TWI600768B - Molecular marker and application for early determining sexes and sex-related traits of papaya - Google Patents

Description

For early identification of papaya sex and gender-related traits Molecular markers and their applications

The present invention relates to a molecular marker and its use, and more particularly to a molecular marker and method and system for early identification of papaya sex and gender-related traits.

Papaya ( Carica papaya L.) is commonly known as papaya, belonging to the Caricaceae family and the Carica plant. Papaya can be rich in nutritional value throughout the year and is an important crop in the tropics. Papaya also has dioecious, which are female (F), male (Male), and hermaphrodite (Hermaphrodite, H).

In general, male plants are not fruitful and have no economic value. Female plants need artificial pollination to get results. Amphiphilic strains can be self-pollinated and have high economic value, so commercial growers prefer to grow pure amphoteric strains.

Before the formation of papaya from the growth to the flower bud, no external characteristics such as leaf type, stem color, plant type and so on, but no external characteristics linked to the sex of the plant were found, and the external type could not be used to judge the sex of the plant. Farmers get For the more economically valuable amphoteric strains, 2-3 papaya seedlings should be planted in each planting hole. After the flower buds are formed for 6-8 months, the flower structure can be confirmed by the flower structure. An amphibious strain, the rest of the plants were cut off to achieve the long oval fruit of the whole garden. However, such cultivation methods not only cause nutrient competition and waste of papaya seedlings, but also cause a large amount of labor expenditure for the growers, and increase seedling costs and management costs. In addition, some of the acupoints may be caused by lack of planting to the amphipathic strain, and it is difficult to control the normal plant spacing.

In order to produce papaya seedlings of the same sex, papaya seedlings often use asexual reproduction methods, such as tissue culture, cutting, grafting and other techniques. Among these methods, the commercial production of tissue culture and reproduction, the multiplication ratio is high and all the plants are amphoteric strains, but the technical threshold of this production method is high, the production cost is relatively increased, and the price of tissue culture seedlings is about 7-15 times the seedlings. Grafted seedlings need to separately cultivate the mother ears and rootstocks. There are many production steps, which require large production space and humidification equipment, and the seedling production speed is slow. As for the breeding rate of cutting seedlings, although it is higher than that of grafted seedlings, because there are no seed roots as well as tissue culture seedlings, some farmers think that their tolerance to the adversity environment is poor and they do not like planting. Therefore, for most farmers, seedlings of whole amphipathic strains are considered to be more developmental seed production methods.

In recent years, biotechnology-related technologies have advanced rapidly. At present, molecular markers have been proven to be early detection in biology, solve many problems, and have excellent application value. Common types of molecular marker detection can be, for example, Simple Sequence Repeat (SSR), Inter-Simple Sequence Repeats (ISSR), and Random Amplified DNA (Random Amplified). Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Sequence Characterized Amplified Region (Sequence Characterized Amplified Region, SCAR) and so on.

For example, in the past studies, the above SSR can be used to distinguish the identification of Dendrobium species and the identification of papaya. ISSR can be applied to the identification and analysis of papaya and eggplant. RAPD, AFLP and RFLP can be applied to distinguish papaya sex. SCAR is derived from RAPD and can be used as a molecular marker to distinguish the sex of papaya.

Some molecular markers have been developed and can be used to identify sex in cases where papaya seedlings cannot be visually identified. However, most of the current molecular markers of papaya sex-related genes, a single molecular marker is only for the identification of a single gender, can not identify three genders at the same time. Secondly, the flowering of papaya young plants under different environmental conditions will be unstable, especially in the case of unsuitable environment or lack of nutrients, even the phenomenon of degeneration and deformity will affect the gender-related traits. Affect the accuracy of identifying gender and gender-related traits.

In view of this, it is urgent to develop a molecular marker for early identification of sex and gender-related traits of papaya to improve the shortcomings of conventional molecular markers.

Therefore, one aspect of the present invention is to provide an early use Identification of molecular markers of papaya sex and gender-related traits.

Another aspect of the present invention provides a method for early identification of sex and sex-related traits in papaya, which is composed of a plurality of single nucleotide polymorphic sites, a gene sequence, and a transcript sequence. A sex-related gene establishes a correlation model, which simultaneously and accurately identifies gender and gender-related traits in the early development of flowering of papaya samples.

Yet another aspect of the present invention provides a system for identifying an early identification of sex and gender-related traits of papaya, comprising the above molecular markers.

A further aspect of the invention provides a recombinant vector comprising the molecular markers described above.

Yet another aspect of the present invention is to provide a transformed plant cell comprising the recombinant vector described above.

According to the above aspect of the present invention, a molecular marker for early identification of sex and sex-related traits of papaya is proposed, comprising at least one sequence selected from the group consisting of SEQ ID NOS: 1-64 and SEQ ID NO: 1-51, 53 A group consisting of 55, 57, 59, 61, 63 complementary sequences.

According to another aspect of the present invention, a method for early identification of sex and sex-related traits of papaya is proposed by molecular markers comprising a plurality of single nucleotide polymorphic sites, genome sequences and transcript sequences. A number of gender-related genes establish a correlation model to identify gender and gender-related traits simultaneously in the early development of the flowering of papaya samples.

In the above embodiment, the step of establishing a correlation mode may include, but is not limited to, obtaining a plurality of nucleic acid samples from a plurality of flower organs of a plurality of papayas. this. Next, a plurality of molecular markers are obtained from the nucleic acid sample, wherein the molecular marker comprises at least one sequence selected from the group consisting of SEQ ID NOs: 1-64 and SEQ ID NOs: 1-51, 53, 55, 57, 59, 61 A group of 63 complementary sequences. Thereafter, a correspondence relationship between any of the aforementioned sex-related genes and at least one of the aforementioned molecular markers is defined to establish a correlation pattern.

In the above embodiment, in the step of obtaining the nucleic acid sample, the average length of the flower is not more than 0.2 cm, and the nucleic acid sample comprises a plurality of gene sequences and a plurality of transcript sequences.

In the above embodiment, the step of using the correlation mode to identify the sex of the papaya sample and the plurality of sex-related genes may include, but is not limited to, obtaining the nucleic acid sample to be tested from the flower sample of the papaya sample, and obtaining the plurality of nucleic acid samples from the sample to be tested. The gender typing data is input into the above correlation pattern to compare whether the sex typing data matches at least one of the above molecular markers, and obtain a correlation between at least one of the molecular markers Correct rate. When at least one of the aforementioned sex typing data is consistent with at least one of the above molecular markers, and the correlation correctness rate is 100%, it is determined that the papaya sample has a gender of a corresponding relationship and at least one of the sex-related genes.

