CN106480060B - Recombinant nucleic acid fragment RecCR01BC06 and detection method thereof - Google Patents

Abstract

The present application provides recombinant nucleic acid fragments and methods for their detection. The application also provides a breeding method of the rice plant containing the recombinant nucleic acid segment, and the rice plant containing the recombinant nucleic acid segment is obtained by performing foreground selection and background selection on the recombinant plant by using the molecular marker.

Description

Recombinant nucleic acid fragment RecCR01BC06 and its detection method

technical field

This application relates to genome-wide selective breeding techniques. Specifically, the present application relates to the selection and breeding of rice plants containing recombinant nucleic acid fragments using the genome-wide selective breeding technology, as well as the recombinant nucleic acid fragments obtained therefrom and detection methods thereof.

Background technique

For a long time, the selection method of traditional breeding mainly relied on the evaluation of field phenotype, and the choice was made according to the personal experience of the breeder. The biggest disadvantage was that it was time-consuming and inefficient. To improve the efficiency of selection, the ideal method should be to be able to select directly on the genotype. With the development of molecular biotechnology, molecular markers provide the possibility to realize the direct selection of genotypes. In recent years, molecular marker-assisted selection methods have been applied to improve individual target traits, which can significantly shorten the breeding years.

Rice blast is one of the most serious diseases of rice, and the annual yield loss of rice caused by rice blast accounts for 11% to 30%. Therefore, research on rice blast and its resistance is particularly important. With the gradual deepening of rice blast research, many rice blast resistance gene DNA fragments have been located and cloned one after another. Among them, many rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm, and Pi50, were located and cloned in the Pi2 region of rice chromosome 6, and this region contains a gene cluster of rice blast resistance genes (Qu et al., Genetics. 2006, 172:1331-1914; Wang et al., Phytopathology. 2012, 102:779-786; Xiao et al., Mol Breeding. 2012, 30:1715-1726; Liu et al., Mol Genet Genomics. 2002, 267:472- 480; Jiang et al, Rice. 2012, 5:29-35; Zhu et al, Theor Appl Genet. 2012, 124:1295-1304; Deng et al, Theor Appl Genet. 2006, 113:705-713).

SUMMARY OF THE INVENTION

In one aspect, the present application provides a recombinant nucleic acid fragment selected from: i) a sequence comprising nucleotides 790-2130 of the sequence shown in SEQ ID NO: 1 or a fragment or a variant thereof or a complementary sequence thereof; ii) comprising The sequence of the sequence shown in SEQ ID NO: 1 or a fragment or a variant thereof or its complement; iii) a sequence comprising nucleotides 770-1283 of the sequence shown in SEQ ID NO: 2 or a fragment or a variant thereof or or iv) a sequence comprising the sequence shown in SEQ ID NO: 2 or a fragment thereof or a variant thereof or its complement; and combinations of the above fragments. In one embodiment, the recombinant nucleic acid fragment is a genomic recombinant nucleic acid fragment.

In addition, the present application provides a primer for detecting the recombinant nucleic acid fragment, which is selected from: (1) a forward primer that specifically recognizes the sequence of nucleotides 1-790 of the sequence shown in SEQ ID NO: 1 and a forward primer that specifically recognizes SEQ ID NO: 1 The reverse primer of the sequence of nucleotides 2130-4133 of the sequence shown in ID NO: 1; (II) the following combination of the first set of primer pairs and the second set of primer pairs, which comprises (a) the first set of primer pairs: Forward primer that specifically recognizes the sequence of nucleotides 1-790 of the sequence shown in SEQ ID NO: 1 and reverse primer that specifically recognizes the sequence of nucleotides 791-2129 of the sequence shown in SEQ ID NO: 1 primers; and (b) a second set of primer pairs: a forward primer that specifically recognizes the sequence of nucleotides 791-2129 of the sequence shown in SEQ ID NO:1 and a forward primer that specifically recognizes the sequence shown in SEQ ID NO:1. A reverse primer for the sequence of nucleotides 2130-4133; (III) a forward primer that specifically recognizes a sequence comprising nucleotides 790-791 of the sequence shown in SEQ ID NO: 1 and a forward primer that specifically recognizes the sequence comprising SEQ ID NO: 1 The reverse primer of the sequence of nucleotides 2129-2130 of the sequence shown in NO: 1; (IV) the forward primer that specifically recognizes the sequence of nucleotides 790-791 of the sequence shown in SEQ ID NO: 1 and A reverse primer that specifically recognizes the sequence of the 2130-4133 nucleotides of the sequence shown in SEQ ID NO: 1; (V) the sequence that specifically recognizes the 1-790 nucleotides of the sequence shown in SEQ ID NO: 1 The forward primer and the reverse primer that specifically recognizes the sequence comprising nucleotides 2129-2130 of the sequence shown in SEQ ID NO:1; and/or optionally, (VI) specifically recognizes SEQ ID NO:2 The forward primer of the sequence of the nucleotides at positions 1-770 of the sequence shown and the reverse primers that specifically recognize the sequence of the nucleotides at positions 1283-2244 of the sequence shown in SEQ ID NO: 2; (VII) The following third set of primers A combination of a pair with a fourth set of primer pairs comprising (c) a third set of primer pairs: forward primers that specifically recognize the sequence of nucleotides 1-770 of the sequence shown in SEQ ID NO: 2 and specific recognition A reverse primer for the sequence of nucleotides 771-1282 of the sequence shown in SEQ ID NO: 2; and (d) a fourth set of primer pairs: specifically recognizing nucleotides 771-1282 of the sequence shown in SEQ ID NO: 2 The forward primer of the sequence of the acid and the reverse primer that specifically recognizes the sequence of the 1283-2244th nucleotide of the sequence shown in SEQ ID NO:2; (VIII) The specific recognition includes the sequence shown in SEQ ID NO:2, The forward primer of the sequence of 770-771 nucleotides and the reverse primer that specifically recognizes the sequence comprising the 1282-1283 nucleotides of the sequence shown in SEQ ID NO: 2; (IX) The specific recognition includes The forward primer of the sequence of the 770-771 nucleotides of the sequence shown in SEQ ID NO:2 and the reverse primer that specifically recognizes the sequence of the 1283-2244 nucleotides of the sequence shown in SEQ ID NO:2; ( X) A forward primer that specifically recognizes a sequence comprising nucleotides 1 to 770 of the sequence shown in SEQ ID NO: 2 and a sequence that specifically recognizes nucleotides 1282 to 1283 of the sequence shown in SEQ ID NO: 2 the reverse primer.

