CN106893769B

CN106893769B - Recombinant nucleic acid fragment RecCR012602 and detection method thereof

Info

Publication number: CN106893769B
Application number: CN201510958735.XA
Authority: CN
Inventors: 周发松; 喻辉辉; 曹志; 邱树青; 张学堂; 张龙雨; 雷昉; 姚玥; 李旭; 江峥; 李菁; 韦懿; 何予卿; 张启发
Original assignee: China National Seed Group Co Ltd
Current assignee: China National Seed Group Co Ltd
Priority date: 2015-12-18
Filing date: 2015-12-18
Publication date: 2021-11-19
Anticipated expiration: 2035-12-18
Also published as: CN106893769A

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 RecCR012602 and detection method thereof

Technical Field

The present application relates to whole genome selection breeding techniques. In particular, the application relates to a rice plant containing the recombinant nucleic acid fragment bred by using a whole genome selective breeding technology, the recombinant nucleic acid fragment obtained by the breeding technology and a detection method thereof.

Background

For a long time, the traditional breeding selection method mainly depends on the evaluation of field phenotype, and selection are carried out according to personal experience of breeders, and the biggest defects of the traditional breeding selection method are long time consumption and low efficiency. To improve the efficiency of selection, it is desirable to select directly from the genotype. With the development of molecular biotechnology, molecular markers offer the possibility of enabling direct selection of genotypes. In recent years, molecular marker assisted selection methods have been applied to improve individual traits of interest, enabling significant reductions in the breeding years.

The rice blast is one of the most serious diseases of rice, and the yield loss of rice caused by the rice blast accounts for 11 to 30 percent each year around the world, so the research on the rice blast and the resistance thereof is particularly important. With the progress of the research on rice blast, many DNA fragments of rice blast resistance genes were located and cloned one after another. Wherein, a Pi2 interval of the 6 th chromosome of rice is located and cloned with a plurality of rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm and Pi50, wherein the interval comprises a gene cluster of the rice blast resistance genes (Qu et al, genetics.2006,172: 1901-1914; Wang et al, phytopathology.2012,102: 779-786; Xiao et al, Mol Breeding.2012,30: 1715-1726; Liu et al, Mol Genomics.2002,267: 472-480; Jiang et al, Rice.2012,5: 29-35; Zhu et al, the or Appl Genet.2012,124: 1295-1304; Deng et al, the tool Appl Genet.2006, 705: 713).

Disclosure of Invention

In one aspect, the present application provides a recombinant nucleic acid fragment selected from the group consisting of: i) a sequence comprising nucleotides 368-1702 of the sequence shown in SEQ ID NO. 1 or a fragment or variant or complement thereof; ii) a sequence comprising the sequence shown in SEQ ID NO. 1 or a fragment or variant or complement thereof; iii) a sequence comprising nucleotides 564-968 of the sequence shown in SEQ ID NO. 2 or a fragment or variant or complement thereof; iv) a sequence comprising the sequence shown in SEQ ID NO. 2 or a fragment or variant or complement thereof; 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 primers for detecting the recombinant nucleic acid fragment selected from the group consisting of: (I) a forward primer which specifically recognizes the sequence of nucleotides 1 to 368 in the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 1702-2839 in 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 368 in the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 369 and 1701 in the sequence shown by SEQ ID NO. 1; and (b) a second set of primer pairs: a forward primer which specifically recognizes the sequence represented by the sequence 369-1701 nucleotide sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence represented by the sequence 1702-2839 nucleotide sequence shown by SEQ ID NO. 1; (III) a forward primer specifically recognizing the sequence comprising nucleotides 367 and 368 or 368 and 369 of the sequence shown by SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 1701 and 1702 or 1702 and 1703 of the sequence shown by SEQ ID NO. 1; (IV) a forward primer specifically recognizing the sequence comprising nucleotides 367 and 368 or 368 and 369 of the sequence shown by SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence of nucleotides 1702 and 2839 of the sequence shown by SEQ ID NO. 1; (V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 368 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence comprising nucleotides 1701 and 1702 or 1702 and 1703 of the sequence shown in SEQ ID NO. 1; and/or optionally, (VI) a forward primer specifically recognizing the sequence of nucleotides 1 to 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer specifically recognizing the sequence of nucleotides 968 and 1748 of the sequence shown in SEQ ID NO. 2; (VII) the following third and fourth sets of primer pairs, comprising (c) the third set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 1 to 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 565 and 967 of the sequence shown in SEQ ID NO. 2; and (d) a fourth set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 565 and 967 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 968 and 1748 of the sequence shown by SEQ ID NO. 2; (VIII) a forward primer specifically recognizing the sequence comprising nucleotides 564 and 565 of the sequence indicated by SEQ ID NO:2 and a reverse primer specifically recognizing the sequence comprising nucleotides 967 and 968 or nucleotides 968 and 969 of the sequence indicated by SEQ ID NO: 2; (IX) a forward primer which specifically recognizes a sequence comprising nucleotides 564 and 565 of the sequence indicated by SEQ ID NO. 2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 968 and 1748 of the sequence indicated by SEQ ID NO. 2; (X) a forward primer which specifically recognizes the sequence of nucleotides 1 to 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence comprising nucleotides 967 and 968 or 968 and 969 of the sequence shown in SEQ ID NO. 2.

