CN106480057B - Recombinant nucleic acid fragment RecCR012083 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 RecCR012083 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 153-727 of the sequence shown in SEQ ID NO. 1 or a fragment or variant or complementary sequence 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 356-1008 of the sequence indicated by 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 for specifically recognizing the sequence of nucleotides 1-153 of the sequence shown in SEQ ID NO. 1 and a reverse primer for specifically recognizing the sequence of nucleotides 727-1161 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 153 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 154 and 726 of the sequence shown by SEQ ID NO. 1; and (b) a second set of primer pairs: a forward primer for specifically recognizing the sequence of nucleotides 154-726 of the sequence shown in SEQ ID NO. 1 and a reverse primer for specifically recognizing the sequence of nucleotides 727-1161 of the sequence shown in SEQ ID NO. 1; (III) a forward primer specifically recognizing the sequence comprising nucleotides 153 and 154 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 726 and 727 of the sequence shown in SEQ ID NO. 1; (IV) a forward primer specifically recognizing the sequence comprising nucleotides 153-154 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 727-1161 of the sequence shown in SEQ ID NO. 1; (V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 153 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence comprising nucleotides 726 and 727 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 356 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 1008-1278 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 356 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 357 and 1007 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 represented by the sequence 357-1007 nucleotides of the sequence represented by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence represented by the sequence 1008-1278 nucleotides of the sequence represented by the SEQ ID NO. 2; (VIII) a forward primer specifically recognizing a sequence comprising nucleotides 356 and 357 of the sequence indicated by SEQ ID NO:2 and a reverse primer specifically recognizing a sequence comprising nucleotides 1007 and 1008 of the sequence indicated by SEQ ID NO: 2; (IX) a forward primer which specifically recognizes a sequence comprising nucleotides 356 and 357 of the sequence indicated by SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 1008 and 1278 of the sequence indicated by SEQ ID NO: 2; (X) a forward primer which specifically recognizes a sequence of nucleotides 1 to 356 of the sequence shown in SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 1007 and 1008 of the sequence shown in SEQ ID NO: 2.

In one embodiment, the primer pair used to amplify the sequence shown in SEQ ID NO. 1 is, for example, 5'-TTCGTGAACTAAACAGGTCCTAA-3', 5'-GAGCAAATAGTGAGTGCGTAAGG-3'. Sequencing primers for detecting the sequence shown in SEQ ID NO. 1 are, for example, 5'-GCATTTGAATTGGACCTAG-3'; 5'-GTGGAACTTGGAAGGGACAG-3', respectively; and 5'-GCTCGCCTAGAACTTGTGAC-3'.

In another embodiment, the primer pair used to amplify the sequence shown in SEQ ID NO. 2 is, for example, 5'-GATTCTATTTCCCATCCTCCTTC-3', 5'-GACTGCTAAACGCTACTCCGTAA-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO. 2 are, for example, 5'-CTCCTGGCTGCCTGTAGTGC-3'; 5'-AAGTCTAAAGAGGGAGTGGG-3', respectively; and 5'-AAGACTGAACCAACCACCAT-3'.

In another aspect, the present application provides a method for breeding a rice plant containing a recombinant nucleic acid fragment, comprising the steps of crossing a rice recipient plant parent not containing a target gene fragment as a recurrent parent with a rice donor plant containing the target gene fragment, backcrossing the resultant hybrid with the recurrent parent, and then selfing the resultant backcrossed, wherein the resultant backcrossed and selfed are subjected to foreground selection and background selection, respectively, 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 Pi2-4, Pi2S67, 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 resistant to rice blast, 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 generation, performing single-sided homologous recombination fragment screening on the RICE blast resistant genome fragment by using a positive selection marker Pi2-4 and negative selection markers Pi2S67 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 Pi2-4, selecting the recombinant single plant containing a RICE blast resistant genome fragment, 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 Pi2-4 and negative markers Pi2S67 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 a 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 Pi2-4, and performing background selection on the selfed seed by using a RICE whole genome breeding chip, such as RICE60K, so as to finally obtain a RICE plant which contains the RICE blast resistant genome recombinant segment and is homozygous and 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 Pi2-4, wherein the forward primer is 5'-CGGTAAGAGTAACACCAAGC-3' and the reverse primer is 5'-GACGTGCGAGTTGTGACAGCT-3'; a primer pair for amplifying the molecular marker Pi2S67, wherein the forward primer is 5'-CCGATGCAAGAACAAGCTAA-3' and the reverse primer is 5'-CCACCACATCACCAGTGTTT-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: an amplification primer pair comprising a forward primer: 5'-TTCGTGAACTAAACAGGTCCTAA-3', reverse primer: 5'-GAGCAAATAGTGAGTGCGTAAGG-3', respectively; sequencing primers, including reverse primers: 5'-GCATTTGAATTGGACCTAG-3', forward primer: 5'-GTGGAACTTGGAAGGGACAG-3', and forward primer: 5'-GCTCGCCTAGAACTTGTGAC-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: an amplification primer pair comprising a forward primer: 5'-GATTCTATTTCCCATCCTCCTTC-3', reverse primer: 5'-GACTGCTAAACGCTACTCCGTAA-3', respectively; sequencing primers, including a forward primer: 5'-CTCCTGGCTGCCTGTAGTGC-3', forward primer: 5'-AAGTCTAAAGAGGGAGTGGG-3', and forward primer: 5'-AAGACTGAACCAACCACCAT-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.