According to an embodiment of the present invention, the papaya and papaya samples may include, but are not limited to, Carica papaya L., Florida, Risheng, Kuala Lumpur, Risheng 13th, Risheng 23, Risheng Hybrid with Tai Nong No. 7.

According to an embodiment of the present invention, when the second sex classification is performed The materials include SEQ ID NO: 1~16, 18, 20~21, 25~26, 30~31, 35~36, 40~41, 45~46, 49~50, 53~58, 63~64 and SEQ. ID NO: at least one of the complementary sequences of 1~16, 18, 20~21, 25~26, 30~31, 35~36, 40~41, 45~46, 49~50, 53~58, 63~64 When judging, the gender of the papaya sample is determined to be gender.

According to an embodiment of the invention, when the second sex classification data includes SEQ ID NOs: 1-17, 19, 23-24, 28-29, 33-34, 38-39, 43-44, 48, 51~ 52, 61~62 and sequences complementary to SEQ ID NO: 1~17, 19, 23~24, 28~29, 33~34, 38~39, 43~44, 48, 51~52, 61~62 In at least one, the sex of the papaya sample is judged to be male.

According to an embodiment of the present invention, when the second sex classification data includes SEQ ID NOs: 1 to 16, 22, 27, 32, 37, 42, 47, 59 to 60, and SEQ ID NOs: 1 to 16, 22 When at least one of the sequences of 27, 32, 37, 42, 47, and 59 to 60 is complementary, the sex of the papaya sample is determined to be female.

According to an embodiment of the invention, the sex-related trait genes are related to a plurality of gender-related traits, and the gender-related traits comprise a plurality of flower shapes and/or a plurality of fruit shapes.

According to still another aspect of the present invention, a system for early identification of sex and gender-related traits of papaya is proposed, which comprises the above molecular markers for early identification of sex and gender-related traits of papaya.

According to still another aspect of the present invention, there is provided a recombinant vector comprising the above molecular marker.

According to still another aspect of the present invention, there is provided a transforming plant cell comprising the recombinant vector described above.

The molecular marker of the invention for early identification of sex and gender-related traits of papaya and its application can be established by using a specific molecular marker and a gender-related trait gene to establish a correlation pattern, which can be identified in the early development of papaya flower The gender and gender-related traits of this papaya sample were used to obtain papaya strains effectively, quickly and accurately, and to help reduce the production of unconsolidated flowers and malformed flowers, and thus applied in various applications, such as early identification of papaya sex and gender correlation. A system for traits, a recombinant vector comprising the above-described molecular marker, a transformed plant cell containing the recombinant vector, and the like are prepared.

100‧‧‧ method

120/130/140/150/160/170/180/190‧‧‧ steps

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Part of the flow chart of the method of gender-related traits.

2A to 2B are diagrams showing the cDNA sequence of CpSVP-LIKE in different papatype expressions (Fig. 2A) and intron/exon according to Y ^h chromosome BAC 71E16 according to an embodiment of the present invention. The difference in the BAC position (Figure 2B).

[Figure 3A] to [] FIG 3B illustrates the analysis based on the cDNA sequence CpSERK type exhibit different properties of papaya (FIG. 3A) and the intron / exon Y ^h BAC position in accordance with an embodiment of the chromosome BAC71E16 present invention (Figure 3B) the difference.

[Fig. 4A] to Fig. 4E are diagrams showing HRM analysis results of molecular markers of different SNP genotypes as papaya sex and sex-related trait genes according to several embodiments of the present invention, wherein Fig. 10A shows the use of CpSVP-LIKE 88. The HRM analysis results of the primer pair, FIG. 10B is the HRM analysis result using the CpSERK 30704 primer pair, FIG. 10C is the HRM analysis result using the CpSERK 34072 primer pair, FIG. 10D is the HRM analysis result using the CpSERK 34760 primer pair, and FIG. 10E is the utilization result. The results of HRM analysis of CpSERK 34787 primer pairs, F for females, M for males, and H for both sexes.

[Fig. 5A] to [Fig. 5B] are PCR-RFLP analysis results of molecular markers of different SNP genotypes as papaya sex and sex-related trait genes according to several embodiments of the present invention, wherein F represents female and M represents Male, H stands for amphoteric, M on the left stands for DNA marker (Marker), H stands for amphibian papaya strain, and FM stands for Florida male papaya strain.

6 is a three-type gene linkage map and a molecular expression pattern of a papaya sex and sex-related trait gene according to an embodiment of the present invention, wherein F represents a female, M represents a male, and H represents a sex.

As stated above, the present invention provides a molecular marker for early identification of a sex and sex-related trait gene of papaya and its use, which utilizes a plurality of specific single nucleic acid polymorphic sites, genome sequences and transcript sequences. Molecular markers establish a correlation model with gender-related trait genes, so as to accurately identify the sex of papaya samples in the early stage of flower development of papaya samples. And gender-related traits.

The papaya and papaya samples referred to herein are papaya ( Carica papaya L.), and the variety thereof is not particularly limited and may include, but is not limited to, the Florida species of papaya ( Carica papaya L.). Risheng, Kuala Lumpur, Risheng 13th, Risheng 23, Risheng and Tainong 7 hybrids.

The term "papaya" as used herein refers to male, female and amphoteric. According to the theory that Storey proposed in 1938 and 1953, the papaya sex (male, female, and amphoteric) was controlled by three alleles: M1 (male, dominant), M2 (bisexual, dominant), and m (female, recessive), and all combinations of dominant alleles (MM, MMh, MhMh) are embryonic lethal. Usually, the amphipathic strains are selfed, and the proportion of the amphoteric and female plants in the offspring is 2:1. The ratio of the amphoteric and female plants in the offspring of the amphoteric and female plants is 1:1.

The term "gender-related trait" as used herein means that the sex-related trait gene expression and/or trait expression is related to gender. In one example, the gender-related trait comprises a plurality of floral patterns and/or a plurality of fruit shapes or gene expression associated with the traits.

The molecular marker for identifying the sex and sex-related traits of papaya in the early stage of the present invention includes at least one sequence selected from 1 to 64 and SEQ ID NO: 1 to 51, 53, 55, 57, 59, 61, 63. A group of complementary sequences. In one embodiment, the above molecular marker comprises the above sequence itself, or the product derived from the above sequence and contains the above sequence, such as a PCR product, a product of HRM, a product of RFLP, and is transferred from the above sequence. Recorded RNA (or mRNA), a polypeptide translated from the above sequence, and the like.

The term "early development" as used herein means that the average length of the papaya flower is not more than 0.2 cm. In general, flowers with an average length of no more than 0.2 cm cannot visually identify sex, but the molecular markers of the present invention can effectively and quickly and accurately distinguish papaya sex and gender-related traits.