In one embodiment, the primer pairs used to amplify the sequences shown in SEQ ID NO. 1 are, for example, 5'-TATGCTTGGCGTCATATTGCTCTT-3', and 5'-TATTTTGCTATCGGATCTAAACCCT-3'. Sequencing primers for detecting the sequence shown in SEQ ID NO. 1 are, for example, 5'-ATGGAACGGTCCCATACTTG-3', 5'-GAATATCGGTTTCGGTTGAT-3', 5'-GGACAATAACATTCCACCAG-3', 5'-TGAAGAGGACGGATTGTGAG-3', 5'-GGGATCGTACTCGCACAGAG-3' and 5'-CGGACAGATGATTACCCACA-3'.

In another embodiment, the primer pairs used to amplify the sequences shown in SEQ ID NO. 2 are, for example, 5'-GAAGTCGCATAATAGTAACCACG-3', and 5'-GAAACCGCCTACGATACATACCC-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO. 2 are, for example, 5'-TGGGAGACAGGATTCATACC-3', 5'-CGAATTTACTACCCGTGAG-3', 5'-CCCCAGTTGGGTCGGAAAG-3', 5'-GGGTCAGAGCCAAAGTGCG-3' and 5'-GAAACCGCCTACGATACATACCC-3'.

In another aspect, the present application provides a method for breeding rice plants containing recombinant nucleic acid fragments, comprising the steps of crossing a rice recipient plant parent not containing a target genomic fragment as a recurrent parent with a rice donor plant containing the target genomic fragment, backcrossing the resultant hybrid with the recurrent parent, and then selfing the resultant backcrossed, wherein the recombinant plants are subjected to foreground selection and background selection using molecular markers. For example, the recombinant nucleic acid fragments are as described above.

In the above method, the molecular marker used for the foreground selection is selected from one or more of Pi31, W068C06B, and Pi2S 122; and/or performing the background selection by using a rice whole genome breeding chip.

In one embodiment, the present application provides a method for breeding rice plants containing a recombinant nucleic acid fragment of a rice blast resistant genome, comprising the steps of: 1) hybridizing the recurrent parent and a donor plant, performing backcross on the obtained hybrid and the recurrent parent to obtain a backcross first generation, performing single-side homologous recombination fragment screening on the RICE blast resistant genome fragment by using a positive selection marker Pi31 and negative selection markers W068C06B and Pi2S122, and performing background selection on the RICE blast resistant genome fragment by using a RICE whole genome breeding chip, such as RICE 6K; 2) selecting a recombinant single plant with better background recovery (the background recovery value of the generation exceeds 75 percent) to carry out backcross with recurrent parents again to obtain a second backcross generation, detecting the second backcross generation by using a forward selection marker Pi31, selecting the recombinant single plant containing a RICE blast resistant genome segment, and then carrying out background selection on the recombinant single plant by using a RICE whole genome breeding chip, such as RICE 6K; 3) selecting a recombinant single plant with a restored background (the background restoration value of the generation exceeds 87.5 percent) and recurrent parents for backcross again to obtain three backcross generations, screening homologous recombination fragments on the other side of a RICE blast resistant genome fragment by using a positive selection marker Pi31 and negative selection markers W068C06B and Pi2S122, and performing background selection on the recombinant single plant by using a RICE whole genome breeding chip, such as RICE 60K; and 4) selecting a recombinant single plant with small introduced segment and good background recovery (the background recovery value exceeds 93.75%), selfing the selected recombinant single plant once to obtain a selfed seed, detecting the selfed seed by using a forward selection marker Pi31, and performing background selection on the selfed seed by using a RICE whole genome breeding chip, such as RICE60K, to finally obtain a RICE plant which contains the RICE blast resistant genome recombinant nucleic acid segment and is homozygous and has good background recovery (the background recovery value exceeds 99%).

