CN114525300A - Application of polynucleotide and protein and haploid inducing line thereof - Google Patents

Abstract

The present invention provides applications of polynucleotides and proteins and haploid inducible lines thereof, the polynucleotides comprising or consisting of the following sequences: 1) the nucleotide sequence shown in SEQ ID NO: 1; or 2) SEQ ID NO : a complementary sequence, a degenerate sequence or a homologous sequence of the sequence shown in 1; or 3) a polynucleotide or its complementary sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions; 4) the The cDNA sequence or CDS sequence of any one of the sequences 1)-3); when the expression and/or activity of the nucleotide is suppressed, or the protein activity expressed by the nucleotide is reduced or inactive, the Plants containing nucleotides can be selfed or crossed with other plants to produce haploid progeny.

Description

Applications of polynucleotides and proteins and their haploid inducible lines

technical field

The present invention relates to the field of biological breeding, in particular to the application of polynucleotides and proteins and their haploid inducible lines.

Background technique

Potato is the most important tuber crop in the world, and unlike other major crops, the complexities of tetraploid inheritance and clonal reproduction greatly hinder the genetic improvement of cultivated potatoes. To obtain hybrids with sufficient heterosis and uniformity, pure inbred lines with high genomic homozygosity are essential.

Selfing recession is the phenomenon of decreased viability, weakened fitness, or degradation of economic traits due to selfing or inbreeding. The reason is that selfing increases the homozygous ratio in the population and reduces the dominance. Potato must undergo inbreeding or selfing in order to obtain a homozygous inbred line, but it has accumulated a large number of harmful mutations through long-term asexual reproduction, and it is difficult to eliminate these harmful mutations by recombination. Selfing decline is also one of the structural obstacles that limit the cultivation of potato elite lines. Homozygous lines are obtained by inducing plants to produce haploids by doubling, which has become one of the methods to quickly obtain inbred lines. In recent years, the induction of haploid plants by induction lines has been achieved in maize, Arabidopsis and tomato.

Self-incompatibility and the decline of inbred lines make the development of pure inbred lines challenging. Potato cross-breeding is still at an early stage, and there is an urgent need to develop an efficient method for obtaining pure inbred lines. In contrast to traditional breeding methods that utilize multiple generations of selfing or backcrossing, the doubled haploid production allows for the fixation of the haploid genome of a single inbred line within a single generation. Haploid gametophytes can develop into double haploid sporophytes by in vitro tissue culture, in vivo inducible line hybridization and intraspecific hybridization or CENH3 centromeric histone induction. Among these methods, haploid induction (HI) triggering is the most effective method, but HI genes such as PLA1/MTL/NLD and DMP have only been demonstrated in individual species, and due to differences in genetic background between species, these species The difference in induction efficiency (HIR) between them was significant. Currently, there is no evidence that these HI genes are universal across species, especially in difficult-to-transform genotypes.

Currently, partially tetraploid potatoes can be induced to become haploid (diploid level). However, whether diploid potatoes can be induced to become haploid (at the monoploid level) has not yet been reported.

SUMMARY OF THE INVENTION

In view of this, the present application provides an application of polynucleotides and proteins and a haploid inducible line thereof. This nucleotide is associated with potato haploid formation. When the expression and/or activity of the nucleotide is inhibited, or the activity of the protein expressed by the nucleotide is affected, for example, the activity is reduced or lost, the plant containing the nucleotide will be selfed or crossed with other plants. Haploid offspring can be produced.

In order to realize the purpose of the present invention, the present invention adopts following technical scheme:

One aspect of the present invention provides the application of a polynucleotide in potato haploid induction, the polynucleotide being at least one of the following sequences:

1) the nucleotide sequence shown in SEQ ID NO: 1, namely the StDMP gene sequence;

2) The complementary sequence, degenerate sequence or homologous sequence of the sequence shown in SEQ ID NO: 1, wherein the homologous sequence is 75% or more, 76% or more of the nucleotide shown in SEQ ID NO. Above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above Above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above Above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, Polynucleotides of 99.8% or more, or 99.9% or more identity;

3) a polynucleotide or its complementary sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions;

4) The cDNA sequence or CDS sequence of any of the sequences 1) to 3).

In a specific embodiment of the present invention, the CDS sequence is shown in SEQ ID NO:3.

Another aspect of the present invention provides the application of a protein in potato haploid induction, the protein being at least one of the following sequences:

1) the protein shown in SEQ ID NO: 2, that is, the amino acid sequence expressed by the StDMP gene sequence;

2) a fusion protein obtained by linking the N-terminal and/or C-terminal of the protein shown in SEQ ID NO: 2 with a tag;

3) a protein with the same function obtained by the amino acid sequence shown in SEQ ID NO: 2 through the substitution and/or deletion and/or addition of one or several amino acid residues;

4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having the same function.

wherein said 75% or more homology is 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more , 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more , 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5 % or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

Another aspect of the present invention provides a potato haploid inducible line, which reduces the expression and/or activity of the above-mentioned polynucleotide or protein through gene insertion, substitution and/or deletion mutation.

Preferably, the mutation method is one of CRISPR/Cas9, TELLEN technology, T-DNA insertion, EMS mutagenesis, and ZFN technology.

In a specific embodiment of the present invention, the mutation mode is CRISPR/Cas9 technology.

In a specific embodiment of the present invention, the StDMP gene is knocked out.

In a specific embodiment of the present invention, the expression and/or activity of the StDMP gene in the genome of the potato haploid inducible line is reduced.

In a specific embodiment of the present invention, the StDMP gene is not expressed.

In a specific embodiment of the present invention, the expression level of the StDMP gene is reduced compared to when the StDMP gene was found.

In a specific embodiment of the present invention, the activity of the StDMP gene is reduced.

In a specific embodiment of the present invention, the activity of the protein expressed by the StDMP gene is reduced.

In a specific embodiment of the present invention, the protein expressed by the StDMP gene loses the original biological activity of the protein, so that the haploid inducible line is self-bred or the offspring of its cross is halved. haploid.

In a specific embodiment of the present invention, the haploid inducible line serves as the male parent.

The present invention should also include the use of RNAi interference, overexpression or promoter editing and other prior art means to reduce or even lose the expression and/or activity of the StDMP gene.

Another aspect of the present invention provides a method for preparing a haploid inducible line, comprising the steps of: reducing the expression and/or activity of the StDMP gene in the biological material containing the StDMP gene, preferably, the StDMP gene does not expression, or the protein expressed by the StDMP gene is inactive.

Another aspect of the present invention provides the application of the above-mentioned haploid induction line in potato haploid breeding or cross-breeding.

Another aspect of the present invention provides a gene knockout cassette, which is a knockout cassette for knocking out the above-mentioned polynucleotide.

Another aspect of the present invention provides a knockout vector, which is a knockout vector for knocking out the above-mentioned polynucleotide.

Another aspect of the present invention provides a recombinant microorganism, which is a recombinant vector for knocking out the above-mentioned polynucleotide.