In one embodiment, the primer pair used to amplify the sequence shown in SEQ ID NO: 1 is, for example, 5'-CTCCATACCACATCGGTGACTCT-3', and 5'-CCTCATTGTGGGCTGTGTTCA-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO: 1 are, for example, 5'-TGCATTCGTCTAGCATGTGT-3', 5'-GATCTTGTCCGGTGCTAGCA-3', 5'-GTTGGCACACCACGTAAGCT-3', 5'-ATACCTCCAGGCTCTAGTCA-3', 5'-GATGAGAGGGATTCCTACCT-3', 5'-AGTCACCTCAGTTGTTAGTG-3', 5'-AGATGGGGTGAACATTGAGGA-3', 5'-CAGGAGGTCTGGTTCTGGTT-3', and 5'-CCTCATTGTGGGCTGTGTTCA-3'.

In another embodiment, the primer pair used to amplify the sequence shown in SEQ ID NO: 2 is, for example, 5'-ACGGGCTCAAGTGAACGAGT-3', and 5'-CGTCTTGGTATCTCTCAAGGCAT-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO: 2 are, for example, 5'-GGGAGAAAGGGGATCGATCT-3', 5'-ACGCCGAGAAGAAAACTCCA-3', 5'-TGAACGCTGCCGGTGAGAGT-3', 5'-CCTGACCTACCACCAACACA-3', 5'-GTGCAGTCTTTGGCGAAGGT-3', and 5'-CGTCTTGGTATCTCTCAAGGCAT-3'.

In another aspect, the present application provides a method for breeding rice plants containing recombinant nucleic acid fragments, which comprises using a rice recipient plant parent without a target genome fragment as a recurrent parent, and combining it with a rice donor plant containing the target genome fragment Carrying out hybridization, then backcrossing the obtained hybrid with the recurrent parent, and then performing self-crossing on the obtained backcross, wherein the recombinant plants are subjected to foreground selection and background selection using molecular markers. For example, the recombinant nucleic acid fragments are as previously described.

In the above method, the molecular marker used for the foreground selection is selected from one or more of Pi31, Pi2ID02 and Pi2S91; and/or the background selection is performed using a rice genome-wide breeding chip.

In one embodiment, the method for breeding rice plants containing a rice blast-resistant recombinant nucleic acid fragment provided by the present application comprises the following steps: 1) hybridizing a recurrent parent with a donor plant, and mixing the obtained hybrid with the recurrent The parent is backcrossed to obtain the first generation of backcrossing, which is screened for the unilateral homologous recombination fragment of the rice blast resistance genome fragment using the positive selection marker Pi31 and the negative selection marker Pi2ID02, Pi2S91, and using the whole genome breeding chip of rice, such as RICE6K was used for background selection; 2) The recombinant individual plant with better background recovery (the background recovery value of this generation was over 75%) was backcrossed with the recurrent parent again to obtain the second generation of backcrossing, and the positive selection marker Pi31 was used to pair It is detected, and the recombinant single plant containing the rice blast-resistant genome fragment is selected, and then the background selection is performed on the rice whole genome breeding chip, such as RICE6K; 3) The recombinant single plant with good background recovery is selected (the background recovery value of this generation exceeds more than 87.5%) were backcrossed with the recurrent parent again, and three generations of backcross were obtained. The positive selection marker Pi31 and the negative markers Pi2ID02 and Pi2S91 were used to screen the homologous recombination fragment on the other side of the rice blast-resistant genomic fragment, and use A rice whole-genome breeding chip, such as RICE60K, is used for background selection; and 4) a recombinant individual plant with a small introduction fragment and a good background recovery (the background recovery value exceeds 93.75%) is selected, and the selected recombinant individual plant is selfed once, The inbred seeds were obtained, detected using the positive selection marker Pi31, and background selection was carried out using a rice whole-genome breeding chip, such as RICE60K, and finally a recombinant nucleic acid fragment containing a rice blast resistant genome was obtained and the background was recovered (background). rice plants with a recovery value of more than 99%).