In another embodiment, the amplification primers used in the foreground selection of recombinant plants using molecular markers comprise: a primer pair for amplifying the molecular marker Pi31, wherein the forward primer is 5'-ATCCAAACCCGTTGTTGCAC-3' and the reverse primer is 5'-CGGCAATTGCCACGATGATA-3'; a primer pair for amplifying a molecular marker W068C06B, wherein the forward primer is 5'-CCTATCGCTGACAAAGAG-3', and the reverse primer is 5'-CACCCAGCCAGTTCATCTA-3'; and a primer pair for amplifying the molecular marker Pi2S122, wherein the forward primer is 5'-GACTTGAAAACCAGTGCGTG-3' and the reverse primer is 5'-CCTACCTAATGGAAAGGATTGC-3'.

In still another aspect, the present application provides a method for detecting a recombinant nucleic acid fragment, which comprises the steps of designing specific primers based on the recombinant nucleic acid fragment as described above, performing a PCR reaction using a genome to be detected as a template, and analyzing the PCR amplification product. Specifically, for example, the primer is as described above. Alternatively, PCR amplification products were analyzed using Sanger sequencing.

Specifically, in the method for detecting the recombinant nucleic acid fragment provided by the application, the primer combination for amplifying and detecting the sequence shown in SEQ ID NO. 1 is as follows: amplification primers, including a forward primer: 5'-TATGCTTGGCGTCATATTGCTCTT-3', reverse primer: 5'-TATTTTGCTATCGGATCTAAACCCT-3', respectively; sequencing primers, including a forward primer: 5'-ATGGAACGGTCCCATACTTG-3', reverse primer: 5'-GAATATCGGTTTCGGTTGAT-3', forward primer: 5'-GGACAATAACATTCCACCAG-3', forward primer: 5'-TGAAGAGGACGGATTGTGAG-3', forward primer: 5'-GGGATCGTACTCGCACAGAG-3' and forward primer: 5'-CGGACAGATGATTACCCACA-3' are provided. The method comprises the steps of taking genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the amplification primer, sequencing an obtained amplification product by using the sequencing primer, and if a sequencing result is consistent with or complementary to a sequence of SEQ ID NO. 1, determining that the sample to be detected contains a homologous recombination fragment shown in SEQ ID NO. 1.

In addition, in the method for detecting the recombinant nucleic acid fragment provided by the application, the primer combination for amplifying and detecting the sequence shown in SEQ ID NO. 2 is as follows: amplification primers, including a forward primer: 5'-GAAGTCGCATAATAGTAACCACG-3', reverse primer: 5'-GAAACCGCCTACGATACATACCC-3', respectively; sequencing primers, including a forward primer: 5'-TGGGAGACAGGATTCATACC-3', forward primer: 5'-CGAATTTACTACCCGTGAG-3', forward primer: 5'-CCCCAGTTGGGTCGGAAAG-3', forward primer: 5'-GGGTCAGAGCCAAAGTGCG-3' and reverse primer: 5'-GAAACCGCCTACGATACATACCC-3' are provided. The method comprises the steps of taking genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the amplification primer, sequencing an obtained amplification product by using the sequencing primer, and if a sequencing result is consistent with or complementary to a sequence of SEQ ID NO. 2, determining that the sample to be detected contains a homologous recombination fragment shown in SEQ ID NO. 2.

The recombinant nucleic acid fragment containing the sequence shown by SEQ ID NO. 1 and/or SEQ ID NO. 2 in the sample to be detected is determined through detection, so that the recombinant nucleic acid fragment containing the resistance gene in the sample to be detected can be determined.

In addition, the present application also provides a kit for detecting a recombinant nucleic acid fragment, which comprises the primer as described above.

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

In yet another aspect, the present application provides rice plants or seeds thereof selected by the methods comprising the recombinant nucleic acid fragments disclosed herein.

The method for breeding the rice plant containing the rice blast resistant genome recombinant nucleic acid segment based on the whole genome selective breeding technology has the advantages of rapidness, accuracy and stability. Only through the transformation of five generations, only the target genome segment can be introduced into the acceptor material, and the reversion of the background can be realized at the same time. The improved receptor material is 'HD 9802S', and is a rice early indica type temperature-sensitive sterile line. By using the method, the rice blast resistance can be greatly improved under the condition of keeping the original characteristics of 'HD 9802S'. Meanwhile, the genome recombinant nucleic acid fragment provided by the application is closely related to rice blast resistance, and can be used as a resistance resource to be applied to cultivation of other varieties.