Another aspect of the present invention provides a transgenic plant cell line, which is a transgenic plant cell line in which the above-mentioned polynucleotide is knocked out.

Another aspect of the present invention provides a potato plant, which is one of the following plants:

1) a plant formed by the growth of the above-mentioned transgenic plant cell line;

2) the progeny formed by the selfing of the plant in the 1), and the plant formed by the growth of the progeny;

3) The progeny formed by crossing the plant in 1) with other varieties, and the plant formed by the growth of the progeny.

The present invention also provides a method for preparing a potato haploid plant, comprising the following steps:

1) with the above-mentioned transgenic plant knocking out the StDMP gene as the male parent, it is self-crossed or crossed with other varieties to obtain seeds;

2) The seeds are treated with 1-5 mg/mL gibberellin for 24-72 hours, then sterilized and sown in 1/2 MS medium to obtain seedlings;

3) Haploid identification of seedlings to obtain haploid plants.

In the specific embodiment of the present invention, the concentration of gibberellin treatment is 2 mg/mL, and the time is 48 hours.

In the present invention, the haploid identification method includes one or more of fluorescent marker identification technology, molecular marker identification technology, mature plant phenotype identification technology, and flow cytometry leaf identification technology.

The present invention also provides a method for preparing a potato homozygous diploid plant, comprising the following steps: using the above screening method to obtain a haploid plant; Chromosomal doubling was carried out for at least 2 days to obtain homozygous diploid plants.

In a specific embodiment of the present invention, the concentration of colchicine treatment is 0.4%-0.5%, and the time is at least 3 days.

Exemplarily, the present invention has at least one of the following advantages:

In one aspect, the polynucleotides provided by the invention are related to potato haploid formation. When the expression and/or activity of the nucleotide is inhibited, or the activity of the protein expressed by the nucleotide is reduced or inactive, the plant containing the nucleotide can be selfed or hybridized with other plants to produce haploids offspring. Therefore, plants containing this polynucleotide can be used for cross-breeding or haploid breeding of potatoes.

On the other hand, due to the existence of a large number of harmful mutation sites in the potato itself, these sites change from recessive to dominant during the formation of haploid, resulting in poor plant development or even death. The germination rate of fluorescent seeds was only 6%. The invention utilizes gibberellin to treat for 48 hours and then sown in 1/2MS medium, which increases its germination rate from 6% to 40%, significantly improves the preparation efficiency of potato haploids, and is a monoploid of diploid potato plants. The ploidy preparation provides a practical and effective method with practical application and operational value.

In addition, the preparation method of potato homozygous diploid provided by the present invention takes about half a year to obtain a haploid through induction, and it only takes about a month to double from a haploid to a diploid. Years of self-crossing and backcrossing can obtain homozygous strains, and the method provided by the present invention saves a lot of time, manpower and material resources. The induced haploid and doubled diploid will also become important materials for basic potato research.

Whether it is actual production or basic research, the present invention has important functions and application values.

Description of drawings

Figure 1 shows the genotype of the potato StDMP gene biallelic mutant line; the two sets of chromosomes of the mutant line are both deleted and mutant compared with the wild type, which is a loss-of-function mutant;

Figure 2 shows the results of fluorescence screening of potato haploid seeds; diploid seeds have red fluorescence in the embryo and endosperm, while haploid seeds have no fluorescence in the embryo and weak fluorescence in the endosperm; scale bar = 1 mm;

Figure 3 shows the results of molecular marker identification of potato haploid plants; I is the parent material of the transgenic potato plant, II is the hybrid potato B material (PG6359, the original number of the International Potato Center CIP705468), III is the StDMP gene biallelic mutant line, IV and V are the haploids induced by the crossing of the StDMP gene biallelic mutant line with the potato B material, VI and VII are the diploids after crossing the StDMP gene biallelic mutant line with the potato B material; haploid The material can only amplify the genotype of the female potato B material;

Figure 4 shows the results of phenotypic identification of potato haploid plants; compared with diploid plants, haploid plants have the characteristics of shorter plants, narrower leaves, and compact plant shapes; scale bar = 1 cm;

Figure 5 shows the identification results of potato haploid plants by flow cytometry; the signal peak of diploid nucleus is set to 50. Since there may be cells that are replicating in the cell, the signal peak of diploid nucleus will appear between 50 and 50. Around 100, the haploid nuclear signal peaks appear around 25 and 50.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Description: StDMP gene in this application is the abbreviation of Soltu.DM.05G005100.1 gene.

StDMP or StDMP representation gene or protein can be determined according to the context.

Example 1 Knockout of StDMP gene using CRISPR/Cas9 system

The StDMP gene in potato was knocked out using the CRISPR/Cas9 system to obtain a StDMP gene knockout potato mutant. Specific steps are as follows:

Step 1. Selection of sgRNA sequence

The target site sequence was designed on the StDMP gene with a length of 20 bp.

The target site is located at positions 4-23 of sequence 1 (genome sequence) and located at positions 4-23 of sequence 3 (CDS sequence), and the sequence of target site 1 is GAGCAAACTAGTGAAGGAAT (sequence 4).

The target site design sgRNA sequence is:

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (sequence 5)

The coding DNA molecule of this sgRNA is:

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 6).

Step 2. Construction of CRISPR/Cas9 vector

The original vector contains the sgRNA sequence, and the target site 1 sequence in step 1 is further inserted into the vector to obtain a CRISPR/Cas9 vector.

Step 3. Obtainment of transgenic plants

The CRISPR/Cas9 vector obtained in step 2 was transferred to Agrobacterium competent cell EHA105 by heat shock transformation (Agrobacterium EHA105 competent cell was purchased from Shanghai Weidi Biotechnology Co., Ltd., and the public can obtain it by purchase) to obtain recombinant strain EHA105/ CRISPR/Cas9.

Then the recombinant strain EHA105/CRISPR/Cas9 was transformed by Agrobacterium to infect potatoes (recombinant Agrobacterium was propagated at 28°C, and the amplified bacterial solution was used for potato infection), and after kana resistance screening, T was obtained. Generation ₀ transgenic potato plants.

Example 2 Identification of Transgenic Plants with StDMP Gene Mutation

The leaves of the T ₀ generation transgenic potato plants obtained in step 3 in Example 1 were collected, and genomic DNA was extracted as a template, and the following primer pairs were used for PCR amplification to obtain PCR amplification products of different lines.

The sequences of the primers for StDMP mutation sequence detection are as follows:

StDMP-test-F: AGAAGATTCAAAACATTTGTAAGTGCATTT (sequence 7)

StDMP-test-R: ACAACTGTCACGATTTATGGTGAAA (sequence 8)

The PCR amplification products of different strains were sequenced by Sanger, and compared with the wild-type StDMP gene according to the sequencing results. The StDMP genotypes were identified according to the following principles.