In a specific embodiment, the above molecular marker can be used for early identification of sex and gender-related traits of papaya, which is based on molecular markers and a plurality of sex-related genes to establish a correlation pattern for flower development in papaya samples. Early, simultaneous and accurate identification of gender and gender-related traits. In this embodiment, the above molecular marker may comprise a plurality of single nucleotide polymorphism sites, a genome sequence, a transcript sequence, and a molecular marker of the polypeptide.

In an illustration, the single Nucleotide Polymorphism (SNP) locus used herein to identify the sex and sex-related traits of papaya is a significant correlation with gender and is sufficiently identified. SNP loci for different genders. The aforementioned site may be located in an exon or an intron of a sex-related trait gene, such as SEQ ID NOS: 17-51, 53, 55, 57, 59, 61, 63 and with SEQ ID NO: at least one of the sequences complementary to 17 to 51, 53, 55, 57, 59, 61, and 63.

The above-described genomic sequence for identifying the sex and sex-related traits of papaya refers to one or all of the exons and/or introns of the sex-related trait gene, which is significantly related to sex, such as SEQ ID NO: At least one of 17 to 50 and a sequence complementary to SEQ ID NOS: 17 to 50.

The above transcription for identifying the sex and gender-related traits of papaya The body sequence refers to the sum of all RNAs transcribed corresponding to the above sex-related trait genes, such as SEQ ID NO: 51, 53, 55, 57, 59, 61, 63 and SEQ ID NO: 51, 53, At least one of the complementary sequences of 55, 57, 59, 61, 63. In other examples, the molecular markers described above for identifying papaya sex and sex-related traits may also comprise the sum of all polypeptides translated from the transcript sequences described above, eg, SEQ ID NOs: 52, 54, 56, 58, 60, 62. At least one of 64.

Please refer to Table 1, which is a comparison table showing the molecular markers, the primer pair sequences, and the sex-related trait genes of the locus according to an embodiment of the present invention.

For example, a SNP molecular marker to which the present invention is applicable may include, for example, the SNP site listed in Table 1, a SNP site comprising the 8511th nucleotide of the intron 3 of the CpSVP-LIKE gene (SNP#88), and the CpSERK gene. SNP site of intron 1 122453 nucleotide (SNP#30704), CpSERK gene intron 7 119290, 119295 nucleotide SNP Site (SNPs #34072a, 34072b), SNP site of 118602 nucleotides of exon 9 of CpSERK gene (SNP#34760) and SNP site of 118575 nucleotides of exon 9 of CpSERK gene (SNP) #34787).

In some embodiments, the above molecular marker may be a probe, a primer pair, a gene sequence or a transcript sequence, and the length thereof is not particularly limited as long as the above SNP site is encompassed. In some embodiments, however, when the above molecular marker is a probe or primer pair, the length can be, for example, 15 to 30 base pairs, or as listed in Table 1, or shorter or longer, thereby detecting the above At least one of the SNP loci, thereby simultaneously identifying gender and gender-related traits.

In other embodiments, when the molecular marker is a gene sequence or a transcript sequence itself, the length may be, for example, 200 to 400 base pairs to detect the above SNP site and detect about 200 to 400 upstream and downstream thereof. A sequence of base pairs to improve the accuracy of detection.

The above molecular markers can be applied to early methods for identifying papaya sex and gender-related traits. Please refer to FIG. 1 , which is a partial flow chart showing a method 100 for early identification of papaya sex and gender-related traits according to an embodiment of the present invention.

In one embodiment, the method 100 can obtain a plurality of nucleic acid samples from a plurality of flower organs of a plurality of papayas, wherein the average length of the flower plants does not exceed 0.2 cm, and the nucleic acid sample comprises a plurality of genes. The body sequence as well as a plurality of transcript sequences. Next, as shown in step 130, a plurality of molecular markers are obtained from the nucleic acid sample to establish a gene library and a transcript database, wherein the molecular marker comprises at least one sequence selected from the group consisting of SEQ ID NOS: 1 to 64 and a group consisting of sequences complementary to SEQ ID NOS: 1 to 51, 53, 55, 57, 59, 61, and 63. Thereafter, as shown in step 140, a correspondence between any of the gender-related genes and at least one of the molecular markers is defined to establish a correlation pattern.

The above sex typing data may comprise a single nucleotide polymorphic site, a genome sequence, a transcript sequence, and a polypeptide, which may utilize, for example, High Resolution Melting (HRM), PCR-RFLP, and/or It is conventionally obtained by analyzing the amino acid sequence. Taking HRM as an example, HRM is a recent development of DNA molecular tagging technology that uses a high resolution melting curve to resolve single nucleotide differences in a sequence. Because HRM has the advantages of accuracy, speed, convenience and low cost, it has been used in the past for the differential analysis of gene sequence traits, such as varietal analysis of trait difference sequences of peach cultivars, scanning analysis of specific genes for tomato mutation breeding, and Analysis of the genetic linkage map of lupins. In other embodiments, the invention may also use other conventional means, such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and/or other conventional methods to obtain sex typing data. And the results can be re-checked with the aforementioned HRM analysis results. The establishment of the aforementioned genomic database and transcript database, HRM, PCR-RFLP and/or other conventional methods are established with reference to conventional methods and should be well known to those of ordinary skill in the art to which the present invention pertains. It will not be repeated here.

In one embodiment, the term "correspondence" as used herein refers to the use of conventional commercial statistical software to correlate the obtained sex-sex data to define at least one of the gender-related genes and at least the molecular markers. The correspondence of one.

After establishing the correlation mode, in an example, the step 150 of using the correlation pattern to identify the sex of the papaya sample and the plurality of sex-related genes may include obtaining the nucleic acid sample to be tested from the flower sample of the papaya sample, as in step 160. Show. In an example, the average length of the flower is not more than 0.2 cm, and the nucleic acid sample also includes a plurality of gene sequences to be tested and a plurality of transcript sequences to be tested.

Then, as shown in step 170, a plurality of sex genotyping data are obtained from the nucleic acid sample to be tested, wherein the sex genotyping data may include, for example, a plurality of single nucleotide polymorphism sites to be tested, and more single nucleotides to be tested. The sequence of the gene to be tested corresponding to the type site and the sequence of the transcript to be tested corresponding to the sequence of the gene to be tested. The above sex typing data is obtained by, for example, HRM analysis, PCR-RFLP analysis, and/or other conventional methods, and will not be further described herein.

Next, as shown in step 180, the sex typing data is input into the correlation mode to compare whether the sex typing data matches at least one of the molecular markers, and obtain a correlation between at least one of the molecular markers. Correct rate. When at least one of the sex typing data matches at least one of the molecular markers and the correlation correct rate is 100%, determining that the papaya sample has one of the genders of the corresponding relationship and at least one of the sex-related genes, as in step 190 Show.

