primer set are designed by using the primer design program BatchPrimer3 v1.0 respectively, wherein the forward primer of the first primer set (the forward primer shown in SEQ ID NO: 2) and the reverse primer (the reverse primer shown in SEQ ID NO: 3) are designed for the Gs allele, and the forward primer of the second primer group The primer (the forward primer shown in SEQ ID NO: 4) and the reverse primer (the reverse primer shown in SEQ ID NO: 5) are designed for the gs allele of the present invention, and the present invention The test kit for identifying hermaphrodite papaya is used to detect whether a papaya sample has the nucleotide sequence shown in SEQ ID NO:1.

Specifically, when papaya samples are identified using the test kit for identifying fully hermaphrodite papaya of the present invention, since papaya seedling No. 7 and hermaphrodite papaya plants of its descendants have a deletion in the gene coding sequence of MDAR4, the positive The forward primer and the reverse primer will not be able to combine with the nucleic acid sample of the papaya sample to obtain the first amplicon product of the first primer set. On the contrary, because the papaya seedling No. 7 and its progeny's fully hermaphrodite papaya plants comprise the nucleotide sequence shown in SEQ ID NO: 1 (i.e. the gs allele of the present invention), the forward primer and reverse primer of the second primer group The primers can be combined with the tested nucleic acid sample of the papaya sample to obtain the second amplicon product of the second primer set, so that the test kit for identifying all-hermaphrodite papaya of the present invention can be quickly and accurately used in the early stage of papaya variety selection. Phase selection of papaya seedlings with better economic value No. 7 all-hermaphrodite papaya plants can be further applied to selection of all-hermaphrodite papaya plants in descendants of papaya seedling No. 7, thereby reducing labor costs for seed selection and planting in the future, and It has application potential in relevant markets.

In addition, the test kit for identifying all-hermaphrodite papaya of the present invention may include reagents required for reverse transcription polymerase chain reaction as required, but the present invention is not limited thereto.

[The method of distinguishing papaya seedling No. 7 of the present invention and its progeny's all-hermaphrodite papaya]

Please refer to FIG. 1 , which is a flow chart showing the steps of a method 100 for identifying papaya seedling No. 7 and its offspring's all-hermaphrodite papaya according to an embodiment of the present invention. The method 100 for identifying the hermaphrodite papaya seedling No. 7 and its descendants includes step 110 , step 120 , step 130 , step 140 and step 150 .

Step 110 is to provide a test nucleic acid sample of a papaya sample. Specifically, the papaya sample can be papaya seeds or papaya leaves, and can also be used as a papaya sample after the flowers appear, but the present invention is not limited thereto. Furthermore, the nucleic acid sample to be tested can be DNA or RNA.

Step 120 is to provide a test kit for identifying all hermaphrodite papayas. Specifically, the test set for identifying all-hermaphroditic papaya can be the test set for identifying all-hermaphroditic papaya according to the present invention, which includes a first primer set and a second primer set, and its details can be found in the preceding paragraph. This will not be repeated.

Step 130 is to perform a first nucleic acid amplification step, which is to use the nucleic acid sample to be detected as a template and use the first primer set to perform a polymerase chain reaction to obtain a first reaction product.

Step 140 is to perform a second nucleic acid amplification step, which is to use the nucleic acid sample to be detected as a template and use the second primer set to perform a polymerase chain reaction to obtain a second reaction product.

Step 150 is to perform a detection step, which is to detect whether the first reaction product has a first amplicon product, and detect whether the second reaction product has a second amplicon product. Specifically, the detection step is performed by a gel electrophoresis system, and the molecular weight of the first amplicon product is equal to 403 bp, and the molecular weight of the second amplicon product is equal to 282 bp. Specifically, the first amplicon product is the PCR amplification product corresponding to the Gs allele, and the second amplicon product is the PCR amplification product corresponding to the gs allele, so that The method 100 of the present invention for identifying papaya seedling No. 7 and its all-hermaphrodite papaya descendants is by detecting whether the first reaction product has the first amplicon product and detecting whether the second reaction product has the second amplicon product , can quickly and effectively identify whether the papaya sample is the hermaphrodite papaya plant of No. 7 papaya seedling, and can be further applied to select the hermaphrodite papaya plant in the descendants of papaya seedling No. 7, thereby reducing the labor cost of seed selection and planting in the future , and has application potential in related markets.

Please refer to FIG. 2 , which is a flow chart showing the steps of a method 200 for identifying papaya seedling No. 7 and its descendants' all-hermaphrodite papaya according to another embodiment of the present invention. The method 200 for identifying papaya seedling No. 7 and its progeny's all-hermaphrodite papaya comprises steps 210, 220, 230, 240, 250 and 260, wherein steps 210, 220, 240, 250 and 260 are the same as Step 110 , step 120 , step 130 , step 140 and step 150 in FIG. 1 are the same and will not be repeated here.