A sequence with bimodal characteristics from the target site sequence, the genotype of the line is a heterozygous genotype (StDMP gene mutation on one of the two homologous chromosomes, and StDMP on the other chromosome The gene is not mutated), the line is a T ₀ generation transgenic potato heterozygous mutant line;

Since the sequence of the target site has a bimodal characteristic sequence, and the StDMP gene is mutated in both homologous chromosomes, the line is a T ₀ generation transgenic potato biallelic mutant line.

If the sequence with specific unimodal characteristics from the target site sequence is the same as the StDMP gene sequence of wild-type potato, the genotype of the line is wild-type, that is, the StDMP gene sequence has no mutation; If the sequence of StDMP gene is different, the genotype of the line is homozygous genotype (the StDMP gene on both homologous chromosomes is mutated), and the line is a homozygous mutant line of T ₀ transgenic potato.

In this case, the T ₀ generation StDMP gene biallelic mutant line (as shown in Figure 1) was identified, which was used for the following haploid inducibility analysis experiment.

Example 3 Application of StDMP gene knockout potato mutant in inducing haploid

The T ₀ generation StDMP gene biallelic mutant lines obtained in Example 2 were respectively crossed with the potato B material (PG6359, the original number of the International Potato Center CIP705468) to obtain offspring, and the haploids in the offspring were identified by the following methods. .

Step 1. Fluorescent label identification

The CRISPR/Cas9 vector carries the promoter OLEO1 to drive the expression element of TagRFP (Entacmaea quadricolor). Since the promoter OLEO1 is specifically expressed in mature seed embryos, the fluorescent signal of TagRFP can be observed by fluorescent light. Therefore, using the mutant carrying the expression element as the male parent and crossing it with other non-fluorescent female parent materials, among the obtained seeds, the embryos of the diploid seeds showed red fluorescence due to the genome of the male parent, while the Embryos of haploid seeds appeared non-fluorescent due to their maternal origin (Figure 2).

Step 2. Haploid Seed Germination

The weak (non-) fluorescent seeds identified in the above step 1 were soaked in 2 mg/mL gibberellin for 48 hours, sterilized with 8% sodium hypochlorite, and then spread in 1/2 MS medium.

Since the haploid-inducing line is in the process of inducing haploid, the genome of the sperm combined with the egg cell will disappear, and the genome of the sperm fused with the central cell will remain, so there is no fluorescence in the roots of the haploid seeds after germination , and only fluoresce in the endosperm.

A week later, the seeds began to germinate one after another, and the germination rate reached 40%. The plants with no fluorescence in the roots and fluorescence in the endosperm were screened out, which were haploid plants.

Step 3. Molecular marker identification

Extract the genomic DNA of the plants with no fluorescence in the root and fluorescence in the endosperm identified in the above step 2, and perform PCR amplification with the polymorphic primers A+B between the maternal material and the transgenic mutant lines, and the amplified products are subjected to PCR amplification. Agarose band detection.

In this example, the primer sequences used are A: CGTGGAAGAACGTCGTG; B: CATGAGTCATATGGGGTGGA. If the size of the amplified product of the single plant to be tested is 117 bp, which appears as one band, the single plant band is considered to be the female parent band type. , there is no banding pattern of the male parent material, then the individual plant is the female parent haploid. If the size of the amplified product of the single plant to be tested is 117bp and 175bp, showing two bands, the single plant band is considered to be the heterozygous band type of the female parent and the transgenic mutant line, and the single plant is the offspring of a normal cross. , is a diploid.

As shown in Figure 3, I is the parent material of the transgenic potato plant, II is the hybrid potato B material (the female parent), III is the StDMP gene biallelic mutant line (the male parent), and IV and V are the StDMP gene biallelic mutation The haploid induced by the hybridization of the line with the potato B material can only amplify the genotype of the female potato B material, and the size of the amplified product is 117 bp, which appears as one band. VI and VII are diploids obtained by crossing the StDMP gene biallelic mutant line with the potato B material. The amplified products are 117 bp and 175 bp in size, showing two bands.

Step 4. Phenotypic identification of mature plants

The weak (non) fluorescent seeds identified in the above step 1 were further planted, and the phenotype of the single plant of the offspring was observed. The leaves are broad (Figure 4).

Step 5. Flow cytometric detection of leaf identification

Perform flow cytometry detection on the plants with haploid traits obtained in the above steps, and the specific method is as follows: extract the nucleus of the young leaves of the plants to be tested, and use the diploid potato leaves as a control; and then use a flow cytometer to detect the signal , firstly detect the diploid nuclear signal, and set the diploid nuclear signal peak position to 50. Since there may be replicating cells in the cell, the diploid nuclear signal peak will appear near 50 and 100. Diploid cells have twice as much genetic material as haploid cells, so the haploid nuclear signal peaks around 25 and 50. If the nuclear signal peak of the plant to be tested appears near 25 and 50, the plant to be tested is considered to be a haploid plant. If the signal peak of the plant to be tested appears near 50 and 100, it is considered to be the same as the enriched position of the signal intensity of the diploid nucleus, and the plant to be tested is a diploid (Fig. 5).

From the above identification results, it can be seen that after the potato StDMP gene is mutated and hybridized with other materials, the maternal haploid can be obtained in the offspring.

Step 6. Homozygous diploid obtained by colchicine doubling

The obtained potato haploids were taken from the apical shoots, transplanted into MS culture with colchicine (0.3%, 0.4%, 0.5% and 0.6%), and transferred to normal MS medium three days later. After a period of culturing, it was found that the terminal buds cultured in the 0.6% colchicine concentration medium appeared to die, and the development of other concentrations was normal. The ploidy test found that no doubling occurred at the colchicine concentration of 0.3% and 0.5%, and doubling occurred at the colchicine concentration of 0.4%, and the doubling efficiency was 5%. Subsequent attempts were made to keep apical buds in 0.4% and 0.5% colchicine MS medium, and it was found that the apical buds could develop normally. And in the 0.4% concentration of colchicine medium, 2 of the 7 terminal buds doubled, and the doubling efficiency was 28.6%; in the 0.5% concentration of colchicine medium, 1 of the 4 terminal buds doubled, and the doubling efficiency was 28.6%. The doubling rate was 25%, and the remaining terminal buds were not doubled. These detected diploid plants were homozygous diploid potatoes.