Drawings

FIG. 1 shows the results of the CR012602 RICE RICE60K whole genome breeding chip test in example 1 of the present application; wherein, the boxes indicated by the abscissa indicate 12 chromosomes of rice in turn, the ordinate indicates the physical position [ in megabases (Mb) ] on the rice genome, the gray line represents the genotype of the receptor parent 'HD 9802S', the black line represents the genotype of the donor parent 'R6', and the white line represents the same genotype of the two parents, i.e., no polymorphic segment. The black dot line of chromosome 6 shows the section which is the introduced rice blast resistant genome recombinant nucleic acid fragment RecCR 012602.

FIGS. 2A and 2B are the sequencing alignment of upstream homologous recombination fragments of RecCR012602 in example 2 of the present application; the asterisks shown in the figure represent the same bases in the alignment results, in the figure, CR012602 is the obtained new strain, HD9802S is the acceptor parent 'HD 9802S', and R6 is the donor parent 'R6'.

FIGS. 3A and 3B are the sequencing alignment results of the homologous recombinant fragments downstream of RecCR012602 in example 2 of the present application; the asterisks shown in the figure represent the same bases in the alignment results, in the figure, CR012602 is the obtained new strain, HD9802S is the acceptor parent 'HD 9802S', and R6 is the donor parent 'R6'.

FIG. 4 is a diagram of the structure of homologous recombination fragments flanking RecCR012602 in example 2 of the present application; wherein (A) is the structure diagram of an upstream homologous recombination fragment; (B) the upper base is SNP or InDel marker of donor 'R6' and the lower base is SNP or InDel marker of acceptor 'HD 9802S' in the structure diagram of downstream homologous recombination fragments. Grey segments are derived from the 'HD 9802S' genome segment, black segments are derived from the 'R6' genome segment, white segments are homologous recombination segments, and the abscissa is the fragment length in base pair numbers (bp).

FIG. 5 shows the results of indoor identification of CR012602 blast resistance in example 3 of the present application; the blades shown in the figure are in the order: (A) the rice blast susceptible variety Lijiang Xinjiang black rice; (B) original variety 'HD 9802S'; (C) improved new line CR 012602; (D) no. 4 of rice blast disease-resistant variety of flos Pruni mume.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art.

As used herein, "nucleotide sequence" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, nucleotide sequences are written in the 5 'to 3' direction from left to right, and include known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

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 that do not alter the amino acid sequence of the nucleotide sequences of the present application can be made according to monocot codon preferences, e.g., codons encoding the same amino acid sequence can be substituted with monocot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence.

In particular, the application relates to further optimization of the resulting nucleotide sequence 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 for improving the expression of the rice blast resistant genome recombinant nucleic acid segment in rice.

In some embodiments, the present application also relates to variants of the sequence shown 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 more sequence identity, or the complement thereof, to the particular nucleotide sequence. 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 the corresponding amino acid residue. Sequence identity is determined by sequence alignment programs known in the art, including hybridization techniques. Nucleotide sequence variants of the embodiments may differ from the sequences of the present application by as little as 1-15 nucleotides, as little as 1-10 (e.g., 6-10), as little as 5, as little as 4, 3, 2, or even 1 nucleotide.

The application also relates to sequences comprising the specified positions in the sequence indicated under SEQ ID NO 1 or SEQ ID NO 2 or fragments or variants thereof or complements thereof, for example sequences comprising nucleotides 368 and 961702 of the sequence indicated under SEQ ID NO 1 or fragments or variants thereof or complements thereof or sequences comprising nucleotides 564 and 968 of the sequence indicated under SEQ ID NO 2 or fragments or variants thereof or complements thereof. Based on the fragment containing the specific site, the corresponding sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 can be specifically identified. Furthermore, the recombinant nucleic acid fragment containing the resistance gene in the sample to be tested can be determined by identifying the recombinant nucleic acid fragment containing the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

As used herein, "rice" is any rice plant and includes all plant varieties 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 plant cells that are intact in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.

The method can be applied to any rice variety needing breeding. That is, any elite variety lacking a favorable trait (i.e., a variety having a good comprehensive trait and expected to have a future development) can be used as a recurrent parent. Another variety having the advantageous trait lacking in the recipient is used as the donor parent and the advantageous trait provided is preferably dominantly monogenically controlled. In the embodiment of the present application, rice 'HD 9802S' was used as a recurrent parent, and rice 'R6' that had been confirmed to have good rice blast resistance was used as a donor.