In another embodiment, the amplification primers used in the foreground selection of recombinant plants using molecular markers include: a primer pair for amplifying the molecular marker Pi31, wherein the forward primer is 5'-ATCCAAACCCGTTGTTGCAC-3', the reverse The primer was 5'-CGGCAATTGCCACGATGATA-3. The primer pair used to amplify the molecular marker Pi2ID02, wherein the forward primer is 5'-AAGTAATAATCCCCGCTGTTGT-3', and the reverse primer is 5'-TTTCAAGCGAAGGAGGATGT-3'. And a primer pair for amplifying the molecular marker Pi2S91, wherein the forward primer is 5'-AAGGTGAAGGAGATGATGGC-3', and the reverse primer is 5'-GTGCACCCACTCATCAAGAC-3'.

In another aspect, the present application provides a method for detecting recombinant nucleic acid fragments, which includes the steps of designing specific primers according to the aforementioned recombinant nucleic acid fragments, performing PCR reaction with the genome to be tested as a template, and analyzing the amplification products. Specifically, for example, the primers are as described above. Alternatively, the amplified products are analyzed using Sanger sequencing.

Specifically, in the method for detecting recombinant nucleic acid fragments provided in the present application, the combination of primers used to amplify and detect the sequence shown in SEQ ID NO: 1 is as follows: amplification primer, including forward primer: 5'-CTCCATACCACATCGGTGACTCT-3' , and reverse primer: 5'-CCTCATTGTGGGCTGTGTTCA-3'; sequencing primers, including reverse primer: 5'-TGCATTCGTCTAGCATGTGT-3', reverse primer: 5'-GATCTTGTCCGGTGCTAGCA-3', forward primer: 5'-GTTGGCACACCACGTAAGCT -3', Forward primer: 5'-ATACCTCCAGGCTCTAGTCA-3', Forward primer: 5'-GATGAGAGGGATTCCTACCT-3', Reverse primer: 5'-AGTCACCTCAGTTGTTAGTG-3', Forward primer: 5'-AGATGGGGTGAACATTGAGGA-3 ', forward primer: 5'-CAGGAGGTCTGGTTCTGGTT-3', and reverse primer: 5'-CCTCATTG

TGGGCTGTGTTCA-3'. The method uses the genomic DNA of the sample to be tested as a template, uses the above-mentioned amplification primers to carry out PCR amplification, and then uses the above-mentioned sequencing primers to sequence the obtained amplification product, if the sequencing result is consistent with or complementary to the sequence of SEQ ID NO: 1, The sample to be tested contains the homologous recombination fragment shown in SEQ ID NO: 2.

In addition, in the method for detecting genomic recombinant nucleic acid fragments provided in the present application, the combination of primers used to amplify and detect the sequence shown in SEQ ID NO: 2 is as follows: amplification primer, including forward primer: 5'-ACGGGCTCAAGTGAACGAGT-3' , and reverse primer: 5'-CGTCTTGGTATCTCTCAAGGCAT-3'; sequencing primers, including reverse primer: 5'-GGGAGAAAGGGGATCGATCT-3', reverse primer: 5'-ACGCCGAGAAGAAAACTCCA-3', forward primer: 5'-TGAACGCTGCCGGTGAGAGT -3', forward primer: 5'-CCTGACCTACCACCAACACA-3', forward primer: 5'-GTGCAGTCTTTGGCGAAGGT-3', and reverse primer: 5'-CGTCTTGGTA

TCTCTCAAGGCAT-3'. The method uses the genomic DNA of the sample to be tested as a template, uses the above-mentioned amplification primers to carry out PCR amplification, and then uses the above-mentioned sequencing primers to sequence the obtained amplification product, if the sequencing result is consistent with or complementary to the sequence of SEQ ID NO: 2, The sample to be tested contains the homologous recombination fragment shown in SEQ ID NO: 2.

By detecting and confirming that the sample to be tested contains the recombinant nucleic acid fragment of the sequence shown in SEQ ID NO: 1 and/or SEQ ID NO: 2, it can be determined that the sample to be tested contains the recombinant nucleic acid fragment containing the resistance gene.

In addition, the present application also provides a kit for detecting recombinant nucleic acid fragments, which includes the aforementioned primers.

Further, the present application also provides a method for screening rice plants or seeds containing recombinant nucleic acid fragments, which includes the step of detecting whether the genome of the rice plant to be tested contains the aforementioned recombinant nucleic acid fragments. In one embodiment, the aforementioned primers are used to detect whether the genome of the rice plant to be tested contains the aforementioned recombinant nucleic acid fragment. In another embodiment, the aforementioned method for detecting recombinant nucleic acid fragments is used to detect whether the genome of the rice plant to be tested contains the aforementioned recombinant nucleic acid fragments. In yet another embodiment, the aforementioned kit is used to detect whether the genome of the rice plant to be tested contains the aforementioned recombinant nucleic acid fragment.

In yet another aspect, the present application provides a rice plant or its seeds screened by the method and containing the recombinant nucleic acid fragment disclosed in the present application.