Therefore, the method for breeding the rice plants containing the rice blast resistant genome recombinant nucleic acid fragments based on the whole genome selective breeding technology has at least one of the following advantages: (one) quick: the high-density molecular marker technology is used for selecting the whole genome background, so that the breeding efficiency is obviously improved, the breeding process is accelerated, and the target plant can be obtained through five generation transformation at the fastest speed. (II) accurate: accurate screening can be carried out on the target genome segment by utilizing an accurate prospect selection marker, so that the introduced target genome segment is very small (theoretically, the introduced target genome segment can be accurate to the level of a single gene), and the encumbrance of the close linkage with the target genome segment is removed; and (3) carrying out whole genome background selection by using a high-density molecular marker technology, and removing non-target genome fragments introduced in the transfer process of the donor parent. Finally, a target plant which has the background completely consistent with that of the receptor parent and only contains the target genome segment of the donor, namely, the existing advantages of the receptor parent are completely kept and the excellent characteristics of the donor parent are introduced can be obtained. (III) stabilizing: because the breeding process is accurately controlled by using a high-density molecular marker whole genome background selection technology, the horizontal genotype of the whole genome of each screened single plant can be clearly understood, and the obtained target plant can be stably inherited.

Drawings

FIG. 1 shows the results of the CR012083 RICE RICE60K whole genome breeding chip assay 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, and the line display section at the black dot of the No. 6 chromosome in the figure is the introduced rice blast resistant genome recombinant nucleic acid fragment RecCR 012083.

FIG. 2 shows the sequencing alignment of upstream homologous recombinant fragment RecCR012083 in example 2 of the present application; the asterisks shown in the figure represent the same base in the comparison result, wherein CR012083 is an obtained new line, KY131 is an acceptor parent 'empty breeding 131', and 'BL 6' is a donor parent.

FIG. 3 shows the sequencing alignment of the homologous recombinant fragments downstream of RecCR012083 in example 2 of the present application.

FIG. 4 is a diagram of the structure of homologous recombination fragments flanking RecCR012083 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 'BL 6', and the lower base is SNP or InDel mark of acceptor 'empty breeding 131'. The gray segments are derived from the ` null-bred 131 ` genome segment, the black segments are derived from the ` BL6 ` genome segment, the white segments are homologous recombination segments, and the abscissa is the fragment length in base pair number (bp) units.

FIG. 5 shows the results of indoor identification of resistance to CR012083 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 'air breeding 131'; (C) improving a new strain CR 012083; (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 resistance gene 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 fragment comprising the specified position in the sequence indicated by SEQ ID NO. 1 or SEQ ID NO. 2 or a variant or complement thereof, for example, a sequence comprising nucleotides 153-727 of the sequence indicated by SEQ ID NO. 1 or a fragment or variant or complement thereof, or a sequence comprising nucleotides 356-1008 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 'air-bred 131' was used as a recurrent parent, and rice 'BL 6' 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 an embodiment of the present application, the foreground selection marker used includes a positive selection marker and a negative selection marker, wherein the positive selection marker is a polymorphic molecular marker selected in a range of 50kb (genetic distance of 0.2cM in rice) upstream and downstream from the target genomic fragment (containing the rice blast resistance gene). The negative selection marker is a polymorphic molecular marker screened in the range of 500kb (genetic distance of 2cM in rice) upstream and downstream from the target genome fragment. In a specific embodiment, the positive foreground selection marker used in the optimized screening is marker Pi2-4 that is closely linked to the target genomic fragment, the negative selection marker is marker Pi2S67 that is about 170kb upstream of the target fragment, and marker Pi2S122 that is about 380kb 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 Pi2-4 detects the same band type as that of 'BL 6', and Pi2S67 or Pi2S122 detects the same band type as that of 'null-grown 131'; the criteria for judging bilateral or bilateral homologous recombination were that Pi2-4 detected the same band type as ` BL6 ` and Pi2S67 and Pi2S122 detected the same band type as ` blank 131 `.