In the breeding method of the recombinant plant provided by the application, the molecular marker is used for carrying out prospect selection on the recombinant plant. The reliability of the foreground selection mainly depends on the closeness degree of linkage between the markers and the target gene, and in order to improve the accuracy of selection, the target gene is generally tracked and selected by two adjacent markers on two sides at the same time.

In embodiments of the present application, the foreground selection markers employed include positive selection markers and negative selection markers. In a specific embodiment, the positive foreground selection marker used in the optimized screen is marker Pi31 that is closely linked to the target genomic fragment, the negative selection marker is marker W068C06B located upstream of the target fragment, and marker Pi2S122 located downstream of the target fragment.

In the present embodiment, in the detection of homologous recombination using the above-mentioned foreground selection marker, the criteria for judging one-sided or one-sided homologous recombination are that Pi31 detects the same band type as that of 'R6', and W068C06B or Pi2S122 detects the same band type as that of 'HD 9802S'; the criteria for judging bilateral or bilateral homologous recombination were that Pi31 detected the same band type as ` R6 ` and W068C06B and Pi2S122 detected the same band type as ` HD9802S `.

In the present application, any available chip can be used for background selection in the breeding method provided in the present application. In a preferred embodiment, the RICE whole genome breeding chip RICE6K disclosed in the present applicant's 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 both of these applications are incorporated herein by reference in their entirety.

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

The rice plant material information used in the application can be seen in Chinese rice varieties and pedigree databases thereof (http:// www.ricedata.cn/variety/index. htm).

The physical location of the rice genome referred to in this application is referred to the rice Nipponbare genome MSU/TIGR annotation, version 6.1 (http:// rice plant. MSU. edu /).

Example 1Breeding recombinant plants introduced with rice blast resistant genome fragments

The materials used in this example were rice 'HD 9802S' and rice 'R6'.

Rice 'R6' has good resistance to rice blast and it is assumed that the regions of the gene cluster where Pi2, Pi9 and Pigm of chromosome 6 are located may play a key role in the resistance of the material to rice blast.

In the process of breeding the recombinant plants, the molecular markers are used for carrying out prospect selection on the recombinant plants, and the adopted prospect selection molecular markers are screened. The DNA sequence of chromosome 69,559,000 to 10,990,000 was downloaded with reference to the Rice Nipponbare genome MSU/TIGR annotation, version 6.1. SSR sites in the above sequences were scanned using SSRLOCATOR software. Primers are designed for the found SSR loci by using Primer Premier 3.0 software, and a Primer 162 pair is designed in total. The polymorphisms of the primer pair in 'R6' and 'HD 9802S' were screened by PCR, and finally the foreground selection molecular markers with polymorphism and high amplification efficiency in the two materials, namely positive selection marker Pi31 and negative selection markers W068C06B and Pi2S122, were selected. The specific primer information for PCR amplification of the above molecular markers is shown in Table 1.

TABLE 1 Foreground selection of molecular marker primer information

The genome segment of the rice 'R6' where the gene cluster is located is introduced into the rice 'HD 9802S', and the specific process is as follows:

taking 'HD 9802S' as a recurrent parent and 'R6' as a donor parent to carry out hybridization, carrying out backcross on the obtained hybrid and the recurrent parent 'HD 9802S', and obtaining BC₁F₁After seed breeding, recombinant individual selection was carried out using positive selection marker Pi31 and negative selection markers W068C06B and Pi2S122, 24 individuals that were homologously recombined on the target genomic DNA fragment side, i.e., Pi31 detected the same band type as ` R6 ` and W068C06B or Pi2S122 detected the same band type as ` HD9802S `, were selected for background selection using RICE whole genome breeding chip RICE6K (CN102747138A) (Yu et al, Plant Biotechnology journal.2014,12: 28-37).

Comparing the chip results in 24 selected unilaterally homologous recombinant individuals, selecting the recombinant individual with best background recovery (the background recovery value of the generation exceeds 75%), backcrossing the recombinant individual with the recurrent parent 'HD 9802S' again to obtain BC₂F₁After the seeds are grown, the positive selection marker Pi31 is used for detecting the seeds, recombinant individuals containing target genome DNA fragments, namely Pi31 with the same band type as that of 'R6' are selected, and the RICE whole genome breeding chip RICE6K is used for background selection.