The method for breeding rice plants containing recombinant nucleic acid fragments of rice blast-resistant genome based on the whole-genome selective breeding technology provided by the present application has the advantages of rapidity, accuracy and stability. Only through five generations of transfection, only the target genome fragment can be introduced into the recipient material, and the background recovery can be achieved at the same time. The receptor material improved in this application is 'Shen 95B', which is a maintainer line for the widely used sterile line 'Shen 95A'. Using the above method, under the premise of retaining the 'deep 95B' cytoplasmic genome, only the rice blast resistance fragment can be introduced into the nuclear genome, and other sites on the nuclear genome are not changed. Further, a stable 'Shen 95A' containing rice blast resistant fragments can be obtained through at least one generation of hybridization and one generation of backcrossing, and then through mating, the blast resistance of hybrid varieties can be greatly improved. At the same time, the genome recombination fragment provided in this application is closely related to rice blast resistance, and can be used as a resistance resource for the cultivation of other varieties.

Description of drawings

1 is the detection result of the CR01BC06 rice RICE60K whole-genome breeding chip in Example 1 of the application; wherein, the boxes indicated by the numbers on the abscissa represent 12 rice chromosomes in turn, and the numbers on the ordinate are the physical positions on the rice genome [in megabases]. (Mb)], the gray line represents the recipient parent 'Shen 95B' genotype, the black line represents the donor parent 'Hua 3418B' genotype, and the white line represents the same genotype of the two parents, i.e. no polymorphic segment. The line at the black dot of chromosome 6 in the figure shows that the segment is the imported rice blast-resistant genome recombinant nucleic acid fragment RecCR01BC06.

Figure 2A and Figure 2B are the sequencing comparison results of the upstream homologous recombination fragments of RecCR01BC06 in Example 2 of the application; the asterisks shown in the figure represent the same bases in the comparison results, CR01BC06 in the figure is the obtained new line, and T001 is the recipient The body parent 'Shen 95B', R002 is the donor parent 'Hua 3418B' is the donor parent.

FIG. 3 is the sequencing comparison result of the downstream homologous recombination fragments of RecCR01BC06 in Example 2 of the application.

4 is a structural diagram of the homologous recombination fragments on both sides of RecCR01BC06 in Example 2 of the application; wherein (A) is a structural diagram of an upstream homologous recombination fragment; (B) is a structural diagram of a downstream homologous recombination fragment, and the base above is The SNP or InDel marker of the donor 'Hua 3418B', the base below is the SNP or InDel marker of the acceptor 'Deep 95B'. The gray segment is derived from the 'Deep 95B' genome segment, the black segment is derived from the 'Hua 3418B' genome segment, the white segment is the homologous recombination segment, the abscissa is the fragment length, and the number of base pairs ( bp) is the unit.

Fig. 5 is the indoor identification result of CR01BC06 rice blast resistance in Example 3 of the application; the leaves shown in the figure are: (A) rice blast susceptible variety Lijiang Xintuan Heigu; (B) original variety 'Shen 95B'; (C) Improved new line CR01BC06; (D) Rice blast resistant variety Gumei 4.

Detailed ways

The following definitions and methods are provided to better define this application and to guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

As used herein, "nucleotide sequence" includes references to deoxyribonucleotides or ribonucleotide polymers in single- or double-stranded form, and unless otherwise limited, a nucleotide sequence in a 5' to 3' orientation Written from left to right, known analogs (eg, peptide nucleic acids) with essential properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides are included.

In some embodiments, changes may be made to the nucleotide sequences of the present application to make conservative amino acid substitutions. In certain embodiments, substitutions of the nucleotide sequences of the present application that do not alter the amino acid sequence can be made in accordance with monocot codon preferences, for example, codons encoding the same amino acid sequence can be replaced with codons preferred by monocots, while The amino acid sequence encoded by the nucleotide sequence is not altered.

In particular, the present application relates to nucleotide sequences obtained by further optimization of SEQ ID NO:1 or SEQ ID NO:2. More details of this method are described in Murray et al. (1989) Nucleic Acids Res. 17:477-498. The optimized nucleotide sequence can be used to improve the expression of rice blast resistance gene in rice.

In some embodiments, the application also relates to variants of the sequences set forth in SEQ ID NO:1 or SEQ ID NO:2. Generally, a variant of a particular nucleotide sequence will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or higher sequence identity, or the complement of the above. Such variant sequences include additions, deletions or substitutions of one or more nucleic acid residues, which may result in the addition, removal or substitution of corresponding amino acid residues. Sequence identity is determined by sequence alignment procedures known in the art, including hybridization techniques. The nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as few as 1-15 nucleotides, as few as 1-10 (eg, 6-10), as few as 5, as few as 4, 3, 2 or even 1 nucleotide.

The present application also relates to a fragment comprising a specific position in the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof or its complement, for example, comprising positions 790-2130 of the sequence shown in SEQ ID NO: 1 A sequence of nucleotides or fragments or variants or complements thereof, or a sequence comprising nucleotides 770-1283 of the sequence shown in SEQ ID NO:2, or fragments or variants or complements thereof. The corresponding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 can be specifically identified according to the fragment containing the above-mentioned specific site. Further, by identifying the recombinant nucleic acid fragment containing the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, it can be determined that the sample to be tested contains the recombinant nucleic acid fragment containing the resistance gene.

