CN106609274B - Recombinant nucleic acid fragment RecCR010066 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 RecCR010066 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 the Pi1 and Pik cluster alleles are located in the long-arm proximal terminal region of rice chromosome 11 (Hua et al, therapeutic and Applied genetics.2012,125: 1047-1055; Li et al, Molecular Breeding.2007,20: 179-188; Alok et al, Functional & Integrated genetics.2012, 12: 215-228; Yuan et al, therapeutic and Applied genetics.2011,122: 1017-1028).

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 331-462 of the sequence shown in SEQ ID NO. 1 or a fragment or variant thereof or the 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 487-513 as 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 1-331 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 462-1042 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 331 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 332 and 461 of 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 332 and 461 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 462 and 1042 of the sequence shown in SEQ ID NO. 1; (III) a forward primer specifically recognizing the sequence comprising nucleotides 331 and 332 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 461 and 462 of the sequence shown in SEQ ID NO. 1; (IV) a forward primer specifically recognizing the sequence comprising nucleotides 331 and 332 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 462 and 1042 of the sequence shown in SEQ ID NO. 1; (V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 331 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence comprising nucleotides 461-462 of the sequence shown in SEQ ID NO. 1; and/or optionally, (VI) a forward primer which specifically recognizes the sequence of nucleotides 1 to 487 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 513-1174 of the sequence shown by 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 487 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 488-512 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 the 488-512 th nucleotide of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of the 513-1174 th nucleotide of the sequence shown in SEQ ID NO. 2; (VIII) a forward primer specifically recognizing a sequence comprising nucleotides 487 and 488 of the sequence shown in SEQ ID NO. 2 and a reverse primer specifically recognizing a sequence comprising nucleotides 512 and 513 of the sequence shown in SEQ ID NO. 2; (IX) a forward primer which specifically recognizes a sequence comprising nucleotides 487 and 488 of the sequence shown in SEQ ID NO:2 and a reverse primer which specifically recognizes nucleotides 513 and 1174 of the sequence shown in SEQ ID NO: 2; (X) a forward primer which specifically recognizes the sequence of nucleotides 1 to 487 of the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes the sequence comprising nucleotides 512 to 513 of the sequence shown by 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'-TGCCGAACGGTGAATAATGTAA-3', and 5'-GCCTTGATCTAGGAGGGAATGT-3'. Sequencing primers for detecting the sequence shown in SEQ ID NO. 1 are, for example, 5'-TGCCGAACGGTGAATAATGTAA-3', and 5'-GCCTTGATCTAGGAGGGAATGT-3'.

In another embodiment, the primer pairs used to amplify the sequences shown in SEQ ID NO. 2 are, for example, 5'-CCTATGTCTAGTCGCTGAAATCC-3', and 5'-GAGAACGAATCCCTGTCTAACTG-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO. 2 are, for example, 5'-CCTATGTCTAGTCGCTGAAATCC-3', 5'-GTACTCGGGCGATGCTTTAC-3', and 5'-GAGAACGAATCCCTGTCTAACTG-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 rice 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 for the foreground selection is selected from one or more of PiC11ID17, PiC11S122 and PiC11S 166; 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 Pic11ID17 and negative selection markers Pic11S122 and Pic11S166, 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 Pic11ID17, 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) carrying out backcross again on a recombinant single plant with a good background (the background reversion value of the generation exceeds 87.5%) and recurrent parents to obtain three backcross generations, carrying out screening on homologous recombination fragments on the other side of a RICE blast resistant genome fragment by utilizing a positive selection marker Pic11ID17, a negative selection marker Pic11S122 and a Pic11S166, and carrying out background selection on the recombinant single plant by utilizing 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 Pic11ID17, 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 is homozygous and has a background recovery (the background recovery value exceeds 99%) and contains RICE blast resistant genome recombinant nucleic acid segments.