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 'air-raised 131' and rice 'BL 6'.

Rice 'BL 6' has been shown to have good resistance to rice blast and it is speculated 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 this 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 6 9,559,000 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 'BL 6' and 'empty breeding 131' were screened by PCR, and finally the foreground selection molecular markers with polymorphism and high amplification efficiency in both materials, namely positive selection marker Pi2-4 and negative selection markers Pi2S67 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 'BL 6' where the gene cluster is located is introduced into the rice 'empty breeding 131', and the specific process is as follows:

hybridizing the 'empty breeding 131' as recurrent parent and 'BL 6' as donor parent, backcrossing the obtained hybrid with recurrent parent to obtain BC₁F₁After seed breeding, recombinant individuals were selected using positive selection marker Pi2-4 and negative selection markers Pi2S67 and Pi2S122, 5 individuals that were homologously recombined on the target genome fragment side, i.e., Pi2-4 detected the same band type as ` BL6 ` and Pi2S67 or Pi2S122 detected the same band type as ` null-bred 131 `, were selected and subjected to 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 5 selected unilateral 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 'air-bred 131' again to obtain BC₂F₁After the seeds are raised, the positive selection marker Pi2-4 is used for detecting the seeds, namely Pi2-4 detects the same band type as 'BL 6', recombinant single plants containing target genome fragments are selected, and the RICE whole genome breeding chip RICE6K is used for carrying out background selection on the recombinant single plants.

Selecting the individual plant with better background recovery (the background recovery value of the generation exceeds 87.5 percent), and carrying out backcross again on the individual plant and the recurrent parent 'empty breeding 131' to obtain BC₃F₁Seeds, after seedling raising, the other side of the target genome fragment is carried out on the harvested seeds by using a positive selection marker Pi2-4 and negative selection markers Pi2S67 and Pi2S122Screening of homologous recombination fragments, 2 individuals recombined at both sides of the target fragment are obtained, namely Pi2-4 detects the same band type as 'BL 6', and Pi2S67 and Pi2S122 detect the same band type as 'sterile 131'.

The 2 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, the positive selection marker Pi2-4 was used to detect it, and the individual plant containing the target genome fragment, i.e., Pi2-4, was selected to detect the same band type as that of 'BL 6', and was background-selected using RICE whole genome breeding chip RICE 60K.

Finally, one of the strains which is homozygous for the target fragment and has recovered background (the background recovery value exceeds 99%) is obtained and named as CR 012083. The recombinant nucleic acid fragment of the blast-resistant genome contained in CR012083 was designated as RecCR 012083. 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 ` null-bred 131 ` introduced fragment was subjected to sequencing of homologous recombination fragments flanking the genomic fragment of interest.

The RecCR012083 is located between two SNP markers F0610271281GA and R0610407176TC as preliminarily determined by the detection result of a RICE whole genome breeding chip RICE 60K.

Meanwhile, Miseq sequencing technology is used for whole genome sequencing of three samples of 'empty breeding 131', 'BL 6' and CR 012083. 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 V2Reagent Kit (illumina) Kit. 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 RecCR012083 is preliminarily positioned in the interval from 10276433bp to 10277592bp of the 6 th chromosome, and the downstream homologous recombination fragment is positioned in the interval from 10398827bp to 10400108 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 the receptor parent 'empty breeding 131' and the donor parent 'BL 6' as controls, amplification primers are respectively designed for upstream and downstream homologous recombination fragments of RecCR012083, 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 ℃,30 sec at 58 ℃, 120sec at 68 ℃ for 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, 3 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 the CR012083 rice blast resistant genome recombination nucleic acid fragment is 1161bp (SEQ ID NO: 1). 1-153bp is the genome segment of acceptor 'sterile 131', and compared with donor 'BL 6', there are 3 SNPs and 1 Indel. The 573bp segment of 154-726bp is a homologous recombination segment. 727 + 1161bp is a donor 'BL 6' genome fragment, and compared with 'sterile 131', 3 SNPs exist.