Selecting the single plant with better background recovery (the background recovery value of the generation exceeds 87.5 percent), and backcrossing the single plant with the recurrent parent 'HD 9802S' again to obtain BC₃F₁And (3) screening homologous recombination fragments on the other side of the target genome DNA fragment of the harvested seeds by using a positive selection marker Pi31 and negative selection markers W068C06B and Pi2S122 after seedling raising, and obtaining 24 individuals recombined on both sides of the target fragment, namely Pi31 detects the same banding pattern as 'R6', and W068C06B and Pi2S122 detect the same banding pattern as 'HD 9802S'.

The 24 double-sided cross-over individuals were subjected to background and target fragment selection using a RICE whole genome breeding chip RICE60K (WO/2014/121419) (Chen et al, Molecular plant 2014,7: 541) 553, and one target individual with a smaller introduced target fragment and a good background was selected (the generation background recovery value was more than 93.75%).

Selfing the selected single plant once to obtain BC₃F₂After seedling raising, useThe positive selection marker Pi31 was detected, and individuals containing the target genomic DNA fragments were selected, i.e., individuals with the band pattern same as that of ` R6 ` were selected with Pi31, and were background-selected with the RICE whole genome breeding chip RICE 60K.

Finally, one of the strains which is homozygous for the target fragment and has background reversion (the background reversion value exceeds 99%) is obtained and named as CR 012602. The chip detection results are shown in FIG. 1.

BC in the above process because 'HD 9802S' is a two-line sterile line₁F₁、BC₂F₁、BC₃F₁And BC₃F₂The selected individual plants in the generation are sterile individual plants, and the fertility transformation of the sterile individual plants is realized by adopting the following method for backcrossing or selfing: when the sterile single plant enters the reproductive stage 4, treating in a low-temperature treatment area (21-23 ℃) for about 10 days, and utilizing a plant artificial climate box, a plant growing chamber, a cold water irrigation pool and the like; and (3) carrying out fertility identification on the pollen of the extracted ear by using an iodine-potassium iodide dyeing method, judging that more than 70% of ears which can be bred by the pollen are fertility successfully transformed ears, and then carrying out backcross or selfing.

Example 2Determination of homologous recombination fragments after introduction of Rice blast-resistant genomic fragment

To determine the size of the introduced rice blast resistant genomic fragment, a single strain homozygous for the ` HD9802S ` introduced fragment was subjected to sequencing of homologous recombination fragments flanking the genomic fragment of interest. The recombinant nucleic acid fragment of the blast-resistant genome contained in CR012602 was designated as RecCR 012602.

As preliminarily determined by the detection result of a RICE whole genome breeding chip RICE60K, RecCR012602 is positioned between two SNP markers R0610084130TG and R0610454932 GA.

Meanwhile, three samples of 'HD 9802S', 'R6' and CR012602 were whole genome sequenced using Miseq sequencing technology. Library construction was performed using TruSeq Nano DNA LT Kit (illumina) Kit, Quantification was performed using Library Quantification Kit-Universal (KAPA biosystems) Kit, and sequencing was performed using MiSeq V2 Reagent Kit (illumina). Detection was performed using Miseq bench top sequencer (illumina). The specific steps and methods are shown in each kit and the instruction manual of the sequencer.

According to the SNP chip and the Miseq sequencing result, the upstream homologous recombination fragment of RecCR012602 is positioned in the interval from 10090076bp to 10093784bp of the 6 th chromosome, and the downstream homologous recombination fragment is positioned in the interval from 10437335bp to 10439846 bp.

On the basis, the DNA sequences of the corresponding segments were downloaded with reference to the rice Nipponbare genome MSU/TIGR annotation, version 6.1. Amplification and sequencing primers were designed using Primer Premier 5.0 software, with the design requirements being around 22nt Primer length, 40-60% GC content and no mismatches.

By taking an acceptor parent 'HD 9802S' and a donor parent 'R6' as controls, amplification primers are respectively designed for upstream and downstream homologous recombination fragments of RecCR012602, high-fidelity enzyme KOD FXneo (TOYOBO) is used for amplification, and a two-step method or a three-step method is used for searching for optimal amplification conditions, so that an amplification product is ensured to be displayed as a single bright band in agarose gel electrophoresis detection. Wherein the reaction conditions of the upstream homologous recombination fragment amplification primers are as follows: 94 ℃ for 2 min; 10sec at 98 ℃, 180sec at 68 ℃, 37 cycles; 1min at 20 ℃. The reaction conditions of the downstream homologous recombination fragment amplification primer are as follows: 94 ℃ for 2 min; 10sec at 98 ℃, 180sec at 68 ℃, 37 cycles; 1min at 20 ℃. Thus, a pair of amplification primers is finally selected for amplification of the upstream and downstream homologous recombination fragments.