In another embodiment, the amplification primers used in the foreground selection of recombinant plants using molecular markers comprise: a primer pair for amplifying a molecular marker Pic11ID17, wherein the forward primer is 5'-GTACTGGAGGATCAGGACTGG-3', and the reverse primer is 5'-CTGTTGCCTTGTGACTGTGAG-3'; a primer pair for amplifying the molecular marker Pic11S122, wherein the forward primer is 5'-TACGACCGTGACATGTCCTT-3', and the reverse primer is 5'-ATTAACCACCATGCTCACCA-3'; and a primer pair for amplifying the molecular marker Pic11S166, wherein the forward primer is 5'-TTAGCCCCTCTCTCTCTCCA-3', and the reverse primer is 5'-GCCAGATCTAGCAGAGGTGA-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'-TGCCGAACGGTGAATAATGTAA-3', and reverse primer: 5'-GCCTTGATCTAGGAGGGAATGT-3', respectively; sequencing primers, including a forward primer: 5'-TGCCGAACGGTGAATAATGTAA-3', and reverse primer: 5'-GCCTTGATCTAGGAGGGAATGT-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'-CCTATGTCTAGTCGCTGAAATCC-3', reverse primer: 5'-GAGAACGAATCCCTGTCTAACTG-3', respectively; sequencing primers, including a forward primer: 5'-CCTATGTCTAGTCGCTGAAATCC-3', reverse primer: 5'-GTACTCGGGCGATGCTTTAC-3', and reverse primer 5'-GAGAACGAATCCCTGTCTAACTG-3'. 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 'flourishing B', is a maintainer line of a high-quality early indica sterile line 'flourishing A', and has the outstanding characteristic of good rice quality. By using the method, the rice blast resistance genome fragment can be introduced to improve the rice blast resistance of the rice blast resistance genome fragment while the original advantages of 'flourishing B' are retained. Furthermore, stable 'flourishing A' containing rice blast resistant fragments can be obtained through at least one generation of hybridization and one generation of backcross, and the rice blast resistance of the hybrid seeds is greatly improved through matching. 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 PCR 010066 RICE RICE60K whole genome breeding chip in example 1 of the present application; wherein, the boxes indicated by the abscissa number sequentially represent 12 rice chromosomes, the ordinate number is the physical position [ in megabases (Mb) ] on the rice genome, the gray line represents the acceptor parent 'flourishing B' genotype, the black line represents the donor parent 'Hua 2048B' genotype, and the white line represents the same genotype of the two parents, i.e. no polymorphism segment. The black dot line of chromosome 11 in the figure shows that the segment is the introduced rice blast resistant genomic recombinant nucleic acid fragment RecCR 010066.

FIG. 2 shows the sequencing alignment of upstream homologous recombination fragment RecCR010066 in example 2 of the present application; the asterisks shown in the figure represent the same bases in the alignment results, in the figure, CR010066 is the obtained new strain, T003 is the acceptor parent 'ShengshiB', and R006 is the donor parent 'Hua 2048B'.

FIG. 3 shows the sequencing alignment of the homologous recombination fragment downstream of RecCR010066 in example 2 of this application.

FIG. 4 is a structural diagram of homologous recombination fragments flanking RecCR010066 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 mark of donor 'Hua2048B' and the lower base is SNP or InDel mark of acceptor 'ShengShi B'. The grey segment is derived from the 'flourishing B' genome segment, the black segment is derived from the 'Hua 2048B' genome segment, the white segment is the homologous recombination segment, the abscissa is the segment length, and the unit is the number of base pairs (bp).

FIG. 5 shows the results of indoor identification of resistance to CR010066 blast 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 'flourishing B'; (C) improving a new strain CR 010066; (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 a sequence comprising the specified position in the sequence indicated by SEQ ID NO 1 or SEQ ID NO 2 or a fragment or variant or complement thereof, for example, a sequence comprising nucleotides 331 and 462 of the sequence indicated by SEQ ID NO 1 or a fragment or complement thereof, or a sequence comprising nucleotides 487 and 513 of the sequence indicated by SEQ ID NO 2 or a fragment or variant or complement 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 'heyday B' was used as a recurrent parent, and rice 'hua 2048B' that has 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 the marker PiC11ID17 that is closely linked to the target genomic DNA fragment, the negative selection marker is the marker PiC11S122 located upstream of the target fragment, and the marker PiC11S166 located downstream of the target fragment.