Sequence 1-Sequence 3 in the above embodiment is as follows:

Sequence 1:

ATGGAGCAAACTAGTGAAGGAATTGGAATAAAAATGTACAGTACATCGAAACGCGTCGATAATTCATCTTCTATGTATCCTACTAATTTACCACAAGATGATACAATCCCAGAATTATCTCAACCTGCATTACCAATTGGTGGGAAAAAAAGAAGAGCAATGGCAAATGGTGTACAAAAAACACTTTCAAAAACTTCATTACTTGTTAATTTTCTACCAACAGGCACACTTTTAACATTTGAAATGTTACTTCCATCAGTATTTGGTAAAGGAGATTGTTCACCAATTACTACATTTATGATTTTAACATTACTTGGACTTTGTACTTTGTCATGTTTTTTCTTCCATTTTACCGATAGTTTTCGAGGTCCTGATGGCAAAGTTTACTATGGTTTTGTTACACCAAGAGGTTTGAAAGTTTTCAAGACTGGACTTGGTGTTGATGTGCCAAAAGATGAAAGGTAATTTTTTATGTTGGTAATCGCCCTTAGCCACGAACACTATTTTCTATAAAAAAGAGAAAAATAGAGATTTGTGATAGGAATATTTTTGACTAAAGAGTTTCATACATATATGATTGTGACAAAAGATGAAAGGTAACTTTTAGGGATGGTTTTTGTAATATTTGTGTGTGTGAGAGACGTACATCGCCCTTGGCTATGAACACTATTTTTCTATGAGAAAAGAGAAAAATATTAATTTGTGATGGAATGAATATTTTTTACTAGAGAGTTTTCATTTATGTAAATTTTATGATTGTGACAAAAGATAGAAAGGTAACTTTTTAGTGATGATTTTTTCAATGTTTATGTGTGTGAGATGTAAATTCGCCCTTGGCTATGAACACTATTTTCTATGAGAAAAGAGAAAATATACAGATTTGTGATGGAATATTTTTGACTAAAGATTTTCATCCACATAATTATAAAAAATTTATGATTGTGACAAAAGGTTTTCCGTCACAAAAATTGTGTGTTTTTTTTAAAAATATAGTTATTTG CTTAAAGTCATTGTTTCACCATAAAAGATGACAGTTTTTAGATAAATTATCAATTTCACCCCTCAAAATCATTGTTTCACCATAAATCGTGACAGTTGTATAGCAAAATCTTCAATATTGTGGACGAAATTTGCAATGCTTTCTTTATTACTAATATTGTGTTTTTTCCCCCTTAATTTAGTGTGTTGACATCATTACCTTTTTGTTGGTTGGTTGTACTAGGTACATTGTGGGATTGACAGATTTTGTACATGCAATGATGTCTGTTTTGGTGTTTGTGGCAATTGCATTTTCTGATCATAGAGTGACACTTTGTCTATTTCCTGGACATGCTAAAGAACTTGATGAAATTATGAGGAGTTTTCCATTAATGGTTGGAGTTATTTGTAGTGGACTTTTTCTTGTTTTTCCTAATTCTAGATATGGTGTTGGATGTATGTCTGCTTAGCTATTTTTATTTTATTCTACACTAGACTACATTTCTTTTCTAGTTTTATACTGTTTCATATATATTGTAATTTAAGTTGGTCGATATTTATTTAAAATTATCTATATGATATATTGTTTTCTTTTCTTTTTCAAATTTATATTTCAAAATTTAAATATATTGGTATTTTGCTAGCTGTTCTAAATATGTCCCATATCCCTCCTATTTATTGCACAAAAATAACAGCTATAACTAAATATTTATATTGCTTGGACTTTTTAAATATATCACGTGGAGTATTTCAAATTTTCAATAATACCCCCTCTATTCCAAATGTAATTGTCCTTTTCAAGAGTCGTGAATTTTCCTAATTTTCAAAACTAAATTTAATTAGATAAGATAACTATGTACAAGTTACAACCTCTGCCACACACATCAATAAATCTATCACTTCAACCAGATAAGGTAACTACTTAAAATTTGTACATCCAAATCTCTACACATTTCAAAATTGCCACTATCCAGCTGTAATCTTCTTTGTATCGTTCCAATTAAGTACGTACTTAATCATAT GGCTATGTTAGTCCCAAAAATTATGTTGAATTCTGGTCATGAAATGCCTCTAATAGGCATGGGAACAGCACCAACGGCACCAACATTACCACCAATTGACCAATTAGTCTCCATTTTTATCGATGCTATCGAAGCTGGTTATCGACACTTTGACACCGCTGCGGCCTATGGCTCCGAGGAAGCACTAGGTCGAGCCGTGGCTGACGCGATACAACGCGGACTCATAGAGAGCCGTGAAGATGTTTTTATTACATCTAAGTTATGGTGCACCGAAACACATCATGACCTTGTTCTTCCTGCTCTCAAAAGATCTCTCGCGTAAGTTTGCTCATACTATTGATTTTAAATTCGAATAAAATAAAAAGTTGTGATAGATTAACATCTTATACATGTTTCTACGGGGTGGGGGTTTGGTTCAACCCCTACCATGTAGTAATATATATATATAAGCAAAAAATTCACTTTAATTAGAGGGACATGGATCTTGTCTATAATCTACAAAAATATTGTCATTGGCTCATAGCAATTTATATGTGGTAATATTCCCAACTTCATTTATGTGATTGTATTTCCTTTTAAGTCCATTCTAGATAGAATGACTATTTTCTAATTTTGATAATAATATAATTTTGACTTTCTCCTTTTTTCCATAATGACATATTTGTATAATCACACAAATATTATACAATATATTTAATATCACATATTTCAAAAATTTTATCGTCATATAAATGTTATGATACGTTTAAGATTACGAATTTTATAAGTCTTGATTTCTTTTTAAAACTTTGCATCTGATCAAAATACACTACATAAATTGAAATTACTATATAATTATTGTTGTTATTGGTACAAACACAATACATGCATCAAATTATTATACTTCATATATTTTGTCATCAATTTCATATATTAATCATGAATGTAAAGATATCTCATCCTCTTTCAGATTTCACATAATGTGGAACTATCCTTGTTGTTGTAATCATGAATGTCATAA AATGTTTTGTAGGAGATTAGGGATGGATTACTTGGACTTGTACCATATACATTGGCCAGTGAGGATGAAGAATGGTAGTGACCAAGGTATAAAGTTGGTAAATGAGGATGTAATTCCATTTGACATGAAAGGAACATGGGAAGCTATGGAAGAATGTCACAAATTAGGTTTGGCCAAATCTATTGGTGTATGCAACTTTAGTTGCACAAAACTCTCTCAACTCCTAACTCATGCTACCATTCCTCCTGCTGTAAATCAGGTAAATATAACATACTATACCCTTAGCGATATTTAATTGGGAACGAAATTTAAGAAATTTAAAGCTATAGATATTTATGGCTATAAATCATTTCGTTAATAATAACATGAATTAATTTAAAATTTAGTTTATGATCAAGAAATGATAACATTCTTTTTGTATAGACTAGAGAGGAAAGTACGACATTCTAGTATAGAATCTTTAAATCTTTTGAAATAAGATTTACTTATTTAAAAATTACATAAAAAATATATTATAAAATGCAATAATTGACAGGTGGAAATGCATGTAGCATGGAGACAAGAGAAGATGTTAGCATTTTGTAAAGAGAAAGGAATACATGTAAGTGCATGGTCTCCTCTTGGAGCAAATGGAATACCTTTTTGGGGCAATCATGCTGTTATGCAAAACTCTGTTCTAAAAGACATTGCCTTTCATAGACAAAAGAGCATTCCACAGGTACCAATAAATAATTAAATTATTCACTCAGTGTTCACACTAAATTATATTAGTATATTAACGTTTAATATATCAATAACTTTTTAAATTTGCTCTCAAATTCTATTTTGACACTTGAAGTAAGACTTGTTTCAATTGAACATCTGAACTCATGATAAATTTTTTTTTATTAGTAATCATTAGACACTTTCAGTTCAAATTTTGAAGAAAAAAGATATCTACTAGATAGTTAAGTTAACTCCATAAAATATGACATCTCCTTTAAGTATGCGATTAGCCTCA ATTGAGGTTTTACGACTTATTAAGGCAAATACATTTAATCAGAGGAGGTAGCATGTTTTAAGTGTGACGTTTGAGAACACACGCTAAAAGTTTTTCAAAATTTGAACCGTAAGTCTCTAATAAAACGCTTTACTATGTGTTTTAAGTGATTAGAATAAATATTAGTTCGAACATCCAAATAAAATTGCTCACAAGGATAGTAATTGTTTGATATTTGTAGGTGGCATTGAGATGGGTATATGAGCAAGGTGTTAGTGTATTAGTGAAGAGTTTTAACAAAGATAGAATGAAAGAGAATCTTCAAATTTTGGATTGGGAATTAAGCAATGAAGAAAATGCTAAGATTCAAGAGATTCCTCAATGCAGGGGATTCAAAGGTGAACTTTTTGTTCATCCAGATGGACCATACAAATCCCCAGATGAATTCTGGGATGGTGAACTATAA