For example, when the nucleic acid sample to be tested is subjected to the sex typing data obtained by HRM, and compared with the above correlation pattern, and the SNP#88(T) in the molecular marker is matched, the papaya sample is determined to be an amphoteric strain, and has Corresponding to the intron 3 of the CpSVP-LIKE gene. CpSVP-LIKE gene In the amphoteric strain, there is a polymorphism of (Alternative Splicing, AS), which will be described later.

One of the technical features of the present invention is to define a correspondence relationship between the aforementioned molecular marker and a sex-related trait gene to establish a correlation pattern. It is worth noting that if the papa strain is identified using molecular markers other than the above and/or related patterns other than the above, it is not expected that the papaya sex and specific gender-related traits can be simultaneously and accurately identified in the early development of the flower.

In the above embodiment, the step of using the correlation mode to identify the sex of the papaya sample and the plurality of sex-related genes may include, but is not limited to, obtaining the nucleic acid sample to be tested from the flower sample of the papaya sample, and obtaining the plurality of nucleic acid samples from the sample to be tested. a gender typing data, the gender typing data is input into the correlation mode to match whether the gender typing data matches at least one of the molecular markers, and the at least one of the molecular markers is obtained The correlation correctness of the correspondence. When the at least one of the gender typing data matches the at least one of the molecular markers, and the correlation correctness rate is 100%, determining that the papaya sample has the corresponding relationship of the genders and the gender correlations At least one of the genes.

According to Storey's genetic hypothesis of papaya sex (male, female and amphoteric) in 1938 and 1953, respectively, controlled by three alleles: M1 (male), M2 (bisexual) and m (female). All combinations of dominant alleles (MM, MMh, MhMh) are embryonic lethal. Usually, the amphipathic strains are selfed, and the proportion of the amphoteric and female plants in the offspring is 2:1. The amphipathic strain is crossed with the female strain, and the ratio of the amphoteric strain to the female strain in the offspring is 1:1.

It is stated herein that the molecular markers of the present invention are not only simultaneously Identification of the three sexes of papaya strains, or the variability of alternative splicing (AS) of transcripts, to determine whether the sex-related traits of papaya flowers are affected and degenerate or deformed, thereby based on the results of early detection. Early response.

For example, when the second gender classification data includes SEQ ID NOs: 1 to 16, 18, 20 to 21, 25 to 26, 30 to 31, 35 to 36, 40 to 41, 45 to 46, and 49 to 50 , 53~58, 63~64 and SEQ ID NO: 1~16, 18, 20~21, 25~26, 30~31, 35~36, 40~41, 45~46, 49~50, 53~ When at least one of the sequences of 58 and 64 complements each other, the sex of the papaya sample is determined to be gender.

When the second gender classification data includes SEQ ID NOs: 1-17, 19, 23-24, 28-29, 33-34, 38-39, 43-44, 48, 51-52, 61-62, and When at least one of the complementary sequences of SEQ ID NO: 1 to 17, 19, 23 to 24, 28 to 29, 33 to 34, 38 to 39, 43 to 44, 48, 51 to 52, and 61 to 62, the papaya is judged. The gender of the sample is male.

When the second sex typing data includes SEQ ID NOS: 1 to 16, 22, 27, 32, 37, 42, 47, 59 to 60, and SEQ ID NOS: 1 to 16, 22, 27, 32, 37, When at least one of the complementary sequences of 42, 47, and 59 to 60 is determined, the sex of the papaya sample is determined to be female.

In addition, the above molecular markers are more versatile, for example, a system for early identification of papaya sex and sex-related traits, a molecular marker-containing recombinant vector comprising the same, and a transformed plant cell containing the recombinant vector.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1. Establishment of papaya genome database and transcript database

In this embodiment, a papaya genome database (or a genomic BAC library) and a transcript database (or a transcript BAC library) are established from a plurality of papaya strains.

First, take papaya different sex plants and flowers, including 4 female flowers, 4 male flowers and 4 bisexual flowers, a total of 12 samples, after extracting their genome and transcript, high-throughput sequencing (High-throughput) Sequencing) to obtain a plurality of genome sequences and transcript sequences.

The aforementioned genomic database and transcript database are established by reference to conventional methods. Briefly, the Bacterial Artificial Chromosome (BAC) gene pool was searched on NCBI, and the gene sequence represented by the above transcript was assembled into the template using the BAC sequence as a template, and the expression was based on gender. Different, 15 papaya BACs were selected (not shown).

Example 2. Screening of Papaya Sex Determination Genes

Among the above 15 papaya BACs, a total of 117 sex-related genes were initially screened, of which 27 genes could be expressed in two sex strains, and 28 genes could be expressed in three sex strains, totaling 55 genes.

The 55 sex-related genes obtained above were subjected to nucleic acid and amino acid multiple sequence alignment using commercially available statistical analysis software, such as excel alignment, according to exon jump (Exon Jump), intron jump (Intron) Jump, amino acid shortness (AA Short) and amino acid change (None AA Change) and other amino acid changes, screening for RNA with alternative splicing (Alternative RNA splice) sex-related genes. Among the 55 genes obtained above, a change in amino acid was produced in the gene of papaya BAC-71E16, as shown in Table 2.

The above selected genes are expressed by commercial software, such as ClustalW2 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/) and the multiple sequences of the gene nucleic acid and the amino acid, according to the gene expression The sequence is divided into type_1 (single gender does not show, the other two amino acid amino acid sequences are different), type_2 (all three genders, and amino acid performance sequences are different) and type_3 (all three genders have The expression, in which the amino acid of the two sexes showed no difference in sequence, was used to distinguish the sex-related genes of RNA with alternative RNA splice from papaya BAC-71E16. The selected gene was predicted using a commercial software such as SMART ( http://smart.embl-heidelberg.de/smart/change_mode.pl ), and the results are shown in Table 3.

table 3

From the results of the comparison of Table 3, it was found that in the papaya BAC-71E16, there were two type_1 genes, one type_2 gene, and one type_3 gene.

Example 3. Gene structure analysis of CpSVP-LIKE and CpSERK between different sexes of papaya

This example uses SNP-HRM and PCR-RFLP to evaluate molecular markers for detecting and analyzing the correlation between sex and gender trait genes.

1. Detect and analyze the correlation between sex and gender trait genes using SNP-HRM molecular markers

Analysis by the genome-related information on gender single nucleotide polymorphism (SNP) position, the Beacon Designer ^TM SNP-HRM analysis software design primer, two gene primers were designed 5 groups. 64 known papaya sample DNAs (including parents and progeny) were tested for SNP-HRM. Each HRM reaction reagent contained: Precision melt supermix (Bio-Rad): 6.34 μl DDH ₂ O, 10 μl Precision Melt supermix, 0.4μl 10μM forward primer, 0.4μl 10μM reverse primer, 3μl (1.5ng/μl) DNA sample, total 15μl/well, in 96-well plate (BIOplastics) sealing membrane (BIOplastics), using instant quantitative polymerase The Bio-Rad CFX Connect is equipped with a Bio-Rad CFX Manager Real-time Polymerase chain reaction (PCR) program.