Step 230 is performing a reverse transcription step, which is to perform a reverse transcription reaction on the nucleic acid sample to be tested. In detail, when the nucleic acid sample to be detected is RNA, the RNA will first undergo reverse transcription reaction to obtain corresponding cDNA for subsequent polymerase chain reaction. In this way, the application range of the method 200 of the present invention for identifying papaya seedling No. 7 and hermaphrodite descendants thereof can be further improved.

[Test example]

1. Papaya sample

The papaya plants used in the present invention are grown in the Pingtung Seedling Research Center, wherein the varieties of papaya plants include the male Florida species, the female Sunrise species and Florida species, the normal hermaphrodite Sunrise species and all hermaphrodite seedlings7 No. species, and a single papaya seed or papaya leaf is used as the papaya sample of the present invention to obtain the corresponding nucleic acid sample to be tested for subsequent experiments.

The nucleic acid samples to be tested used in this test can be divided into DNA and RNA. When DNA is selected as the nucleic acid sample to be tested, a single papaya seed or 40 mg of papaya leaf tissue will be treated with TANBead ^® Plant DNA Auto Kit (Taiwan Advanced Nanotech Inc, Taiwan) to extract total genomic DNA (Total genomic DNA), and when the test When RNA is selected as the nucleic acid sample, a single papaya seed or 40 mg of papaya leaves will be treated with TANBead ^® Plant RNA Auto Kit (Taiwan Advanced Nanotech Inc, Taiwan) to extract total RNA for subsequent experiments. In addition, the aforementioned single papaya seed line extracts the DNA or RNA of the kernel to increase the accuracy of the test.

The polymerase chain reaction

Polymerase chain reaction is a nucleic acid synthesis technology that replicates specific DNA fragments outside the body. The nucleic acid sample to be tested must be in the form of DNA before it can react. Therefore, when RNA is selected as the nucleic acid sample to be tested in the present invention, 1 μg of papaya sample total RNA will be processed with Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science, Germany) to obtain the first strand cDNA ( 1 ^st strand cDNA) for subsequent reactions.

In polymerase chain reaction, 10 ng of total genomic DNA obtained from a single papaya seed, 50 ng of total genomic DNA obtained from papaya leaves, or 2 ul of first-strand cDNA will be mixed with 1×PCR buffer, 0.8 units of ProTaq polymerase (Protech Technology Enterprise, Taiwan), 0.1 mM of dNTP and 0.2 mM of the first primer set or the second primer set of the present invention were mixed to form a PCR reaction product with a total volume of 20 μl, and placed in the polymerase The reaction was carried out in an enzyme chain reaction apparatus, and the polymerase chain reaction was carried out under the following conditions: react at 95°C for 3 minutes, then react at 95°C for 30 seconds, and then at an annealing temperature of 55°C or 60°C React for 30 seconds, then react at 72°C for 1 minute; the aforementioned reaction will be carried out for 35 cycles, and finally react at 72°C for 3 minutes to end the reaction, so as to obtain the first reaction corresponding to the first primer group The product or the second reaction product corresponding to the second primer set.

Next, the aforementioned first reaction product or second reaction product will be separated by gel electrophoresis on 1.5% agarose gel to detect whether the first reaction product has the first amplicon product, and to detect the second reaction product Whether the product has a second amplicon product where the first amplicon product corresponds to the PCR amplification product of the Gs allele and the second amplicon product corresponds to the PCR amplification product of the gs allele increase product. In addition, this test also uses the papain gene fragment as an internal control group to ensure the accuracy of this test.

Please refer to Figure 3 and Figure 4, Figure 3 is a colloidal electrophoresis analysis diagram for polymerase chain reaction of the test kit for identifying all amphoteric papayas of the present invention, and Figure 4 is a diagram of the present invention Another gel electrophoresis profile of the polymerase chain reaction of the test kit for differentiating fully hermaphrodite papaya.

As shown in Figure 3, when the first primer set of the present invention is used to carry out the polymerase chain reaction on the nucleic acid sample to be tested in the papaya sample, the male Florida species, the female Sunrise species, the female Florida species and normal hermaphrodites The first reaction products of Risheng species all had the polymerase chain reaction amplification product of the Gs allele with a molecular weight equal to 403 bp, showing that there was no variation in the MDAR4 gene coding sequence. However, since the fully hermaphroditic papaya seedling No. 7 contains three indel SNP sites in the MDAR4 gene coding sequence, it cannot combine with the first primer set and obtain the amplicon product of the first primer set.

Again as shown in Figure 4, when the second primer set of the present invention is used to carry out the polymerase chain reaction on the nucleic acid sample to be tested in the papaya sample, the second reaction product of the all-hermaphrodite papaya seedling No. 7 has a molecular weight equal to 282 bp The polymerase chain reaction amplification product of the gs allele, but no male Florida species, female Sunrise species, female Florida species, and normal hermaphrodite Sunrise species, shows that the second primer set of the present invention is indeed effective Detecting the molecular markers of the present invention for identifying papaya seedling No. 7 and hermaphrodite papaya descendants thereof can effectively distinguish whether a papaya sample is a hermaphrodite papaya plant.