As used herein, "rice" is any rice plant and includes all plant species that can be bred with rice. As used herein, "plant" or "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plants or plant parts Intact plant cells, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.

The method in this application can be applied to any rice variety that needs to be selected. That is, any elite cultivar lacking a certain favorable trait (that is, a cultivar with better comprehensive traits and expected to have a promising future) can be used as the recurrent parent. Another cultivar with a favorable trait that the recipient lacks is used as the donor parent, and the favorable trait provided is preferably controlled by a dominant single gene. In the embodiment of the present application, the rice 'Shen 95B' was used as the recurrent parent, and the rice 'Hua 3418B', which had been confirmed to have good blast resistance, was used as the donor.

In the breeding method for recombinant plants provided in the present application, the prospect selection of recombinant plants is carried out by using molecular markers. The reliability of foreground selection mainly depends on the degree of linkage between the marker and the target gene. To improve the selection accuracy, two adjacent markers on both sides are generally used to track and select the target gene at the same time.

In the embodiment of the present application, the prospective selection markers used include positive selection markers and negative selection markers, wherein the positive selection marker is 50kb upstream and downstream from the target genome segment (containing the rice blast resistance gene) (in rice Among them, the genetic distance is 0.2cM) within the range of screening polymorphic molecular markers. The negative selection marker is a polymorphic molecular marker that is screened in the range of 500kb upstream and downstream from the target genome segment (the genetic distance is 2cM in rice). In a specific embodiment, the positive foreground selection marker used in the optimized screening is the marker Pi31, which is tightly linked to the target genomic fragment, the negative selection marker is the marker Pi2ID02, which is located about 230 kb upstream of the target fragment, and is located about 270 kb downstream of the target fragment. Tag Pi2S91.

In the embodiment of the present application, when the above-mentioned foreground selection marker is used for the detection of homologous recombination, the criterion for one-sided or one-sided homologous recombination is that Pi31 detects the same band type as 'Hua 3418B', and Pi2ID02 or Pi2S91 detects the same band type. The same band type as 'Shen 95B' was detected; the criterion for bilateral or bilateral homologous recombination was that Pi31 detected the same band type as 'Hua 3418B', and Pi2ID02 and Pi2S91 detected the same band type as 'Shen 95B'.

In this application, any of the available chips can be used for background selection in the breeding methods provided in this application. In a preferred embodiment, the rice whole genome breeding chip RICE6K disclosed by the applicant in Chinese patent application CN102747138A or the rice whole genome breeding chip RICE60K disclosed in PCT international application WO/2014/121419 can be used. The entire contents of these two application documents are incorporated herein by reference in their entirety.

The following examples are for purposes of illustration only and not to limit the scope of the present application. Unless otherwise specified, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory manual of Sambrook et al. (Sambrook J&Russell DW, Molecular cloning: a laboratory manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

The information on rice plant materials used in this application can be found in the Chinese Rice Variety and Pedigree Database (http://www.ricedata.cn/variety/index.htm).

The physical location of rice genome mentioned in this application refers to rice Nipponbare genome MSU/TIGR annotation version 6.1 (http://rice.plantbiology.msu.edu/).

Example 1 Breeding of recombinant plants into which rice blast resistant genome fragments were introduced

The materials used in this example are rice 'Shen 95B' and rice 'Hua 3418B'.

Rice 'Hua 3418B' has good blast resistance, and it is speculated that the gene cluster region where Pi2, Pi9 and Pigm of chromosome 6 are located may play a key role in the blast resistance of this material.

During the selection and breeding of recombinant plants, the recombinant plants were selected using molecular markers, and the molecular markers used for foreground selection were screened. Download the 9,559,000 to 10,990,000 DNA sequences of chromosome 6 with reference to the Rice Nipponbare Genome MSU/TIGR Annotation Version 6.1. SSR sites in the above sequences were scanned using SSRLocator software. The Primer Premier 3.0 software was used to design primers for the found SSR sites, and a total of 162 primer pairs were designed. The polymorphisms of the above primer pairs in 'Hua 3418B' and 'Shen 95B' were screened by PCR, and the promising selection molecular markers with polymorphism and high amplification efficiency in the two materials were finally selected. Positive selection marks Pi31 and negative selection marks Pi2ID02, Pi2S91. The specific primer information used for PCR amplification of the above molecular markers is shown in Table 1.

Table 1 Prospect selection molecular marker primer information

The genome fragment containing the aforementioned gene clusters in the rice 'Hua 3418B' was introduced into the rice 'Shen 95B', and the specific process was as follows:

Hybrid with 'Shen 95B' as the recurrent parent and 'Hua 3418B' as the donor parent, the obtained hybrid was backcrossed with the recurrent parent 'Shen 95B' to obtain BC ₁ F ₁ seeds, which were then used for positive selection after seedling growth Marker Pi31 and negative selection markers Pi2ID02 and Pi2S91 were used to select recombinant individual plants, and 9 single plants with homologous recombination on one side of the target genome fragment were screened, that is, Pi31 detected the same band type as 'Hua 3418B', and Pi2ID02 or Pi2S91 The same band type as 'Deep 95B' was detected, and background selection was performed using the rice whole genome breeding chip RICE6K (CN102747138A) (Yu et al., Plant Biotechnology Journal. 2014, 12:28-37).

