CN106480059B - Recombinant nucleic acid fragment RecCR010215 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 RecCR010215 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: 3611-1914; 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: 113-713).

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 361-459 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 nucleotide 289-397 shown in SEQ ID NO 2 or a fragment or variant or complement thereof; or 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 1-361 shown in the sequence SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 459-825 shown in the sequence 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 361 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 362-458 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 362-458 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 459-825 of the sequence shown by SEQ ID NO. 1; (III) a forward primer specifically recognizing the sequence comprising the 361 st and 362 th nucleotides of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising the 458 nd and 459 th nucleotides of the sequence shown in SEQ ID NO. 1; (IV) a forward primer specifically recognizing the sequence comprising nucleotides 361-362 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 459-825 of the sequence shown in SEQ ID NO. 1; (V) a forward primer specifically recognizing the sequence of nucleotides 1 to 361 of the sequence shown in SEQ ID NO. 1 and a reverse primer specifically recognizing the sequence comprising nucleotides 458 and 459 of the sequence shown in SEQ ID NO. 1; and/or optionally, (VI) a forward primer specifically recognizing the sequence of nucleotides 1 to 289 of the sequence shown by SEQ ID NO. 2 and a reverse primer specifically recognizing the sequence of nucleotides 397-764 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 289 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 290 to 396 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 nucleotides 290-; (VIII) a forward primer specifically recognizing a sequence comprising nucleotides 289 and 290 of the sequence shown by SEQ ID NO. 2 and a reverse primer specifically recognizing a sequence comprising nucleotides 396 and 397 of the sequence shown by SEQ ID NO. 2; (IX) a forward primer which specifically recognizes a sequence comprising nucleotide 289-290 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes nucleotide 397-764 of the sequence shown by SEQ ID NO. 2; (X) a forward primer which specifically recognizes the sequence of nucleotides 1 to 289 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence comprising nucleotides 396-397 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'-TCACGACGGTTGACGGGACTTA-3', and 5'-TCGGCTGCTTATGAAATGGAAA-3'. Sequencing primers for detecting the sequence shown in SEQ ID NO. 1 are, for example, 5'-TCACGACGGTTGACGGGACTTA-3', and 5'-TCGGCTGCTTATGAAATGGAAA-3'.

In another embodiment, the primer pairs used to amplify the sequences shown in SEQ ID NO. 2 are, for example, 5'-ACGCAGTCAAGCGAGCCGTTCT-3', and 5'-TTCTTCAACCGCAATGCCAGAT-3'. The sequencing primers used to detect the sequence shown in SEQ ID NO. 2 are, for example, 5'-ACGCAGTCAAGCGAGCCGTTCT-3', and 5'-TTCTTCAACCGCAATGCCAGAT-3'.