In the present embodiment, when the detection of homologous recombination is carried out using the above-mentioned foreground selection marker, the criteria for judging one-sided or one-sided homologous recombination are that PiC11ID17 detects the same band pattern as 'hua 2048B', and PiC11S122 or PiC11S166 detects the same band pattern as 'heyday B'; the criteria for judging bilateral or bilateral homologous recombination were that PiC11ID17 detected the same band pattern as ` Wahua 2048B ` and PiC11S122 and PiC11S166 detected the same band pattern as ` ShengshiB `.

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 'heyday B' and rice 'hua 2048B'.

Rice 'Hua 2048B' has good rice blast resistance, and it is presumed that the region where Pi1 and Pik cluster alleles of chromosome 11 are located plays a key role in the rice blast resistance of the material.

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 11 th chromosome 27,155,000 to 28,495,000 DNA sequences were 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 385 pair is designed in total. The polymorphisms of the primer pair in ` Hua 2048B ` and ` Shengshi B ` were screened by PCR method, and finally the foreground selective molecular markers with polymorphism and high amplification efficiency in the two materials, namely the positive selective marker Pic11ID17 and the negative selective markers Pic11S122 and Pic11S166, 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 gene in the rice 'Hua 2048B' is introduced into the rice 'Shengshi B', and the specific process is as follows:

hybridizing 'flourishing B' as recurrent parent 'Hua2048B' as donor parent, backcrossing the obtained hybrid with recurrent parent 'flourishing B' for first generation to obtain BC₁F₁After breeding, the seeds were selected for recombinant individuals using positive selection marker Pic11ID17 and negative selection markers Pic11S122, Pic11S166, 7 individuals that were homologously recombined on one side of the target genomic DNA fragment were selected, that is, Pic11ID17 showed the same band pattern as ` Hua 2048B ` and Pic11S122 or Pic11S166 showed the same band pattern as ` Shengshi B `, and were background-selected using RICE whole genome breeding chip RICE6K (CN102747138A) (Yu et al, Plant Biotechnology journal.2014,12: 28-37).

Comparing the chip results in 7 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 recurrent parent 'hessian B' again to obtain BC₂F₁After the seeds are raised, the seeds are detected by using a positive selection marker Pic11ID17, a recombinant single plant containing a target genome fragment, namely Pic11ID17 is selected, the banding pattern of the recombinant single plant is detected to be the same as that of 'Hua2048B', and the recombinant single plant is subjected to background selection by using a RICE whole genome breeding chip RICE 6K.

Selecting 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 'flourishing B' again to obtain BC₃F₁Seed, after seedling raising, using positive selection marker Pic11ID17 and negative markers Pic11S122 and Pic11S166 to screen homologous recombination fragments at the other side of target genome fragment on harvested seed, obtaining 8 recombination fragments at both sides of target fragmentThe individuals of (2), Pic11ID17, detected the same band pattern as ` Hua2048B ` and Pic11S122 and Pic11S166 detected the same band pattern as ` ShengshiB `.

The 8 double-sided cross-over individuals were subjected to background and target fragment selection using a rice whole genome breeding chip R ICE60K (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, the forward selection marker Pic11ID17 was used to detect it, and a single plant containing a target genomic DNA fragment, i.e., Pic11ID17, was selected to detect the same band type as that of ` Hua 2048B ` and was background-selected using RICE whole genome breeding chip RICE 60K.

Finally, one strain which is homozygous for the target fragment and has a recovered background (the background recovery value exceeds 99%) is obtained and named as CR 010066. The chip detection results are shown in FIG. 1.

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 ` hessian B ` introduced fragment was subjected to sequencing of homologous recombination fragments flanking the genomic fragment of interest. The rice blast resistant genomic recombinant nucleic acid fragment contained in CR010066 was designated RecCR 010066.

The RecCR010066 is located between two SNP markers F1127345466CT and F1128121185GA as preliminarily determined by the detection result of a RICE whole genome breeding chip RICE 60K.

Meanwhile, Miseq sequencing technology was used to perform whole genome sequencing on three samples of 'flourishing B', 'Hua 2048B' and CR 010066. 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 RecCR010066 is positioned in the interval from 27353258bp to 27354300bp of the 11 th chromosome, and the downstream homologous recombination fragment is positioned in the interval from 28117029bp to 28118200 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.