The microarray results were compared among the screened 9 single-sided homologous recombination plants, and the recombination plant with the best background recovery was selected (the background recovery value of this generation exceeded 75%), and it was backed with the recurrent parent 'Shen 95B' again. Crossed to obtain BC ₂ F ₁ seeds. After seedlings were raised, the positive selection marker Pi31 was used to detect them, and the recombinant single plant containing the target genome fragment was selected, that is, Pi31 detected the same band type as 'Hua 3418B', and the whole genome breeding of rice was used. The chip RICE6K performs background selection on it.

Select a single plant with better background recovery (the background recovery value of this generation exceeds 87.5%), make it backcross with the recurrent parent 'Shen 95B' again, obtain BC ₃ F ₁ seeds, and use the positive selection markers Pi31 and Negative markers Pi2ID02 and Pi2S91 were used to screen the harvested seeds for the homologous recombination fragment on the other side of the target genome fragment, and 5 individual plants recombined on both sides of the target fragment were obtained, that is, Pi31 detected the same band type as 'Hua 3418B', And Pi2ID02 and Pi2S91 detected the same band type as 'Deep 95B'.

The background and target fragments were selected for the above-mentioned 5 double-sided crossover plants using the rice whole-genome breeding chip RICE60K (WO/2014/121419) (Chen et al., Molecular Plant. 2014, 7:541-553), and the target fragments were screened. One target plant with a small size and good background recovery (the background recovery value of this generation exceeds 93.75%).

The selected individual plants were selfed once to obtain BC ₃ F ₂ . After seedlings were raised, the positive selection marker Pi31 was used to detect them, and the individual plants containing the target genome fragment were selected, that is, Pi31 detected the same band type as 'Hua 3418B', Background selection was performed using the rice genome-wide breeding chip RICE60K.

Finally, a line that was homozygous for the target fragment and had a background recovery (over 99% of the background recovery value) was obtained and named CR01BC06. The chip detection results are shown in Figure 1.

Example 2 Determination of homologous recombination fragments after introduction of rice blast resistance genomic fragments

In order to determine the size of the introduced fragment of the rice blast resistance genome, the homozygous single plant of the 'deep 95B' introduced fragment was sequenced with the homologous recombination fragments on both sides of the target genome fragment. The recombinant nucleic acid fragment of the rice blast resistance genome contained in CR01BC06 was named RecCR01BC06.

It was preliminarily determined by the detection results of the rice whole genome breeding chip RICE60K that RecCR01BC06 was located between the two SNP markers R0610372586AG and R0610424502CT.

At the same time, whole-genome sequencing was performed on three samples of 'Deep 95B', 'Hua 3418B' and CR01BC06 using Miseq sequencing technology. The TruSeq Nano DNA LT Kit (illumina) was used for library establishment, the LibraryQuantification Kit–Universal (KAPA Biosystems) was used for quantification, and the MiSeqV2 Reagent Kit (illumina) was used for the sequencing reaction. Detection was performed using a Miseq benchtop sequencer (illumina). For specific steps and methods, please refer to the instruction manual of each kit and sequencer.

According to the aforementioned SNP chip and Miseq sequencing results, the upstream homologous recombination fragment of RecCR01BC06 was initially located in the 10375077bp to 10379192bp range of chromosome 6, and the downstream homologous recombination fragment was located in the 10418697bp to 10420932bp range.

On this basis, referring to the MSU/TIGR annotation version 6.1 of the rice Nipponbare genome, download the DNA sequence of the corresponding segment. Primer Premier 5.0 software was used to design primers for amplification and sequencing, and the design requirements were that the primers were about 22 nt in length, GC content was 40-60%, and there was no mismatch.

Using the acceptor parent 'Shen 95B' and the donor parent 'Hua 3418B' as controls, amplification primers were designed for the upstream and downstream homologous recombination fragments of RecCR01BC06, respectively, and LATaq (TAKARA) was used for amplification, using a two-step or three-step method The optimal amplification conditions were searched by the method to ensure that the amplification product appeared as a single bright band in agarose gel electrophoresis detection. The determined upstream homologous recombination fragment amplification primer reaction conditions are: 94°C for 3 min; 98°C for 10 sec, 68°C for 5 min, 35 cycles; 72°C for 10 min; 25°C for 1 min. The downstream homologous recombination fragment amplification primer reaction conditions were: 94°C for 3 min; 98°C for 10 sec, 68°C for 5 min, 35 cycles; 72°C for 10 min; 25°C for 1 min. Thus, two pairs of amplification primers were finally screened for the amplification of upstream and downstream homologous recombination fragments, respectively.

In addition, using the amplified product as a template, Sanger sequencing was used for sequencing. According to the actual sequencing effect, 9 and 6 sequencing primers were finally screened for the sequencing of upstream and downstream homologous recombination fragments, respectively. The specific amplification primers and sequencing primer sequences are shown in Table 2, and the sequencing results are shown in FIGS. 2 and 3 .