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

In the above method, the molecular marker for the foreground selection is one or more selected from Pi31, Pi2ID01, and Pi2ID 05; 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-side homologous recombination fragment screening on the RICE blast resistant gene fragment by using a positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05, and performing background selection on the RICE blast resistant gene fragment by using a RICE whole genome breeding chip, such as RICE 6K; 2) selecting a recombinant single plant with better background recovery (the background recovery value of the generation exceeds 75 percent) to carry out backcross with recurrent parents again to obtain a second backcross generation, detecting the second backcross generation by using a forward selection marker Pi31, selecting the recombinant single plant containing the RICE blast resistant gene segment, and then carrying out background selection on the recombinant single plant by using a RICE whole genome breeding chip, such as RICE 6K; 3) selecting a recombinant single plant with a restored background (the background restoration value of the generation exceeds 87.5 percent) and recurrent parents for backcross again to obtain three backcross generations, screening homologous recombination fragments on the other side of the RICE blast resistant gene fragment by using a positive selection marker Pi31 and negative markers Pi2ID01 and Pi2ID05, and performing background selection on the recombinant single plant by using a RICE whole genome breeding chip, such as RICE 60K; and 4) selecting a recombinant single plant with small introduced segment and good background recovery (the background recovery value exceeds 93.75%), selfing the selected recombinant single plant once to obtain a selfed seed, detecting the selfed seed by using a forward selection marker Pi31, and performing background selection on the selfed seed by using a RICE whole genome breeding chip, such as RICE60K, to finally obtain a RICE plant which contains the RICE blast resistance gene and has homozygous recombinant nucleic acid segment 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 Pi31, wherein the forward primer is 5'-ATCCAAACCCGTTGTTGCAC-3' and the reverse primer is 5'-CGGCAATTGCCACGATGATA-3'; a primer pair for amplifying the molecular marker Pi2ID01, wherein the forward primer is 5'-CGTAAACTTGTTAGGTGGGTG-3', and the reverse primer is 5'-AAAATATGAGGAACTGGGCA-3'; and a primer pair for amplifying the molecular marker Pi2ID05, wherein the forward primer is 5'-CCTTATCACAGCCACATAGAGC-3' and the reverse primer is 5'-TGGGATTCATTGGGTGAGTAT-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'-TCACGACGGTTGACGGGACTTA-3', and reverse primer: 5'-TCGGCTGCTTATGAAATGGAAA-3', respectively; sequencing primers, including a forward primer: 5'-TCACGACGGTTGACGGGACTTA-3', and reverse primer: 5'-TCGGCTGCTTATGAAATGGAAA-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'-ACGCAGTCAAGCGAGCCGTTCT-3', and reverse primer: 5'-TTCTTCAACCGCAATGCCAGAT-3', respectively; sequencing primers, including a forward primer: 5'-ACGCAGTCAAGCGAGCCGTTCT-3', and reverse primer: 5'-TTCTTCAACCGCAATGCCAGAT-3' are provided. The method comprises the steps of taking genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the amplification primer, sequencing an obtained amplification product by using the sequencing primer, and if a sequencing result is consistent with or complementary to a sequence of SEQ ID NO. 2, determining that the sample to be detected contains a homologous recombination fragment shown in SEQ ID NO. 2.

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

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

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

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

The method for breeding the rice plant containing the rice blast resistant genome recombinant nucleic acid segment based on the whole genome selective breeding technology has the advantages of rapidness, accuracy and stability. Only through the transformation of five generations, only the target genome segment can be introduced into the acceptor material, and the reversion of the background can be realized at the same time. The acceptor material improved by the application is 'Luxiang 618B', which is a maintenance line matched with an odor type sterile line 'Luxiang 618A' with low-medium amylose content. By using the method, the rice blast resistance of the rice can be greatly improved under the condition of keeping the original advantages of the Luxiang 618B'. Furthermore, stable Luxiang 618A' containing the rice blast resistant fragment 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. Meanwhile, the 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 chip detection of CR010215 RICE RICE60K whole genome breeding in example 1 of the present application; wherein, the boxes indicated by the abscissa number sequentially represent 12 chromosomes of rice, the ordinate number is the physical position [ in megabases (Mb) ] on the rice genome, the gray line represents the genotype of the receptor parent Luxiang 618B ', the black line represents the genotype of the donor parent Hua3418B', and the white line represents the same genotype of the two parents, namely no polymorphic segment. The black dot line of chromosome 6 in the figure shows that the section is the introduced rice blast resistant genomic recombinant nucleic acid fragment RecCR 010215.

FIG. 2 shows the sequencing alignment of the upstream homologous recombination fragment of RecCR010215 in example 2 of the present application; the asterisks shown in the figure represent the same base in the alignment results, in the figure, CR010215 is the obtained new line, T004 is the acceptor parent Luxiang 618B ', R002 is the donor parent Luxiang 618B ', and R002 is the donor parent Hua 3418B ' and is the donor parent.

FIG. 3 shows the sequencing alignment of the homologous recombination fragment downstream of RecCR010215 in example 2 of this application.