Sequence 2:

Met Glu Gln Thr Ser Glu Gly Ile Gly Ile Lys Met Tyr Ser Thr Ser LysArg Val Asp Asn Ser Ser Ser Met Tyr Pro Thr Asn Leu Pro Gln Asp Asp Thr IlePro Glu Leu Ser Gln Pro Ala Leu Pro Ile Gly Gly Lys Lys Arg Arg Ala Met AlaAsn Gly Val Gln Lys Thr Leu Ser Lys Thr Ser Leu Leu Val Asn Phe Leu Pro ThrGly Thr Leu Leu Thr Phe Glu Met Leu Leu Pro Ser Val Phe Gly Lys Gly Asp CysSer Pro Ile Thr Thr Phe Met Ile Leu Thr Leu Leu Gly Leu Cys Thr Leu Ser CysPhe Phe Phe His Phe Thr Asp Ser Phe Arg Gly Pro Asp Gly Lys Val Tyr Tyr GlyPhe Val Thr Pro Arg Gly Leu Lys Val Phe Lys Thr Gly Leu Gly Val Asp Val ProLys Asp Glu Arg Tyr Ile Val Gly Leu Thr Asp Phe Val His Ala Met Met Ser ValLeu Val Phe Val Ala Ile Ala Phe Ser Asp His Arg Val Thr Leu Cys Leu Phe ProGly His Ala Lys Glu Leu Asp Glu Ile Met Arg Ser Phe Pro Leu Met Val Gly ValIle Cys Ser Gly Leu Phe Leu Val Phe Pro Asn Ser Arg Tyr Gly Val Gly Cys MetGly Thr Ala Pro Thr Ala Pro Thr Leu Pro Pro Ile Asp Gln Leu Val Ser Ile PheIle Asp Ala Ile Glu Ala Gly Tyr A rg His Phe Asp Thr Ala Ala Ala Ala Tyr Gly SerGlu Glu Ala Leu Gly Arg Ala Val Ala Asp Ala Ile Gln Arg Gly Leu Ile Glu SerArg Glu Asp Val Phe Ile Thr Ser Lys Leu Trp Cys Thr Glu Thr His His Asp LeuVal Leu Pro Ala Leu Lys Arg Ser Leu Ala Arg Leu Gly Met Asp Tyr Leu Asp LeuTyr His Ile His Trp Pro Val Arg Met Lys Asn Gly Ser Asp Gln Gly Ile Lys LeuVal Asn Glu Asp Val Ile Pro Phe Asp Met Lys Gly Thr Trp Glu Ala Met Glu GluCys His Lys Leu Gly Leu Ala Lys Ser Ile Gly Val Cys Asn Phe Ser Cys Thr LysLeu Ser Gln Leu Leu Thr His Ala Thr Ile Pro Pro Ala Val Asn Gln Val Glu MetHis Val Ala Trp Arg Gln Glu Lys Met Leu Ala Phe Cys Lys Glu Lys Gly Ile HisVal Ser Ala Trp Ser Pro Leu Gly Ala Asn Gly Ile Pro Phe Trp Gly Asn His AlaVal Met Gln Asn Ser Val Leu Lys Asp Ile Ala Phe His Arg Gln Lys Ser Ile ProGln Val Ala Leu Arg Trp Val Tyr Glu Gln Gly Val Ser Val Leu Val Lys Ser PheAsn Lys Asp Arg Met Lys Glu Asn Leu Gln Ile Leu Asp Trp Glu Leu Ser Asn GluGlu Asn Ala Lys Ile Gln Glu Ile Pro Gln Cys Arg Gly Phe Ly s Gly Glu Leu PheVal His Pro Asp Gly Pro Tyr Lys Ser Pro Asp Glu Phe Trp Asp Gly Glu Leu

Sequence 3:

ATGGAGCAAACTAGTGAAGGAATTGGAATAAAAATGTACAGTACATCGAAACGCGTCGATAATTCATCTTCTATGTATCCTACTAATTTACCACAAGATGATACAATCCCAGAATTATCTCAACCTGCATTACCAATTGGTGGGAAAAAAAGAAGAGCAATGGCAAATGGTGTACAAAAAACACTTTCAAAAACTTCATTACTTGTTAATTTTCTACCAACAGGCACACTTTTAACATTTGAAATGTTACTTCCATCAGTATTTGGTAAAGGAGATTGTTCACCAATTACTACATTTATGATTTTAACATTACTTGGACTTTGTACTTTGTCATGTTTTTTCTTCCATTTTACCGATAGTTTTCGAGGTCCTGATGGCAAAGTTTACTATGGTTTTGTTACACCAAGAGGTTTGAAAGTTTTCAAGACTGGACTTGGTGTTGATGTGCCAAAAGATGAAAGGTACATTGTGGGATTGACAGATTTTGTACATGCAATGATGTCTGTTTTGGTGTTTGTGGCAATTGCATTTTCTGATCATAGAGTGACACTTTGTCTATTTCCTGGACATGCTAAAGAACTTGATGAAATTATGAGGAGTTTTCCATTAATGGTTGGAGTTATTTGTAGTGGACTTTTTCTTGTTTTTCCTAATTCTAGATATGGTGTTGGATGCATGGGAACAGCACCAACGGCACCAACATTACCACCAATTGACCAATTAGTCTCCATTTTTATCGATGCTATCGAAGCTGGTTATCGACACTTTGACACCGCTGCGGCCTATGGCTCCGAGGAAGCACTAGGTCGAGCCGTGGCTGACGCGATACAACGCGGACTCATAGAGAGCCGTGAAGATGTTTTTATTACATCTAAGTTATGGTGCACCGAAACACATCATGACCTTGTTCTTCCTGCTCTCAAAAGATCTCTCGCGAGATTAGGGATGGATTACTTGGACTTGTACCATATACATTGGCCAGTGAGGATGAAGAATGGTA GTGACCAAGGTATAAAGTTGGTAAATGAGGATGTAATTCCATTTGACATGAAAGGAACATGGGAAGCTATGGAAGAATGTCACAAATTAGGTTTGGCCAAATCTATTGGTGTATGCAACTTTAGTTGCACAAAACTCTCTCAACTCCTAACTCATGCTACCATTCCTCCTGCTGTAAATCAGGTGGAAATGCATGTAGCATGGAGACAAGAGAAGATGTTAGCATTTTGTAAAGAGAAAGGAATACATGTAAGTGCATGGTCTCCTCTTGGAGCAAATGGAATACCTTTTTGGGGCAATCATGCTGTTATGCAAAACTCTGTTCTAAAAGACATTGCCTTTCATAGACAAAAGAGCATTCCACAGGTGGCATTGAGATGGGTATATGAGCAAGGTGTTAGTGTATTAGTGAAGAGTTTTAACAAAGATAGAATGAAAGAGAATCTTCAAATTTTGGATTGGGAATTAAGCAATGAAGAAAATGCTAAGATTCAAGAGATTCCTCAATGCAGGGGATTCAAAGGTGAACTTTTTGTTCATCCAGATGGACCATACAAATCCCCAGATGAATTCTGGGATGGTGAACTATAA

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. within.

sequence listing

<110> Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences

China Agricultural University

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences

<120> Applications of polynucleotides and proteins and their haploid inducible lines