The sequenced length of the upstream homologous recombination fragment of RecCR01BC06 was 4133 bp (SEQ ID NO: 1). 1-790bp is the genome segment of the acceptor 'Deep 95B', compared with the donor 'Hua 3418B', there are 3 SNPs. The 1339bp segment of 791-2129bp is a homologous recombination segment. 2130-4133bp is the genomic fragment of the donor 'Hua 3418B'. Compared with 'Deep 95B', there are 4 SNPs.

The sequenced length of the downstream homologous recombination fragment of RecCR01BC06 was 2244 bp (SEQ ID NO: 2). 1-770bp is the genome segment of the donor 'Hua 3418B', compared with 'Deep 95B', there are 11 SNPs. The 512bp segment of 771-1282bp is the homologous recombination segment. 1283-2244bp is the genome segment of the acceptor 'Deep 95B'. Compared with the donor 'Hua 3418B', there are 31 SNPs and 3 Indels.

Figure 4 is a structural diagram of the homologous recombination fragments on both sides of RecCR01BC06. Among them, (A) is the structural diagram of the upstream homologous recombination fragment; (B) is the structural diagram of the downstream homologous recombination fragment. The upper base is the SNP or InDel marker of the donor 'Hua 3418B', and the lower base is the SNP or InDel marker of the acceptor 'Deep 95B'. The gray segment is derived from the 'Deep 95B' genome segment, the black segment is derived from the 'Hua 3418B' genome segment, and the white segment is the homologous recombination segment. The abscissa is the fragment length in base pairs (bp).

Table 2 Amplification and sequencing primer information of recombinant nucleic acid fragment of rice blast resistance genome

Example 3 Identification of resistance after 'Shen 95B' was introduced into rice blast-resistant genomic fragments

In order to identify the resistance effect, the new line CR01BC06, the recurrent parent 'Shen 95B', the rice blast resistant variety Gumei No. 4 (as a positive control), and the rice blast susceptible variety Lijiang Xintuan Heigu (as a Negative control) to carry out indoor planting, after it is cultivated to the 3-4 leaf stage, the following method is used to identify:

The strains were selected in 2013, Hubei Enshi strains 13-21K02, 13-21K14 and 13-2104, Jiangxi Jinggangshan strain 13-5121, Sichuan Luzhou strain 13-6102, Fujian strains 13-8101, 13-8108, a total of 7 rice strains Blast strains were used as inoculation strains. . The strains were stored at -20°C by the sorghum grain method. Before use, the preserved sorghum grains were taken out to a potato dextrose medium (PDA) plate for activation (PDA: 200g of peeled potatoes, 20g of glucose, 15g of agar powder, and distilled water to 1L), After 5 days of light culture at 28°C, a fresh mycelium block with a diameter of 5 mm was taken and transferred to the sorghum grain medium (500 g of sorghum grains were added with 1.5 L of distilled water, and the liquid was filtered off after boiling to boiling, and the sorghum grains were taken out and loaded into a 250ml conical flask, 100ml/bottle, sterilized by moist heat for 20 minutes), 10 pieces/bottle, 2 days after inoculation, the sorghum grains were shaken every day, and cultivated in the dark at 28°C until the mycelium covered with sorghum grains. Then spread the sorghum grains on sterile gauze, cover with sterile damp gauze, and culture at 25°C, RH ≥ 95%, and 12 hours of light for 4-5 days until a large number of spores are produced, with sterile water (containing 0.02% Tween 20) washed the spores, mixed the same amount of spores to inoculate the strains, and adjusted the concentration to 5×10 ⁵ cells/ml.

CR01BC06, 'Shen 95B', Gumei No. 4 and Lijiang Xintuan Heigu were inoculated in three replicates with the mixed conidial suspension. After inoculation, cover with a transparent cover, incubate in the dark at 28°C for 24h, and then incubate in the light of 16h for 5 days.

The investigation criteria were grade 0 (high resistance, HR): no symptoms; grade 1 (resistance, R): small brown lesions; grade 2 (moderate resistance, MR): brown lesions about 1 mm in diameter; grade 3 (MS, medium sense): Directly about 2-3mm round lesions with grayish-white center and brown edges; Grade 4 (sensation, S): oval lesions about 1-3cm long, with grayish-white center and brown edges ; Grade 5 (High Sensitivity, HS): Long and wide large oval lesions, the lesions merged into pieces until the leaves died. Among them, grades 0-2 are disease-resistant, and grades 3-5 are susceptible. See Table 3 and Figure 5 for the vaccination results.

Table 3 Resistance performance after inoculation with M. oryzae

Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application fall within the scope of protection claimed in the present application.

Claims (9)

1. A recombinant nucleic acid fragment consisting of the first recombinant nucleic acid fragment and/or the second recombinant nucleic acid fragment of:

-a first recombinant nucleic acid fragment selected from the group consisting of:

i) a sequence comprising the nucleotide sequence 790-2130 of the sequence shown in SEQ ID NO. 1 or a complementary sequence thereof, or

ii) a sequence comprising the sequence shown in SEQ ID NO. 1 or a complementary sequence thereof;

-a second recombinant nucleic acid fragment selected from:

iii) a sequence comprising nucleotides 770-1283 of the sequence indicated by SEQ ID NO 2 or the complement thereof, or

iv) a sequence comprising the sequence shown in SEQ ID NO. 2 or a complementary sequence thereof.