Respectively designing amplification primers for upstream and downstream homologous recombination fragments of RecCR010066 by using acceptor parent 'flourishing B' and donor parent 'Hua2048B' as controls, amplifying by using a high-fidelity enzyme KOD FXneo (TOYOBO), and searching for an optimal amplification condition by using a two-step method or a three-step method to ensure that an amplification product shows 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 ℃, 30sec at 60 ℃, 60sec 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 ℃, 30sec at 61 ℃, 60sec at 68 ℃, 37 cycles; 1min at 20 ℃. Thus, two pairs of amplification primers are finally screened for amplification of upstream and downstream homologous recombination fragments, respectively.

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

The sequencing length of the upstream homologous recombination fragment of RecCR010066 is 1042bp (SEQ ID NO: 1). 1-331bp is the genome segment of the acceptor 'flourishing B', and compared with the donor 'Hua 2048B', 8 SNPs and 1 Indel exist. The 130bp segment of 332-461bp is a homologous recombination segment. 462-1042bp is the donor genome fragment of 'Hua 2048B', and compared with 'ShengShi B', 8 SNPs and 3 indels exist.

The sequencing length of the homologous recombination fragment downstream of RecCR010066 is 1174bp (SEQ ID NO: 2). 1-487bp is the genome segment of donor 'Hua2048B', and compared with 'flourishing B', there are 2 SNPs and 1 Indel. The 25bp segment of 488-512bp is a homologous recombination segment. 513-1174bp is a genome segment of the acceptor 'flourishing B', and compared with the donor 'Hua 2048B', 3 SNPs exist.

FIG. 4 is a diagram of the structure of homologous recombination fragments flanking RecCR 010066. 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 SNP or InDel marker of donor 'Hua 2048B', and the lower base is SNP or InDel marker of acceptor 'ShengShi B'. The grey segment is derived from 'flourishing B' genome segment, the black segment is derived from 'Hua 2048B' genome segment, and the white segment is a homologous recombination segment. 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

In order to identify the resistance effect, the new strain CR010066, recurrent parent 'flourishing B', rice blast disease-resistant variety flos Pruni mume No. 4 (as positive control) and rice blast susceptible variety Lijiang new-ball black valley (as negative control) bred by the method are planted indoors, and the new strain is cultured to 3-4 leaves and then identified by the following method:

5121-1 separated from rice blast disease leaves in West Jiang and 1124-1 separated from rice blast disease leaves in Guangdong disease nursery in 2013 are selected, and 2 rice blast strains in total are used as inoculation strains. The strain is preserved at-20 deg.C by sorghum grain method, the preserved sorghum grains are taken out before use and are activated on potato glucose culture medium (PDA) flat plate (PDA: peeled potato 200g, glucose 20g, agar powder 15g, distilled water constant volume to 1L), after illumination culture at 28 deg.C for 5 days, fresh mycelium blocks with diameter of 5mm are taken and transferred to sorghum grain culture medium (500 g of sorghum grains are added with 1.5L of distilled water, liquid is filtered after boiling,taking out sorghum grains, placing into a 250ml triangular flask, sterilizing for 20 min with 100 ml/bottle, sterilizing with damp heat, 10 blocks/bottle, inoculating bacteria for 2 days, shaking the sorghum grains, and culturing in dark at 28 deg.C until the sorghum grains grow over the hypha. 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.

Spray inoculation of CR010066, 'heuchen B', oryzanol 4 and lijiang new ball black grain with mixed conidia suspension, three replicates of inoculation. 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 (10)

1. A recombinant nucleic acid fragment consisting of a first recombinant nucleic acid fragment and a second recombinant nucleic acid fragment of:

-a first recombinant nucleic acid fragment being the sequence of SEQ ID No. 1 or the complement thereof;

-a second recombinant nucleic acid fragment which is the sequence of SEQ ID No. 2 or the complement thereof.