The above PCR temperature program set conditions were as follows: DNA double strand dissociation (Denaturation): 95 ° C for 2 min. Next, 39 cycles of polymerase chain reaction were performed, including DNA double strand dissociation (Denaturation): 95 ° C for 10 sec; and primer paired binding and single stranded DNA lengthening (Annealing and Elongation): 57 ° C for 30 sec. The above PCR product fluorescence dissociation curve temperature program is set as follows: DNA double strand dissociation (Denaturation): 95 ° C 30 sec, 60 ° C 1 min; and DNA double strand fluorescence detection: 65 ° C to 90 ° C, an increase of 0.2 per 10 seconds °C.

The above Real-time PCR results were subjected to cluster analysis using Bio-Rad Precision Melt Analysis, and the correlation rate (correlection%) related to the sex of papaya was calculated. The SNP-HRM molecular markers with 100% correct rate were selected, and 96 papaya blind samples were selected from the offspring of male and female parents. The sex number of SNP-HRM analysis was calculated and analyzed by chi-square analysis. Whether the sex trait separation rate of the SNP-HRM molecular markers of the progeny group is in agreement with the theoretical value.

2. Detection and analysis of the correlation between sex and sex trait genes using PCR-RFLP molecular markers

PCR-RFLP primers were designed according to SNP sequence. The PCR product size is about 200 bp. The PCR conditions are: 34 μl DDH ₂ O, 5 μl DMSO (Dimethyl sulfoxide), 5 μl 10×PC2 buffer (500 mM Tris-HCl pH=9.1, 160 mM (NH ₄ ) ₂ SO ₄ , 35 mM MgCl ₂ , 1.5 mg/ml BSA), 1 μl of 10 mM dNTP, 1 μl of 10 mM forward primer, 1 μl of 10 mM reverse primer, 1 μl of Taq (2 U/μl), 2 μl of DNA sample, for a total of 50 μl. Divided into 0.2 ml eppendorf by polymerase chain reaction reactor (BIOER-GenePro) PCR temperature program setting: Denaturation: 94 ° C 40 sec; 35 cycles of reaction: primer pairing (Annealing): 61 ° C 40 sec, single-stranded DNA lengthening ( Elongation): 72 ° C 30 sec. The obtained PCR product was purified by a purification reagent set (Geneaid), and the concentration of the PCR product was measured. 400 ng of PCR products and enzymes were used to analyze: the PCR product of MADS-BOX gene was analyzed by Nde I enzyme in combination with buffer; the PCR product of SERK gene was analyzed by Ase I enzyme in combination with buffer for 8 hours at 37 ° C. The PCR products before and after the action of the enzyme were separately subjected to electrophoresis analysis and UV digital photography.

After screening by HRM and PCR-RFLP, a short vegetative phase (CpSVP-LIKE) and Somatic Embryogenesis Receptor Kinase (CpSERK) located at a distance of about 26000 bp from Y ^h chromosome BAC 71E16 were found. These two genes are all related to gender. Secondly, Papaya ( Carica papaya L., Cp) CpSVP-LIKE gene and CpSERK gene and Vitis vinifera (Vv) VvSVP and VvSERK amino acid sequence alignment, respectively, up to 72% and 96% similarity (Information not shown).

Please refer to FIG. 2A and FIG. 2B. FIGS. 2A-2B illustrates a system according to the cDNA sequence of papaya CpSVP-LIKE different manifestations of the type (FIG. 2A) and the intron / exon BAC embodiment of the present invention, a position Y ^h BAC 71E16 embodiment of chromosome analysis ( Figure 2B) is the difference. The figures of the double bottom line of Figures 2A to 2B represent the position on the BAC, F stands for female, M stands for male, H stands for gender, the round figure represents the starting password, the triangle type represents the termination password, and the square represents the explicit The black square represents the untranslated domain, the straight striped square represents the non-specific domain, and the square E1/E2 represents the MADS-BOX domain (GenBank sequence number: BAR64349.1, whose amino acid sequence is SEQ ID NO: 65 Show), the square E3/E4/E5 represents the K-BOX domain, and the horizontal line represents the interpolation. The right side of each structure indicates the number of nucleic acids and amino acids. ORF stands for open reading frame, AA stands for amino acid, UTR stands for untranslated regions, single bottom line numbers are nucleic acid numbers, primers are marked under the structure, and h stands for HRM primers. Dc represents the PCR-RFLP primer and the arrow indicates the SNP position.

The results of Figure 2A indicate that the CpSVP-LIKE gene is predicted to have a protein sequence length and its putative protein sequence contains a K-BOX domain containing six interposons and seven exons. The gene was not expressed in female papyrus 0.2cm calyx; male 0.2cm calyx had a transcriptional phenotype with a predicted translation length of 167 amino acid (cDNA: 4416bp; ORF: 501bp); and amphoteric 0.2cm calyx was three transcriptional phenotypes. The three putative lengths of the two sexes were 98 amino acids (cDNA: 839 bp; ORF: 294 bp; 4th intron alternative splicing), 167 amino acids (cDNA: 5093 bp; ORF: 501 bp) and 203 amino acids ( cDNA: 840 bp; ORF: 609 bp; 2nd intron alternative splicing). Through SNP genotyping analysis of sex traits, it was found that SNP G(M)→T(H) is located in the 3rd intron 3 (MSY Y ^h chromosome BAC 71E16 is the reference position) and has only the CpSVP-LIKE gene. It has a polymorphism of AS in amphipathic strains and does have gender relevance, as shown in Figure 2A. Therefore, the SNP-HRM primer and the PCR-RFLP primer are designed according to the SNP position, and are named as SVP_HRM_88 and SVP_RFLP_Nde I, as shown in Table 1 and SEQ ID NOs: 1-4.

See FIGS. 3A and 3B, the system which is shown in the cDNA sequence analysis CpSERK type exhibit different properties of papaya (FIG. 3A) and the intron / exon The positions Y ^h BAC chromosome BAC71E16 embodiment of the present invention, an embodiment (Figure 3B) the difference.