In addition, the amplification product is used as a template, sequencing is carried out by a Sanger sequencing method, and finally, 6 and 5 sequencing primers are respectively screened out and are respectively used for sequencing the upstream homologous recombination fragment and the downstream homologous recombination fragment according to the actual sequencing effect. The specific sequences of the amplification primers and the sequencing primers are shown in Table 2, and the sequencing results are shown in FIG. 2A, FIG. 2B, FIG. 3A and FIG. 3B.

The sequencing length of the homologous recombination fragment at the upstream of RecCR012602 is 2839bp (SEQ ID NO: 1). 1-368bp is the genome segment of the acceptor 'HD 9802S', and there are 2 SNPs and 1 Indel compared to the donor 'R6'. The 1333bp segment of 369-1701bp is a homologous recombination segment. 1702-2839bp was the donor 'R6' genomic fragment, and there were 4 SNPs compared with 'HD 9802S'.

The sequencing length of the homologous recombination fragment at the downstream of RecCR012602 was 1748bp (SEQ ID NO: 2). 1-564bp is the genome segment of donor 'R6', and there are 2 SNPs and 1 Indel compared with 'HD 9802S'. The 403bp segment of 565-and 967bp is a homologous recombination segment. 968-1748bp is the genome segment of the acceptor 'HD 9802S', and there are 3 SNPs and 2 indels compared with the donor 'R6'.

FIG. 4 shows the structure of homologous recombination fragments flanking RecCR 012602. Wherein (A) is the structure diagram of an upstream homologous recombination fragment; (B) is a structure diagram of a downstream homologous recombination fragment. The upper base is the SNP or InDel marker of the donor 'R6', and the lower base is the SNP or InDel marker of the acceptor 'HD 9802S'. Gray segments are derived from the 'HD 9802S' genome segment, black segments are derived from the 'R6' genome segment, and white segments are homologous recombination segments. The abscissa is the fragment length in base pair number (bp).

TABLE 2 amplification and sequencing primer information for recombinant nucleic acid fragments of rice blast resistant genomes

Example 3Identification of resistance after introduction of genomic fragment against Rice blast ` HD9802S `

In order to identify the resistance effect, the new strain CR012602, the recurrent parent 'HD 9802S', the rice blast disease-resistant variety flos Pruni mume No. 4 (as a positive control) and the rice blast susceptible variety Lijiang new-group black valley (as a negative control) bred by the method are planted indoors and are cultured to 3-4 leaf stages for identification by adopting the following method:

selecting 7 rice blast strains of M15Bb-1-1, M15Bb-1-2, M15Bb-2-1, M15Bb-3-1, M15Bb-4-1, M15Bb-5-1 and M15Bb-6-1 which are separated from rice blast samples of Yichang disease nursery in Hubei in 2015 as inoculation strains. The strain is preserved at-20 deg.C by sorghum grain method, and the preserved sorghum grains are taken out to potato glucose culture medium (PDA) before useActivating a plate (PDA, 200g of peeled potatoes, 20g of glucose, 15g of agar powder and distilled water to a constant volume of 1L), culturing for 5 days at 28 ℃ by illumination, taking a fresh mycelium block with the diameter of 5mm, transferring the mycelium block into a sorghum grain culture medium (500 g of sorghum grains are added with 1.5L of distilled water, filtering out liquid after boiling, taking out sorghum grains into a 250ml triangular flask and 100 ml/flask, carrying out moist heat sterilization for 20 minutes), 10 blocks/flask, inoculating bacteria for 2 days, shaking the sorghum grains out every day, and culturing in the dark at 28 ℃ until the mycelium grows over the sorghum grains. Spreading sorghum grains on sterile gauze, covering with sterile wet gauze, culturing at 25 deg.C and RH of 95% or more for 12 hr under illumination for 4-5 days until a large amount of spores are generated, washing the spores with sterile water (containing 0.02% Tween 20), mixing with the inoculated strains, adjusting concentration to 5 × 10⁵One per ml.

CR012602, 'HD 9802S', oryzanol 4 and lijiang new ball black cereals were spray inoculated with mixed conidia suspensions, three replicates were inoculated. After inoculation, the cells were covered with a transparent hood, incubated at 28 ℃ in the dark for 24 hours, then incubated under light for 16 hours for 5 days, and investigated.