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

-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 1-331 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 462-1042 of the sequence shown in SEQ ID NO. 1; or

(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 331 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 332 and 461 of 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 332 and 461 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 462 and 1042 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:

(III) a forward primer which specifically recognizes the sequence of nucleotides 1 to 487 of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 513-1174 of the sequence shown in SEQ ID NO. 2; or

(IV) the following combination of a third set of primer pairs and a fourth set of primer pairs, comprising

(c) Third set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 1 to 487 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 488-512 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 the 488-512 th nucleotide of the sequence shown in SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of the 513-1174 th nucleotide of the sequence shown in SEQ ID NO. 2.

3. A primer for detecting the recombinant nucleic acid 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’-TGCCGAACGGTGAATAATGTAA-3’，

5’-GCCTTGATCTAGGAGGGAATGT-3’；

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

5’-TGCCGAACGGTGAATAATGTAA-3’，

5’-GCCTTGATCTAGGAGGGAATGT-3’；

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

5’-CCTATGTCTAGTCGCTGAAATCC-3’，

5'-GAGAACGAATCCCTGTCTAACTG-3', respectively; and

(IV) primer for sequencing SEQ ID NO. 2

5’-CCTATGTCTAGTCGCTGAAATCC-3’，

5’-GTACTCGGGCGATGCTTTAC-3’，

5’-GAGAACGAATCCCTGTCTAACTG-3’。

4. A method of breeding a rice plant comprising the recombinant nucleic acid fragment of claim 1, wherein the recombinant nucleic acid fragment comprises a rice blast resistance gene, and the method comprises the steps of:

1) hybridizing 'flourishing B' of recurrent parent rice and 'Hua 2048B' of donor rice, backcrossing the obtained hybrid with recurrent parent to obtain a backcross first generation, screening single-side homologous recombination fragments of rice blast resistance genome fragments by using a positive selective marker Pic11ID17 and negative selective markers Pic11S122 and Pic11S166, and performing background selection on the rice blast resistance genome fragments by using a rice whole genome breeding chip;

2) selecting a recombinant single plant with better background reversion and recurrent parents for backcross again to obtain a second backcross generation, detecting the second backcross generation by using a forward selection marker Pic11ID17, selecting the recombinant single plant containing a rice blast resistant genome segment, and then performing background selection by using a rice whole genome breeding chip;

3) selecting a recombinant single plant with a restored background and recurrent parents for backcross again to obtain three backcross generations, screening homologous recombination fragments on the other side of the rice blast resistant genome fragment by using a positive selection marker Pic11ID17 and negative markers Pic11S122 and Pic11S166, and selecting the background of the recombinant single plant and the recurrent parents by using a rice whole genome breeding chip; and

4) selecting a recombinant single plant with a small introduced segment and good background reversion, selfing the selected recombinant single plant once to obtain a selfed seed, detecting the selfed seed by using a forward selection marker Pic11ID17, and performing background selection on the selfed seed by using a rice whole genome breeding chip to finally obtain a rice plant containing a homozygous rice blast resistant genome recombinant nucleic acid segment and with a background reversion;

wherein, the amplification primers adopted when the molecular marker is used for carrying out the foreground selection on the recombinant plant are as follows:

the primer pair of the amplification molecular marker Pic11ID17 is as follows:

a forward primer: 5'-GTACTGGAGGATCAGGACTGG-3' the flow of the air in the air conditioner,

reverse primer: 5'-CTGTTGCCTTGTGACTGTGAG-3', respectively;

the primer pair of the amplification molecular marker Pic11S122 is as follows:

a forward primer: 5'-TACGACCGTGACATGTCCTT-3' the flow of the air in the air conditioner,

reverse primer: 5'-ATTAACCACCATGCTCACCA-3', respectively; and

the primer pair for amplifying the molecular marker Pic11S166 is as follows:

a forward primer: 5'-TTAGCCCCTCTCTCTCTCCA-3' the flow of the air in the air conditioner,

reverse primer: 5'-GCCAGATCTAGCAGAGGTGA-3' are provided.

5. 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.

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

7. 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.

8. The method of claim 7, 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.

9. The method of claim 7, wherein the method 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.

10. The method of claim 7, wherein the kit of claim 6 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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