The figures of the double bottom line of Figures 3A to 3B represent the position on the BAC, F stands for female, M stands for male, H stands for gender, the round figure represents the starting password, the triangle type represents the termination password, and the square represents the explicit The black square represents the untranslated domain, the square E1 represents the signal peptide (SP domain), and the square E2/E3/E4/E5 represents the Leucine-rich repeats. In the LRR domain, the square E7 represents the Transmembrane region (TM) (E7), and the square E9/E10/E11 represents Serine/Threonine protein kinases. Kinase domain), the horizontal line represents the interpolation. The right side of each structure indicates the number of nucleic acids and amino acids. ORF stands for open reading frame, AA stands for The amino acid, UTR represents the untranslated regions, the single bottom line number is the number of nucleic acids, the primer position is indicated below the structure, h represents the HRM primer, dc represents the PCR-RFLP primer, and the arrow indicates the SNP position.

The results of Fig. 3A, Fig. 3B, Table 3 and Table 4 indicate that the CpSERK gene protein sequence contains a message peptide (SP domain), a leucine-rich repeat (LRR domain), a transmembrane region (TM domain) and silk. The C-terminal (S_TKC-like domain) of the amino acid/threonine protein kinase (Kinase domain) contains 10 interposons and 11 exons, and papaya is different in three sexes: female 593 amino acids (cDNA: 2183 bp; ORF: 1779 bp), male 523 amino acids (cDNA: 1952 bp; ORF: 1569 bp; 10th intron alternative splicing) and amphoteric 628 amino acids (cDNA: 2467 bp; ORF: 1884 bp) ). According to the sex trait SNP genotyping analysis, it was found that the SNP of the 122453th nucleic acid of intron 1 is female if it is A, and male and amphoteric if it is G; the SNP of 119295 nucleic acid of intron 7 is female if it is T, if it is G And male and female; and the SNP of the 119,290th nucleic acid is female and male if it is A, and is amphoteric if it is T; the SNP of Exon 9 118602 nucleic acid is female if it is C, and male if it is T And the sexes; and the SNP of the 118,575 nucleic acids is a female if G, and a male and amphoteric if A. All of the above SNPs have gender-related changes that can be used to identify papaya sex. The SNP-HRM and PCR-RFLP primer pairs are designed according to the SNP position, and are named as SERK_HRM_30704, SERK_HRM_34072, SERK_HRM_34760, respectively. SERK_HRM_34787, SERK_RFLP_Dde I and SERK_RFLP_Spe I are shown in Table 1 and SEQ ID NOs: 1-16.

Please refer to Table 4, which shows the alignment of the trisex CpSERK with the Y ^h chromosome BAC 71E16 amino acid sequence according to an embodiment of the present invention.

The results in Table 4 indicate that the above two genes (CpSVP-LIKE and CpSERK) do have gender relevance. In addition, the above two genes (CpSVP-LIKE and CpSERK) only have the CpSVP-LIKE gene having an AS polymorphism in the amphoteric strain, as shown in Fig. 2A.

Example 4. Detection and Genetic Analysis of Papaya Molecular Markers

In this example, 40 blind population testes were performed using the above molecular markers to evaluate the relative correctness of the above molecular markers for early identification of papaya sex and gender-related traits.

First, a sex trait SNP genotyping analysis was performed on the genome-encoded genome. It was found that the CpSVP-LIKE and CpSERK genes have gender-related genotype SNP distributions, as shown in Figures 2A to 3B and Table 5.

Secondly, in the CpSVP-LIKE gene, the SVP_HRM_88 primer found that there was a significant difference in the DNA dissociation fluorescence curve between the three sexes (Fig. 4A, CpSVP-LIKE 88 primer pair), and the genetic analysis of the gender traits found 24 parents. Tested with 40 progeny samples, the correlation between the three sex SVP-HRM_88 tests was 100%. Furthermore, in the 40 blind sample group tests, the SVP_HRM_88 primer detected the ratio of the three sexes, and the chi-square test met the 1:1:1 theoretical value, as shown in the results of Table 5. The results of PCR-RFLP analysis showed that the SVP_RFLP_Nde I primer group was PCR-based for different genders, and the PCR products produced by the females, while the male and amphoteric PCR products were treated with Nde I enzyme, the SNP could distinguish between males and males. As shown in the results of Figure 5A.

In the CpSERK gene, the SERK_HRM_34072, SERK_HRM_30704, SERK_HRM_34760, and SERK_HRM_34787 four sets of primer pairs can detect females, as shown in the results of Figures 4B to 4E. The SERK_HRM_34072 was found to have a significant difference in the DNA dissociation fluorescence curve between the three sexes, as shown in the results of Figure 4B. Parents and offspring in the genetic analysis The analysis results of SERK_HRM_34072, SERK_HRM_34760 and SERK_HRM_34787 found that the correlation between female and SNP-HRM detection of the two sexes reached 100%, and 40 blind sample groups tested the ratio of females to other sexes in the three groups, and the chi-square test met 1:2. The theoretical values are shown in the results of Table 5. PCR-RFLP analysis of the 119737 nucleic acid, using the SERK_RFLP_Spe I primer group for PCR of different genders, the product has no difference between the sexes, and after Spe I enzyme treatment, the SNP can distinguish between the sexes and other genders, as shown in Figure 5B The results are shown.

In the above embodiment, the number of samples per set of data is at least three (n ≧ 3).

Please refer to FIG. 6 , which illustrates a linkage map of a triple-type gene of a molecular marker of a papaya sex and sex-related trait gene according to an embodiment of the present invention, and a representation pattern thereof, wherein F represents a female, M represents a male, and H represents a sex. For example, when the second sex typing data includes SEQ ID NOS: 1 to 16, 22, 27, 32, 37, 42, 47, 59 to 60, and SEQ ID NOS: 1 to 16, 22, 27, When at least one of the complementary sequences of 32, 37, 42, 47, 59-60, but not SEQ ID NO: 51-58, the flower type and/or fruit shape of the papaya sample is determined to be a stable female type. When the second gender classification data includes SEQ ID NOs: 1-17, 19, 23-24, 28-29, 33-34, 38-39, 43-44, 48, 51-52, 61-62, and When at least one of the complementary sequences of SEQ ID NO: 1 to 17, 19, 23 to 24, 28 to 29, 33 to 34, 38 to 39, 43 to 44, 48, 51 to 52, and 61 to 62, the papaya is judged. The floral pattern and/or fruit shape of the sample is a stable male type. When the second sex classification data includes SEQ ID NOs: 1-16, 18, 20~21, 25~26, 30~31, 35~36, 40~41, 45~46, 49~50, 53~58, 63~64 and SEQ ID NO: 1~16, 18, 20~21 When at least one of 25~26, 30~31, 35~36, 40~41, 45~46, 49~50, 53~58, 63~64 complementary sequences is determined, the flower type of the papaya sample is judged and/or Or the shape of the fruit is unstable and bisexual.

In summary, the above several examples demonstrate that the molecular markers of the present invention for early identification of sex and gender-related traits of papaya can indeed identify gender and gender-related traits in the early stage of flower development of papaya samples, and experimentally It was confirmed that the identification result thus obtained can accurately and accurately identify the sex and gender-related traits of papaya in the early stage.