CR012083 blast resistant genome recombination nucleic acid fragment downstream homologous recombination fragment length of 1278bp (SEQ ID NO: 2). 1-356bp is the genome segment of donor 'BL 6', and compared with 'sterile 131', there are 4 SNPs, 1 Indel. 357 and 1007bp of the 651bp segment are homologous recombination segments. 1008-1278bp is a genome segment of an acceptor 'empty breeding 131', and 5 SNPs exist compared with a donor 'BL 6'.

FIG. 4 shows the structure of homologous recombination fragments flanking RecCR 012083. 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 'BL 6', and the lower base is SNP or InDel marker of acceptor 'empty breeding 131'. The grey segment is derived from 'empty breeding 131' genome segment, the black segment is derived from 'BL 6' genome segment, and the white segment is 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 ` empty-breeding 131 ` -into Rice blast-resistant genomic fragment

In order to identify the resistance effect, the new strain CR012083, the recurrent parent 'empty breeding 131', the rice blast disease-resistant variety flos Pruni mume No. 4 (as a positive control) and the rice blast susceptible variety Lijiang new-ball black valley (as a negative control) are planted indoors and are cultured to 3-4 leaf periods to be identified by the following method:

7 rice blast strains separated from rice blast leaves and disease necks of a Yangtze Jia Mus disease garden in Heilongjiang in 2014 are selected as inoculation strains, and the numbers are 14-7301, 14-7302, 14-7303, 14-7305, 14-7309, 14-7324 and 14-7328 respectively. 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) plate (PDA: peeled potato 200g, glucose 20g, agar powder 15g, distilled water constant volume to 1L), and the strain is cultured for 5 days at 28 deg.C by illuminationThen, fresh hypha blocks with the diameter of 5mm are taken and transferred into a sorghum grain culture medium (500 g of sorghum grains are added with 1.5L of distilled water, the liquid is filtered after boiling, the sorghum grains are fished out and put into a 250ml triangular flask and 100 ml/flask, moist heat sterilization is carried out for 20 minutes), 10 hypha blocks are placed in 10 flasks, after inoculation for 2 days, the sorghum grains are shaken out every day, and dark culture is carried out at 28 ℃ until the hypha 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.

CR012083, 'air-bred 131', oryzanol 4 and lijiang new-ball black grain were inoculated with mixed conidia suspension by spraying, and inoculation was repeated three times. 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 (9)

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 set for detecting the fragments of claim 1, wherein the primer set consists of the following primers for detecting the first recombinant nucleic acid fragment and the second recombinant nucleic acid fragment:

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

(I) a forward primer for specifically recognizing the sequence of nucleotides 1-153 of the sequence shown in SEQ ID NO. 1 and a reverse primer for specifically recognizing the sequence of nucleotides 727-1161 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 153 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 154 and 726 of the sequence shown by SEQ ID NO. 1; and

(b) a second set of primer pairs: a forward primer for specifically recognizing the sequence of nucleotides 154-726 of the sequence shown in SEQ ID NO. 1 and a reverse primer for specifically recognizing the sequence of nucleotides 727-1161 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 specifically recognizing the sequence of nucleotides 1 to 356 of the sequence shown by SEQ ID NO. 2 and a reverse primer specifically recognizing the sequence of nucleotides 1008-1278 of the sequence shown by SEQ ID NO. 2;

(VI) 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 356 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 357 and 1007 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 represented by the sequence 357-1007 nucleotides of the sequence represented by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence represented by the sequence 1008-1278 nucleotides of the sequence represented by SEQ ID NO. 2.

3. A primer set for detecting the fragment of claim 1, wherein the primer set is:

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

5’-TTCGTGAACTAAACAGGTCCTAA-3’，

5’-GAGCAAATAGTGAGTGCGTAAGG-3’；

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

5’-GCATTTGAATTGGACCTAG-3’；

5’-GTGGAACTTGGAAGGGACAG-3’；

5’-GCTCGCCTAGAACTTGTGAC-3’；

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

5’-GATTCTATTTCCCATCCTCCTTC-3’，

5'-GACTGCTAAACGCTACTCCGTAA-3', respectively; and

(IV) primer for sequencing SEQ ID NO. 2

5’-CTCCTGGCTGCCTGTAGTGC-3’；

5’-AAGTCTAAAGAGGGAGTGGG-3’；

5’-AAGACTGAACCAACCACCAT-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 set of claim 2 or 3 and a test genome as a template, and analyzing the PCR product.

5. A kit for detecting the recombinant nucleic acid fragment of claim 1, comprising the primer set 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 primer set of claim 2 or 3 is used to detect whether the genome of the rice plant or seed to be tested contains the recombinant nucleic acid fragment of claim 1.

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

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

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