<130> 21-13620CN

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4445

<212> DNA

<213> Artificial Sequence

<400> 1

atggagcaaa ctagtgaagg aattggaata aaaatgtaca gtacatcgaa acgcgtcgat 60

aattcatctt ctatgtatcc tactaattta ccacaagatg atacaatccc agaattatct 120

caacctgcat taccaattgg tgggaaaaaa agaagagcaa tggcaaatgg tgtacaaaaa 180

acactttcaa aaacttcatt acttgttaat tttctaccaa caggcacact tttaacattt 240

gaaatgttac ttccatcagt atttggtaaa ggagattgtt caccaattac tacatttatg 300

attttaacat tacttggact ttgtactttg tcatgttttt tcttccattt taccgatagt 360

tttcgaggtc ctgatggcaa agtttactat ggttttgtta caccaagagg tttgaaagtt 420

ttcaagactg gacttggtgt tgatgtgcca aaagatgaaa ggtaattttt tatgttggta 480

atcgccctta gccacgaaca ctattttcta taaaaaagag aaaaatagag atttgtgata 540

ggaatatttt tgactaaaga gtttcataca tatatgattg tgacaaaaga tgaaaggtaa 600

cttttaggga tggttttttgt aatatttgtg tgtgtgagag acgtacatcg cccttggcta 660

tgaacactat ttttctatga gaaaagagaa aaatattaat ttgtgatgga atgaatattt 720

tttactagag agttttcatt tatgtaaatt ttatgattgt gacaaaagat agaaaggtaa 780

ctttttagtg atgatttttt caatgtttat gtgtgtgaga tgtaaattcg cccttggcta 840

tgaacactat tttctatgag aaaagagaaa atatacagat ttgtgatgga atatttttga 900

ctaaagattt tcatccacat aattataaaa aatttatgat tgtgacaaaa ggttttccgt 960

cacaaaaatt gtgtgttttt tttaaaaata tagttatttg cttaaagtca ttgtttcacc 1020

ataaaagatg acagttttta gataaattat caatttcacc cctcaaaatc attgtttcac 1080

cataaatcgt gacagttgta tagcaaaatc ttcaatattg tggacgaaat ttgcaatgct 1140

ttctttatta ctaatattgt gttttttccc ccttaattta gtgtgttgac atcattacct 1200

ttttgttggt tggttgtact aggtacattg tgggattgac agattttgta catgcaatga 1260

tgtctgtttt ggtgtttgtg gcaattgcat tttctgatca tagagtgaca ctttgtctat 1320

ttcctggaca tgctaaagaa cttgatgaaa ttatgaggag ttttccatta atggttggag 1380

ttatttgtag tggacttttt cttgtttttc ctaattctag atatggtgtt ggatgtatgt 1440

ctgcttagct atttttattt tattctacac tagactacat ttcttttcta gttttatact 1500

gtttcatata tattgtaatt taagttggtc gatatttatt taaaattatc tatatgatat 1560

attgttttct tttctttttc aaatttatat ttcaaaattt aaatatattg gtattttgct 1620

agctgttcta aatatgtccc atatccctcc tatttattgc acaaaaataa cagctataac 1680

taaatattta tattgcttgg actttttaaa tatatcacgt ggagtatttc aaattttcaa 1740

taataccccc tctattccaa atgtaattgt ccttttcaag agtcgtgaat tttcctaatt 1800

ttcaaaacta aatttaatta gataagataa ctatgtacaa gttacaacct ctgccacaca 1860

catcaataaa tctatcactt caaccagata aggtaactac ttaaaatttg tacatccaaa 1920

tctctacaca tttcaaaatt gccactatcc agctgtaatc ttctttgtat cgttccaatt 1980

aagtacgtac ttaatcatat ggctatgtta gtcccaaaaa ttatgttgaa ttctggtcat 2040

gaaatgcctc taataggcat gggaacagca ccaacggcac caacattacc accaattgac 2100

caattagtct ccatttttat cgatgctatc gaagctggtt atcgacactt tgacaccgct 2160

gcggcctatg gctccgagga agcactaggt cgagccgtgg ctgacgcgat acaacgcgga 2220

ctcatagaga gccgtgaaga tgtttttatt acatctaagt tatggtgcac cgaaacacat 2280

catgaccttg ttcttcctgc tctcaaaaga tctctcgcgt aagtttgctc atactattga 2340

ttttaaattc gaataaaata aaaagttgtg atagattaac atcttataca tgtttctacg 2400

gggtgggggt ttggttcaac ccctaccatg tagtaatata tatatataag caaaaaattc 2460

actttaatta gagggacatg gatcttgtct ataatctaca aaaatattgt cattggctca 2520

tagcaattta tatgtggtaa tattcccaac ttcatttatg tgattgtatt tccttttaag 2580

tccattctag atagaatgac tattttctaa ttttgataat aatataattt tgactttctc 2640

cttttttcca taatgacata tttgtataat cacacaaata ttatacaata tatttaatat 2700

cacatatttc aaaaatttta tcgtcatata aatgttatga tacgtttaag attacgaatt 2760

ttataagtct tgatttcttt ttaaaacttt gcatctgatc aaaatacact acataaattg 2820

aaattactat ataattattg ttgttattgg tacaaacaca atacatgcat caaattatta 2880

tacttcatat attttgtcat caatttcata tattaatcat gaatgtaaag attctcatc 2940

ctctttcaga tttcacataa tgtggaacta tccttgttgt tgtaatcatg aatgtcataa 3000

aatgttttgt aggagattag ggatggatta cttggacttg taccatatac attggccagt 3060

gaggatgaag aatggtagtg accaaggtat aaagttggta aatgaggatg taattccatt 3120

tgacatgaaa ggaacatggg aagctatgga agaatgtcac aaattaggtt tggccaaatc 3180

tattggtgta tgcaacttta gttgcacaaa actctctcaa ctcctaactc atgctaccat 3240

tcctcctgct gtaaatcagg taaatataac atactatacc cttagcgata tttaattggg 3300

aacgaaattt aagaaattta aagctataga tatttatggc tataaatcat ttcgttaata 3360

ataacatgaa ttaatttaaa atttagttta tgatcaagaa atgataacat tctttttgta 3420

tagactagag aggaaagtac gacattctag tatagaatct ttaaatcttt tgaaataaga 3480

tttacttatt taaaaattac ataaaaaata tattataaaa tgcaataatt gacaggtgga 3540

aatgcatgta gcatggagac aagagaagat gttagcattt tgtaaagaga aaggaataca 3600

tgtaagtgca tggtctcctc ttggagcaaa tggaatacct ttttggggca atcatgctgt 3660

tatgcaaaac tctgttctaa aagacattgc ctttcataga caaaagagca ttccacaggt 3720

accaataaat aattaaatta ttcactcagt gttcacacta aattatatta gtatattaac 3780

gtttaatata tcaataactt tttaaatttg ctctcaaatt ctattttgac acttgaagta 3840

agacttgttt caattgaaca tctgaactca tgataaattt ttttttatta gtaatcatta 3900

gacactttca gttcaaattt tgaagaaaaa agatatctac tagatagtta agttaactcc 3960

ataaaatatg acatctcctt taagtatgcg attagcctca attgaggttt tacgacttat 4020

taaggcaaat acatttaatc agaggaggta gcatgtttta agtgtgacgt ttgagaacac 4080

acgctaaaag tttttcaaaa tttgaaccgt aagtctctaa taaaacgctt tactatgtgt 4140

tttaagtgat tagaataaat attagttcga acatccaaat aaaattgctc acaaggatag 4200

taattgtttg atatttgtag gtggcattga gatgggtata tgagcaaggt gttagtgtat 4260

tagtgaagag ttttaacaaa gatagaatga aagagaatct tcaaattttg gattgggaat 4320

taagcaatga agaaaatgct aagattcaag agattcctca atgcagggga ttcaaaggtg 4380

aactttttgt tcatccagat ggaccataca aatccccaga tgaattctgg gatggtgaac 4440

tataa 4445

<210> 2

<211> 529

<212> PRT

<213> Artificial Sequence

<400> 2

Met Glu Gln Thr Ser Glu Gly Ile Gly Ile Lys Met Tyr Ser Thr Ser

1 5 10 15

Lys Arg Val Asp Asn Ser Ser Ser Met Tyr Pro Thr Asn Leu Pro Gln

20 25 30

Asp Asp Thr Ile Pro Glu Leu Ser Gln Pro Ala Leu Pro Ile Gly Gly

35 40 45

Lys Lys Arg Arg Ala Met Ala Asn Gly Val Gln Lys Thr Leu Ser Lys

50 55 60

Thr Ser Leu Leu Val Asn Phe Leu Pro Thr Gly Thr Leu Leu Thr Phe

65 70 75 80

Glu Met Leu Leu Pro Ser Val Phe Gly Lys Gly Asp Cys Ser Pro Ile

85 90 95

Thr Thr Phe Met Ile Leu Thr Leu Leu Gly Leu Cys Thr Leu Ser Cys

100 105 110

Phe Phe Phe His Phe Thr Asp Ser Phe Arg Gly Pro Asp Gly Lys Val

115 120 125

Tyr Tyr Gly Phe Val Thr Pro Arg Gly Leu Lys Val Phe Lys Thr Gly

130 135 140

Leu Gly Val Asp Val Pro Lys Asp Glu Arg Tyr Ile Val Gly Leu Thr

145 150 155 160

Asp Phe Val His Ala Met Met Ser Val Leu Val Phe Val Ala Ile Ala

165 170 175

Phe Ser Asp His Arg Val Thr Leu Cys Leu Phe Pro Gly His Ala Lys

180 185 190

Glu Leu Asp Glu Ile Met Arg Ser Phe Pro Leu Met Val Gly Val Ile

195 200 205

Cys Ser Gly Leu Phe Leu Val Phe Pro Asn Ser Arg Tyr Gly Val Gly

210 215 220

Cys Met Gly Thr Ala Pro Thr Ala Pro Thr Leu Pro Pro Ile Asp Gln

225 230 235 240

Leu Val Ser Ile Phe Ile Asp Ala Ile Glu Ala Gly Tyr Arg His Phe

245 250 255

Asp Thr Ala Ala Ala Tyr Gly Ser Glu Glu Ala Leu Gly Arg Ala Val