Survey criteria were grade 0 (high resistance, HR): no symptoms; grade 1 (anti, R): very small brown lesions; grade 2 (medium, MR): brown lesions with a diameter of about 1 mm; grade 3 (MS, in feeling): directly taking 2-3mm round scab with gray center and brown edge; grade 4 (feeling, S): oval lesion spots about 1-3cm long, gray-white in the center, brown at the edge; grade 5 (high, HS): the long and wide large oval lesion spots are fused into pieces until the leaves die. Wherein the disease is resistant in 0-2 grade, and susceptible in 3-5 grade. The results of the inoculation are shown in Table 3 and FIG. 5.

TABLE 3 resistance Performance after inoculation with Pyricularia oryzae

Although the present application has been described in detail with respect to the general description and specific embodiments, it will be apparent to those skilled in the art that certain modifications or improvements may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.

Claims

1. A recombinant nucleic acid fragment selected from the group consisting of:

i) 1 or the complementary sequence thereof;

ii) the sequence shown in SEQ ID NO. 2 or the complementary sequence thereof; and

combinations of the above fragments.

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

(I) a forward primer which specifically recognizes the sequence of nucleotides 1 to 368 in the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 1702-2839 in 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 368 in the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 369 and 1701 in the sequence shown by SEQ ID NO. 1; and

(b) a second set of primer pairs: a forward primer which specifically recognizes the sequence represented by the sequence 369-1701 nucleotide sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence represented by the sequence 1702-2839 nucleotide sequence shown by SEQ ID NO. 1;

(III) a forward primer specifically recognizing the sequence comprising nucleotides 367 and 368 or 368 and 369 of the sequence shown by SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 1701 and 1702 or 1702 and 1703 of the sequence shown by SEQ ID NO. 1;

(IV) a forward primer specifically recognizing the sequence comprising nucleotides 367 and 368 or 368 and 369 of the sequence shown by SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence of nucleotides 1702 and 2839 of the sequence shown by SEQ ID NO. 1;

(V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 368 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence comprising nucleotides 1701 and 1702 or 1702 and 1703 of the sequence shown in SEQ ID NO. 1; and/or, optionally, the one or more of,

(VI) a forward primer which specifically recognizes the sequence of nucleotides 1 to 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 968-1748 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 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 565 and 967 of the sequence shown in SEQ ID NO. 2; and

(d) a fourth set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 565 and 967 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 968 and 1748 of the sequence shown by SEQ ID NO. 2;

(VIII) a forward primer specifically recognizing the sequence comprising nucleotides 564 and 565 of the sequence indicated by SEQ ID NO:2 and a reverse primer specifically recognizing the sequence comprising nucleotides 967 and 968 or nucleotides 968 and 969 of the sequence indicated by SEQ ID NO: 2;

(IX) a forward primer which specifically recognizes a sequence comprising nucleotides 564 and 565 of the sequence indicated by SEQ ID NO. 2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 968 and 1748 of the sequence indicated by SEQ ID NO. 2;

(X) a forward primer which specifically recognizes the sequence of nucleotides 1 to 564 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence comprising nucleotides 967 and 968 or 968 and 969 of the sequence shown in 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’-TATGCTTGGCGTCATATTGCTCTT-3’，

5'-TATTTTGCTATCGGATCTAAACCCT-3', respectively; and

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

5’-ATGGAACGGTCCCATACTTG-3’，

5’-GAATATCGGTTTCGGTTGAT-3’，

5’-GGACAATAACATTCCACCAG-3’，

5’-TGAAGAGGACGGATTGTGAG-3’，

5’-GGGATCGTACTCGCACAGAG-3’，

5'-CGGACAGATGATTACCCACA-3', respectively; and/or, optionally, the one or more of,

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

5’-GAAGTCGCATAATAGTAACCACG-3’，

5'-GAAACCGCCTACGATACATACCC-3', respectively; and

(IV) primer for sequencing SEQ ID NO. 2

5’-TGGGAGACAGGATTCATACC-3’，

5’-CGAATTTACTACCCGTGAG-3’，

5’-CCCCAGTTGGGTCGGAAAG-3’，

5’-GGGTCAGAGCCAAAGTGCG-3’，

5’-GAAACCGCCTACGATACATACCC-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 genome to be detected as a template, and analyzing the PCR amplification 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 detection is performed using the primer of claim 2 or 3, or using the method of claim 4, or using the kit of claim 5.