From the above results, it can be seen that the test kit for identifying papaya seedlings of the present invention and the method for identifying papaya seedling No. 7 and its descendants are hermaphroditic papayas by detecting whether the papaya samples contain the papaya seedling No. 7 and its descendants of the present invention. Molecular markers of the hermaphrodite papaya descendants can quickly and accurately select the hermaphrodite papaya plants with better economic value in the early stage of papaya variety selection, and can be further applied to the selection of descendants of papaya seedling No. 7 All hermaphrodite papaya plants in the plant, thereby reducing the labor cost of seed selection and planting in the future, and has application potential in related markets.

Although the present invention has been disclosed above in terms of implementation, it is not intended to limit the present invention. Anyone skilled in this art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be defined by the appended patent application scope.

100,200: The method for identifying papaya seedling No. 7 and its all-hermaphroditic papaya descendants 110,120,130,140,150,210,220,230,240,250,260: steps

Figure 1 is a flow chart showing the steps of a method for identifying papaya seedling No. 7 and its descendants' all-hermaphrodite papaya according to an embodiment of the present invention; Fig. 2 is a flow chart showing the steps of the method for identifying papaya seedling No. 7 and its descendants' fully hermaphrodite papaya according to another embodiment of the present invention; Fig. 3 is a gel electrophoresis analysis diagram for polymerase chain reaction of the test kit for distinguishing whole hermaphrodite papaya of the present invention; and Fig. 4 is another gel electrophoresis analysis diagram for the polymerase chain reaction of the test kit for differentiating fully hermaphrodite papaya according to the present invention.

100: The method for identifying papaya seedling No. 7 and its descendants of the hermaphrodite papaya

110,120,130,140,150: steps

Claims (9)

A molecular marker for distinguishing papaya seedling No. 7 and its descendants of fully hermaphrodite papaya, comprising a nucleotide sequence shown in SEQ ID NO: 1, wherein the nucleotide sequence shown in SEQ ID NO: 1 Contains a single nucleotide polymorphism (SNP) locus of monodehydroascorbate reductase 4 (MDAR4). A test kit for identifying hermaphrodite papaya, which is used to identify hermaphrodite papaya seedling No. 7 and its descendants, comprising: A first primer set, including: A forward primer as shown in SEQ ID NO: 2; and A reverse primer as shown in SEQ ID NO: 3; and A second set of primers, including: A forward primer as shown in SEQ ID NO: 4; and A reverse primer as shown in SEQ ID NO: 5; Wherein, the test kit for identifying all hermaphrodite papaya is used to detect whether a papaya sample has the nucleotide sequence shown in SEQ ID NO: 1. The test kit for identifying all hermaphrodite papaya as described in claim 2 further includes: A reagent required for reverse transcription polymerase chain reaction. A method for identifying papaya seedling No. 7 and its all-hermaphroditic papaya descendants, comprising: Provide a test nucleic acid sample of a papaya sample; Provide a test kit for identifying all-hermaphrodite papaya as described in claim 2; Carrying out a first nucleic acid amplification step, which is to use the nucleic acid sample to be tested as a template and use the first primer set to perform a polymerase chain reaction to obtain a first reaction product; Carrying out a second nucleic acid amplification step, which is to use the nucleic acid sample to be tested as a template and use the second primer set to perform a polymerase chain reaction to obtain a second reaction product; and performing a detection step of detecting whether the first reaction product has a first amplicon product and detecting whether the second reaction product has a second amplicon product; Wherein, when the first reaction product does not have the first amplicon product and the second reaction product has the second amplicon product, the papaya sample will be determined as a fully amphipathic papaya sample. The method for identifying papaya seedling No. 7 and its offspring's all-hermaphrodite papaya as described in claim 4, wherein the molecular weight of the first amplicon product is equal to 403 bp, and the molecular weight of the second amplicon product is equal to 282 bp. The method for identifying papaya seedling No. 7 and hermaphrodite papaya of its descendants as described in claim 4, wherein the nucleic acid sample to be tested is DNA or RNA. The method for identifying papaya seedling No. 7 and its all-hermaphrodite papaya of its descendants as described in claim 6, wherein when the nucleic acid sample to be tested is RNA, the method for identifying papaya seedling No. 7 and its all-hermaphrodite papaya of its descendants further includes : A reverse transcription step is carried out, which is to perform a reverse transcription reaction on the nucleic acid sample to be tested. The method for identifying papaya seedling No. 7 and its offspring's all-hermaphrodite papaya as described in claim 4, wherein the detection step is performed by a gel electrophoresis system. The method for identifying papaya seedling No. 7 and hermaphrodite papaya of its descendants as described in claim 4, wherein the papaya sample is papaya seeds, papaya leaves or papaya flowers.

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