2. The primer for detecting the fragment of claim 1, wherein the primer is selected from the group consisting of a primer for detecting a first recombinant nucleic acid fragment and/or a primer for detecting a second recombinant nucleic acid fragment:

-a primer for detecting the first recombined nucleic acid fragment, selected from the group consisting of:

(I) a forward primer which specifically recognizes the sequence of nucleotides 1 to 790 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 2130 and 4133 of the sequence shown in SEQ ID NO. 1;

(II) a combination of a first set of primer pairs and a second set of primer pairs comprising

(a) The first set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 1 to 790 in the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 791-2129 in the sequence shown in SEQ ID NO. 1; and

(b) a second set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 791 and 2129 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 2130 and 4133 of the sequence shown by SEQ ID NO. 1;

(III) a forward primer which specifically recognizes the sequence of SEQ ID NO:1 comprising nucleotides 790 and 791 of the sequence of SEQ ID NO:1 and a reverse primer which specifically recognizes the sequence of SEQ ID NO:1 comprising nucleotides 2129 and 2130 of the sequence of SEQ ID NO: 1;

(IV) a forward primer which specifically recognizes the sequence comprising nucleotides 790 and 791 of the sequence shown by SEQ ID NO. 1 in the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 2130 and 4133 of the sequence shown by SEQ ID NO. 1; or

(V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 790 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 2129 and 2130 of the sequence shown in SEQ ID NO. 1;

-a primer for detecting the second recombinant nucleic acid fragment selected from the group consisting of:

(VI) a forward primer which specifically recognizes the sequence of nucleotides 1 to 770 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 1283-2244 of the sequence shown in SEQ ID NO. 2;

(VII) the following combination of a third group of primer pairs and a fourth group of primer pairs, which comprises

(c) Third set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 1 to 770 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 771-1282 of the sequence shown by SEQ ID NO. 2; and

(d) a fourth set of primer pairs: a forward primer which specifically recognizes the sequence of 771-1282 th nucleotide of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of 1283-2244 th nucleotide of the sequence shown by SEQ ID NO. 2;

(VIII) a forward primer which specifically recognizes a sequence comprising nucleotides 770 and 771 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 1282 and 1283 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO: 2;

(IX) a forward primer which specifically recognizes a sequence comprising nucleotides 770 and 771 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 1283 and 2244 of the sequence shown by SEQ ID NO: 2; or

(X) a forward primer which specifically recognizes a sequence of nucleotides 1 to 770 of the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 1282 and 1283 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO: 2.

3. A primer for detecting the fragment of claim 1, wherein the primer is selected from the group consisting of:

(I) primer pair for amplifying sequence shown in SEQ ID NO. 1

5’-CTCCATACCACATCGGTGACTCT-3’，

5’-CCTCATTGTGGGCTGTGTTCA-3’；

(II) primer for sequencing sequence shown in SEQ ID NO. 1

5’-TGCATTCGTCTAGCATGTGT-3’，

5’-GATCTTGTCCGGTGCTAGCA-3’，

5’-GTTGGCACACCACGTAAGCT-3’，

5’-ATACCTCCAGGCTCTAGTCA-3’，

5’-GATGAGAGGGATTCCTACCT-3’，

5’-AGTCACCTCAGTTGTTAGTG-3’，

5’-AGATGGGGTGAACATTGAGGA-3’，

5’-CAGGAGGTCTGGTTCTGGTT-3’，

5’-CCTCATTGTGGGCTGTGTTCA-3’；

(III) primer pairs for amplifying sequences shown in SEQ ID NO. 2

5’-ACGGGCTCAAGTGAACGAGT-3’，

5'-CGTCTTGGTATCTCTCAAGGCAT-3', respectively; and

(IV) primer for sequencing SEQ ID NO. 2

5’-GGGAGAAAGGGGATCGATCT-3’，

5’-ACGCCGAGAAGAAAACTCCA-3’，

5’-TGAACGCTGCCGGTGAGAGT-3’，

5’-CCTGACCTACCACCAACACA-3’，

5’-GTGCAGTCTTTGGCGAAGGT-3’，

5’-CGTCTTGGTATCTCTCAAGGCAT-3’。

4. A method for detecting the recombinant nucleic acid fragment of claim 1, which comprises the steps of performing a PCR reaction using the primer of claim 2 or 3 and a test genome as a template, and analyzing the PCR product.

5. A kit for detecting the recombinant nucleic acid fragment of claim 1, comprising the primer of claim 2 or 3.

6. A method of screening rice plants or seeds containing the recombinant nucleic acid fragment of claim 1, comprising the step of detecting whether the genome of a test rice plant or seed contains the recombinant nucleic acid fragment of claim 1.

7. The method of claim 6, wherein the primers of claim 2 or 3 are used to detect whether the genome of a test rice plant or seed contains the recombinant nucleic acid fragment of claim 1.

8. The method of claim 6, wherein the method of claim 4 is used to detect whether the genome of a test rice plant or seed contains the recombinant nucleic acid fragment of claim 1.

9. The method of claim 6, wherein the kit of claim 5 is used to detect whether the genome of a test rice plant or seed contains the recombinant nucleic acid fragment of claim 1.

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