In conclusion, the above several examples show that, in terms of genome, CpSVP-LIKE has a difference in the X, Y ^m and Y ^h genomes in papaya, and the CpSVP-LIKE and CpSERK genes are in The X genome is closely linked to genotypic changes in the same female type. In terms of transcripts, there are three types of CpSVP-LIKE on the bisexual flower at the locus, and only one of the males has a sequence of expression, which can explain not only the locus X, Y ^m and Y ^h genomes. The difference may cause CpSVP-LIKE to differ in gender performance. In particular, the polymorphism of the CpSVP-LIKE sequence of the amphipathic strain may be related to the phenomenon of papaya variability (as shown in Figure 6).

Secondly, the above several examples also confirmed that using HRM to analyze blind-type genetic data, SNP#34072, SNP#34760 and SNP#34787 can achieve 100% female correlation; and PCR-RFLP analysis of blind-like CpSVP-LIKE gene SNP#88 and CpSERK genes SNP #34072 can distinguish between three sexes and female sex. In addition, using HRM to analyze blind transcript data, CpSVP-LIKE gene can not be expressed in female plants, males are single expression sequences, and amphipathic genes exhibit three sequences of alternative RNA splicing; CpSERK gene The transcript sequences of the expression sequences between different genders also differ. From the above results, it can be judged whether the gender and gender-related traits of the papaya flower are affected and there is the possibility of degeneration and deformity, thereby early response according to the results of early detection.

Furthermore, the above several examples further demonstrate that the above molecular markers of the present invention can be used in various applications, such as a system for early identification of sex and sex-related traits of papaya, a molecular marker-containing recombinant vector comprising the same, and a transformation comprising the recombinant vector. Plant cells, etc.

It should be noted that the present invention exemplifies a molecular marker of the early identification of sex and sex-related traits of papaya by using a specific variety, a specific molecular marker, a specific sex-related trait gene or a specific evaluation method as an example. It is to be understood that the invention is not limited thereto, and the present invention is intended to identify molecules of sex and sex-related traits of papaya in the early stage without departing from the spirit and scope of the present invention. Markers and their applications can also be carried out using other varieties, other molecular markers, other gender-related trait genes or other assessment methods.

It can be seen from the above examples that the molecular marker of the present invention for early identification of sex and gender-related traits of papaya and its application utilize a specific molecular marker to establish a correlation pattern with any of the sex-related trait genes, which can be used in papaya flowers. Identification of the sex and gender of this papaya sample during early development The traits are used to obtain the papaya strains effectively, quickly and accurately, and help to reduce the production of unconsolidated flowers and deformed flowers and fruits.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

<110> National Pingtung University of Science and Technology

<120> Molecular markers for early identification of sex and gender-related traits in papaya and their applications

<130> None

<160> 65

<210> 1

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward SVP_HRM_88

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<210> 2

<211> 23

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<213> Artificial sequence

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<223> SVP_HRM_88 reverse primer

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<213> Artificial sequence

<220>

<223> SVP_RFLP_Nde I forward of I

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<213> Artificial sequence

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<223> SVP_RFLP_Nde I reverse primer

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<212> DNA

<213> Artificial sequence

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<223> Forward introduction of SERK_HRM_30704

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<213> Artificial sequence

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<223> Reverse Inversion of SERK_HRM_30704

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<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction of SERK_HRM_34072 (detection of 119290 base pairs)

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<213> Artificial sequence

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<223> Reverse primer for SERK_HRM_34072 (detects 119290 base pairs)

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<213> Artificial sequence

<220>

<223> Forward introduction of SERK_HRM_34072 (detection of 119295 base pairs)

<400> 9

<210> 10

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for SERK_HRM_34072 (detection of 119295 base pairs)

<400> 10

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Positive thrust of SERK_HRM_34760

<400> 11

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse inversion of SERK_HRM_34760

<400> 12

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction of SERK_HRM_34787

<400> 13

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse introduction of SERK_HRM_34787

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<210> 15

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> SERK_RFLP_Spe I forward primer

<400> 15

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> SERK_RFLP_Spe I reverse primer

<400> 16

<210> 17

<211> 231

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 primer pair (SNP# 88) male strain PCR product

<400> 17

<210> 18

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 primer pair (SNP# 88) amphiphilic PCR product

<400> 18

<210> 19

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_RFLP_Nde I primer pair (SNP# 88) male strain PCR product

<400> 19

<210> 20

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_RFLP_Nde I primer pair (SNP# 88) amphipathic PCR product

<400> 20

<210> 21

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_RFLP_Nde I primer pair (SNP# 88) amphipathic PCR product

<400> 21

<210> 22

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30704 primer pair (SNP# 30704) female strain PCR product

<400> 22

<210> 23

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30704 primer pair (SNP# 30704) male strain PCR product

<400> 23

<210> 24

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30704 primer pair (SNP# 30704) male strain PCR product

<400> 24

<210> 25

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30704 primer pair (SNP# 30704) amphiphilic PCR product

<400> 25

<210> 26

<211> 88

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30704 primer pair (SNP# 30704) amphiphilic PCR product

<400> 26

<210> 27

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072a) female strain PCR product

<400> 27

<210> 28

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072a) male strain PCR product

<400> 28

<210> 29

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072a) male strain PCR product

<400> 29

<210> 30

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072a) amphiphilic PCR product

<400> 30

<210> 31

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072a) amphiphilic PCR product

<400> 31

<210> 32

<211> 90

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072b) female strain PCR product

<400> 32

<210> 33

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072b) male strain PCR product

<400> 33

<210> 34

<211> 92

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072b) male strain PCR product

<400> 34

<210> 35

<211> 90

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072b) amphipathic PCR product

<400> 35

<210> 36

<211> 90

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34072 primer pair (SNP# 34072b) amphipathic PCR product

<400> 36

<210> 37

<211> 65

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34760 primer pair (SNP# 34760) female strain PCR product

<400> 37

<210> 38

<211> 65

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34760 primer pair (SNP# 34760) male strain PCR product

<400> 38

<210> 39

<211> 65

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34760 primer pair (SNP# 34760) male strain PCR product

<400> 39

<210> 40

<211> 65

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34760 primer pair (SNP# 34760) amphiphilic PCR product

<400> 40

<210> 41

<211> 65

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34760 primer pair (SNP# 34760) amphiphilic PCR product

<400> 41

<210> 42

<211> 85

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34787 Inducible pair (SNP# 34787) female strain PCR product

<400> 42

<210> 43

<211> 85

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34787 Primer pair (SNP# 34787) male strain PCR product