260 265 270

Ala Asp Ala Ile Gln Arg Gly Leu Ile Glu Ser Arg Glu Asp Val Phe

275 280 285

Ile Thr Ser Lys Leu Trp Cys Thr Glu Thr His His Asp Leu Val Leu

290 295 300

Pro Ala Leu Lys Arg Ser Leu Ala Arg Leu Gly Met Asp Tyr Leu Asp

305 310 315 320

Leu Tyr His Ile His Trp Pro Val Arg Met Lys Asn Gly Ser Asp Gln

325 330 335

Gly Ile Lys Leu Val Asn Glu Asp Val Ile Pro Phe Asp Met Lys Gly

340 345 350

Thr Trp Glu Ala Met Glu Glu Cys His Lys Leu Gly Leu Ala Lys Ser

355 360 365

Ile Gly Val Cys Asn Phe Ser Cys Thr Lys Leu Ser Gln Leu Leu Thr

370 375 380

His Ala Thr Ile Pro Pro Ala Val Asn Gln Val Glu Met His Val Ala

385 390 395 400

Trp Arg Gln Glu Lys Met Leu Ala Phe Cys Lys Glu Lys Gly Ile His

405 410 415

Val Ser Ala Trp Ser Pro Leu Gly Ala Asn Gly Ile Pro Phe Trp Gly

420 425 430

Asn His Ala Val Met Gln Asn Ser Val Leu Lys Asp Ile Ala Phe His

435 440 445

Arg Gln Lys Ser Ile Pro Gln Val Ala Leu Arg Trp Val Tyr Glu Gln

450 455 460

Gly Val Ser Val Leu Val Lys Ser Phe Asn Lys Asp Arg Met Lys Glu

465 470 475 480

Asn Leu Gln Ile Leu Asp Trp Glu Leu Ser Asn Glu Glu Asn Ala Lys

485 490 495

Ile Gln Glu Ile Pro Gln Cys Arg Gly Phe Lys Gly Glu Leu Phe Val

500 505 510

His Pro Asp Gly Pro Tyr Lys Ser Pro Asp Glu Phe Trp Asp Gly Glu

515 520 525

Leu

<210> 3

<211> 1590

<212> DNA

<213> Artificial Sequence

<400> 3

atggagcaaa ctagtgaagg aattggaata aaaatgtaca gtacatcgaa acgcgtcgat 60

aattcatctt ctatgtatcc tactaattta ccacaagatg atacaatccc agaattatct 120

caacctgcat taccaattgg tgggaaaaaa agaagagcaa tggcaaatgg tgtacaaaaa 180

acactttcaa aaacttcatt acttgttaat tttctaccaa caggcacact tttaacattt 240

gaaatgttac ttccatcagt atttggtaaa ggagattgtt caccaattac tacatttatg 300

attttaacat tacttggact ttgtactttg tcatgttttt tcttccattt taccgatagt 360

tttcgaggtc ctgatggcaa agtttactat ggttttgtta caccaagagg tttgaaagtt 420

ttcaagactg gacttggtgt tgatgtgcca aaagatgaaa ggtacattgt gggattgaca 480

gattttgtac atgcaatgat gtctgttttg gtgtttgtgg caattgcatt ttctgatcat 540

agagtgacac tttgtctatt tcctggacat gctaaagaac ttgatgaaat tatgaggagt 600

tttccattaa tggttggagt tatttgtagt ggactttttc ttgtttttcc taattctaga 660

tatggtgttg gatgcatggg aacagcacca acggcaccaa cattaccacc aattgaccaa 720

ttagtctcca ttttttatcga tgctatcgaa gctggttatc gacactttga caccgctgcg 780

gcctatggct ccgaggaagc actaggtcga gccgtggctg acgcgataca acgcggactc 840

atagagagcc gtgaagatgt ttttattaca tctaagttat ggtgcaccga aacacatcat 900

gaccttgttc ttcctgctct caaaagatct ctcgcgagat tagggatgga ttacttggac 960

ttgtaccata tacattggcc agtgaggatg aagaatggta gtgaccaagg tataaagttg 1020

gtaaatgagg atgtaattcc atttgacatg aaaggaacat gggaagctat ggaagaatgt 1080

cacaaattag gtttggccaa atctattggt gtatgcaact ttagttgcac aaaactctct 1140

caactcctaa ctcatgctac cattcctcct gctgtaaatc aggtggaaat gcatgtagca 1200

tggagacaag agaagatgtt agcattttgt aaagagaaag gaatacatgt aagtgcatgg 1260

tctcctcttg gagcaaatgg aatacctttt tggggcaatc atgctgttat gcaaaactct 1320

gttctaaaag acattgcctt tcatagacaa aagagcattc cacaggtggc attgagatgg 1380

gtatatgagc aaggtgttag tgtattagtg aagagtttta acaaagatag aatgaaagag 1440

aatcttcaaa ttttggattg ggaattaagc aatgaagaaa atgctaagat tcaagagatt 1500

cctcaatgca ggggattcaa aggtgaactt tttgttcatc cagatggacc atacaaatcc 1560

ccagatgaat tctgggatgg tgaactataa 1590

<210> 4

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 4

gagcaaacta gtgaaggaat 20

<210> 5

<211> 76

<212> DNA/RNA

<213> Artificial Sequence

<400> 5

guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu 60

ggcaccgagu cggugc 76

<210> 6

<211> 76

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 7

<211> 30

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

agaagattca aaacatttgt aagtgcattt 30

<210> 8

<211> 25

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

acaactgtca cgatttatgg tgaaa 25

Claims (10)

1. The application of polynucleotide in haploid induction of potato, wherein the polynucleotide is at least one of the following sequences:

1) SEQ ID NO: 1;

2) SEQ ID NO: 1, wherein the homologous sequence is a sequence that is complementary to, degenerate as, or homologous to the sequence set forth in SEQ ID NO: 1 or more than 75% identity;

3) under stringent conditions with SEQ ID NO: 1 or a complementary sequence thereof;

4) a cDNA sequence or CDS sequence of any one of the sequences 1) to 3).

2. The application of a protein in potato haploid induction is characterized in that the protein is at least one of the following sequences:

1) SEQ ID NO: 2;

2) SEQ ID NO: 2, the N end and/or the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

3) SEQ ID NO: 2 by substitution and/or deletion and/or addition of one or more amino acid residues to obtain the protein with the same function;

4) and SEQ ID NO: 2 has homology of 75% or more than 75% and has the same function.

3. A potato haploid inducer line, characterized in that the haploid inducer line reduces the expression and/or activity of the polynucleotide of claim 1 or the protein of claim 2 by means of mutation by insertion, substitution and/or deletion of a gene.

4. The haploid inducer line of claim 3, wherein the mutation is one of CRISPR/Cas9, TELLEN technology, T-DNA insertion, EMS mutagenesis, ZFN technology.

5. Use of the haploid inducer line of claim 3 or 4 for haploid breeding or crossbreeding of potatoes.

6. A biomaterial, characterized in that it is any of the following 1) to 4):

1) a knock-out cassette for knocking out the polynucleotide of claim 1;

2) a knock-out vector for knocking out the polynucleotide of claim 1;

3) a recombinant microorganism which knockdown the polynucleotide of claim 1;

4) a transgenic plant cell line wherein the polynucleotide of claim 1 is knocked out.

7. A potato plant grown from the transgenic plant cell line of claim 6.

8. A potato plant, which is a progeny resulting from selfing of the plant of claim 7, and a plant grown from said progeny;

or progeny thereof formed by crossing the plant of claim 7 with another variety, and plants grown from said progeny.

9. A preparation method of a potato haploid plant is characterized by comprising the following steps:

1) selfing or crossing with other varieties to obtain seeds by using the haploid inducer line as a male parent in claim 3;

2) treating the seeds with gibberellin of 1-5 mg/mL for 24-72 hours, disinfecting, and sowing in 1/2MS culture medium to obtain seedlings;

3) and (4) carrying out haploid identification on the seedlings to obtain haploid plants.

10. A method for preparing a potato homozygous diploid plant, comprising the steps of: obtaining haploid plants by the screening method of claim 9, taking the top shoots of the haploid plants, and performing chromosome doubling treatment with 0.35% -0.55% colchicine for at least 2 days to obtain homozygous diploid plants.

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