<400> 43

<210> 44

<211> 85

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34787 Primer pair (SNP# 34787) male strain PCR product

<400> 44

<210> 45

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34787 primer pair (SNP# 34787) amphiphilic PCR product

<400> 45

<210> 46

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_34787 primer pair (SNP# 34787) amphiphilic PCR product

<400> 46

<210> 47

<211> 238

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_RFLP_Spe I primer pair (SNP# 34072a) female strain PCR product

<400> 47

<210> 48

<211> 238

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_RFLP_Spe I primer pair (SNP# 34072a) male strain PCR product

<400> 48

<210> 49

<211> 248

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_RFLP_Spe I primer pair (SNP# 34072a) amphiphilic PCR product

<400> 49

<210> 50

<211> 248

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_RFLP_Spe I primer pair (SNP# 34072a) amphiphilic PCR product

<400> 50

<210> 51

<211> 4416

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript cDNA sequence of the corresponding male strain CpSVP-LIKE gene

<400> 51

<210> 52

<211> 166

<212> PRT

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript amino acid sequence of the corresponding male strain CpSVP-LIKE gene

<400> 52

<210> 53

<211> 840

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript cDNA sequence of the corresponding amphoteric strain H1 CpSVP-LIKE gene

<400> 53

<210> 54

<211> 202

<212> PRT

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript amino acid sequence of the corresponding amphoteric strain H1 CpSVP-LIKE gene

<400> 54

<210> 55

<211> 5093

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript cDNA sequence of the corresponding amphoteric strain H2 CpSVP-LIKE gene

<400> 55

<210> 56

<211> 166

<212> PRT

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript amino acid sequence of the corresponding amphoteric strain H2 CpSVP-LIKE gene

<400> 56

<210> 57

<211> 843

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript cDNA sequence of the corresponding amphoteric strain H3 CpSVP-LIKE gene

<400> 57

<210> 58

<211> 98

<212> PRT

<213> Papaya ( Carica papaya L.)

<220>

<223> SVP_HRM_88 transcript amino acid sequence of the corresponding amphoteric strain H3 CpSVP-LIKE gene

<400> 58

<210> 59

<211> 2183

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 transcript cDNA sequence of the corresponding female strain CpSERK gene

<400> 59

<210> 60

<211> 593

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 Introduction to the transcript amino acid sequence of the corresponding female strain CpSERK gene

<400> 60

<210> 61

<211> 1952

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 transcript cDNA sequence of the corresponding male strain CpSERK gene

<400> 61

<210> 62

<211> 523

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 The transcript amino acid sequence of the corresponding male strain CpSERK gene

<400> 62

<210> 63

<211> 2392

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 transcript cDNA sequence of the corresponding amphoteric strain CpSERK gene

<400> 63

<210> 64

<211> 627

<212> DNA

<213> Papaya ( Carica papaya L.)

<220>

<223> SERK_HRM_30702 The transcript amino acid sequence of the corresponding amphoteric strain CpSERK gene

<400> 64

<210> 65

<211> 266

<212> PRT

<213> Papaya ( Carica papaya )

<220>

<223> MADS-box protein

<400> 65

100‧‧‧ method

120/130/140/150/160/170/180/190‧‧‧ steps

Claims (10)

A molecular marker for early identification of papaya sex and sex-related traits, comprising at least one sequence selected from the group consisting of SEQ ID NOS: 1-4 and 17-21. A system for early identification of sex and gender-related traits of papaya, comprising a molecular marker for early identification of sex and gender-related traits of papaya as described in claim 1 of the scope of the patent application. An early method for identifying sex and gender-related traits of papaya comprises: establishing a correlation pattern comprising: obtaining a plurality of nucleic acid samples from a plurality of flower organs of a plurality of papayas, wherein an average length of one of the flowers is not more than 0.2 cm, and The nucleic acid samples comprise a plurality of gene body sequences and a plurality of transcript sequences; and the plurality of molecular markers are obtained from the nucleic acid samples, wherein the molecular markers comprise at least one sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and 17 a group of ~21 sequences; and defining a correspondence between any one of the gender-related genes and at least one of the molecular markers to establish the correlation pattern; and using the correlation pattern to identify a sex of a papaya sample and a plurality of sex-related genes, comprising: obtaining a nucleic acid sample to be tested from one of the papaya samples, wherein an average length of one of the flowers to be measured does not exceed 0.2 cm, and the test is to be tested The nucleic acid sample comprises a sequence of the gene to be tested and a sequence of the transcript to be tested; Obtaining a plurality of sex genotyping data from the nucleic acid sample to be tested, wherein the sex genotyping data comprises a plurality of polymorphic sites of the single nucleotide to be tested, and the polymorphic sites of the single nucleotides to be tested correspond to The sequence of the gene to be tested and the sequence of the transcript to be tested corresponding to the sequence of the gene; and inputting the sex typing data into the correlation pattern to compare whether the sex typing data is related to the molecular markers At least one of the matches, and obtaining one of the correspondences of the at least one of the molecular markers is associated with a correct rate, and when the at least one of the sex-typing data matches the at least one of the molecular markers and When the correlation correct rate is 100%, it is determined that the papaya sample has one of the genders of the correspondence relationship and at least one of the sex-related genes. A method for early identification of sex and gender-related traits of papaya according to the third aspect of the patent application, wherein the papaya and the papaya sample comprise papaya ( Carica papaya L.) of Florida species, Japanese ascending species, Kuala Lumpur species, and Japanese Rising No. 13 species, Risheng No. 23 species, Risheng and Tai Nong No. 7 hybrids. The method for early identification of sex and gender-related traits of papaya according to item 3 of the patent application scope, wherein the second sex classification data comprises SEQ ID NOS: 1 to 4, 18, 20 to 21, 53 to 58 In at least one of the sequences, the gender of the papaya sample is determined to be amphoteric. A method for early identification of sex and sex-related traits of papaya according to item 3 of the scope of the patent application, wherein the second sex-sex data comprises sequences of SEQ ID NOS: 1-4, 17, 19, 51-52 In at least one, the sex of the papaya sample is judged to be male. The method for early identification of sex and gender-related traits of papaya according to item 3 of the patent application scope, wherein the papaya is judged when the second sex type data includes at least one of the sequences of SEQ ID NOS: 1-4 The gender of the sample is female. According to the method for identifying early sex and gender-related traits of papaya according to item 3 of the patent application scope, wherein the gender-related trait genes are related to a plurality of gender-related traits, and the gender-related traits comprise a plurality of flower patterns and/or Or a plurality of fruit shapes. A recombinant vector comprising the molecular marker for early identification of papaya sex and sex-related traits as described in claim 1 of the scope of the patent application. A transgenic plant cell comprising the recombinant vector of claim 9 of the patent application.