FIG. 4 is a structural diagram of homologous recombination fragments flanking RecCR010215 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 'Hua3418B', and the lower base is SNP or InDel mark of acceptor 'Luxiang 618B'. The grey segment is derived from 'Luxiang 618B' genome segment, the black segment is derived from 'Hua3418B' genome segment, the white segment is homologous recombination segment, the abscissa is the fragment length, and the unit is the number of base pairs (bp).

FIG. 5 shows the results of indoor identification of resistance to CR010215 rice 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) the original variety Luxiang 618B'; (C) improving a new strain CR 010215; (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 site in the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or a variant or complement thereof, for example, a sequence comprising nucleotides 361-459 of the sequence shown in SEQ ID NO. 1 or a fragment or variant or complement thereof, or a sequence comprising nucleotides 289-397 of the sequence shown in SEQ ID NO. 2 or a fragment or variant or complement thereof. According to 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 'Luxiang 618B' was used as a recurrent parent, and rice 'Hua3418B' that has been confirmed to have good resistance to rice blast 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 Pi31 that is closely linked to the target genomic fragment, the negative selection marker is marker Pi2ID01 located about 360kb upstream of the target fragment, and marker Pi2ID05 located about 450kb 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 Pi31 detects the same band type as that of 'Hua 3418B', and Pi2ID01 or Pi2ID05 detects the same band type as that of 'Luxiang 618B'; the criteria for judging bilateral or bilateral homologous recombination were that Pi31 detected the same band pattern as ` Hua3418B `, and Pi2ID01 and Pi2ID05 detected the same band pattern as ` Luxiang 618B `.

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 Manual, 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 'luxiang 618B' and rice 'hua 3418B'.

Rice 'Hua 3418B' has good rice blast resistance, and it is presumed that the regions of the gene cluster in which Pi2, Pi9 and Pigm of chromosome 6 are located may play a key role in the resistance of the material to rice blast.

In the process of breeding the recombinant plants, the molecular markers are used for carrying out prospect selection on the recombinant plants, and the adopted prospect selection molecular markers are screened. The DNA sequence of chromosome 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 'Hua 3418B' and 'Luxiang 618B' are screened by a PCR method, and finally, the foreground selective molecular markers which have polymorphism and high amplification efficiency in two materials are selected, namely a positive selective marker Pi31 and negative selective markers Pi2ID01 and Pi2ID 05. 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 cluster in the rice 'Hua3418B' is introduced into the rice 'Luxiang 618B', and the specific process is as follows:

taking 'Luxiang 618B' as a recurrent parent and 'Hua3418B' as a donor parent for hybridization, and backcrossing the obtained hybrid with the recurrent parent 'Luxiang 618B' to obtain BC₁F₁After the seed was grown, 10 individuals were selected for recombination by using the positive selection marker Pi31 and the negative selection markers Pi2ID01 and Pi2ID05, and 10 individuals were selected for homologous recombination on the target genome fragment side, i.e., Pi31 showed the same band pattern as ` Hua3418B ` and Pi2ID01 or Pi2ID05 showed the same band pattern as ` Luxiang 618B `, and were selected for background by using the RICE whole genome breeding chip RICE6K (CN102747138A) (Yu et al, plant Biotechnology journal.2014,12: 28-37).

Comparing the chip results in 10 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 the receptor parent Luxiang 618B again to obtain BC₂F₁After the seeds are raised, the positive selection marker Pi31 is used for detecting the seeds, a recombinant individual strain containing a target genome fragment, namely Pi31 detects the same band type as 'Hua3418B', and the RICE whole genome breeding chip RICE6K is used for carrying out background selection on the recombinant individual strain.

Selecting the single plant with better background recovery (the background recovery value of the generation exceeds 87.5 percent), and backcrossing the single plant with the recurrent parent Luxiang 618B again to obtain BC₃F₁And (3) screening homologous recombination fragments on the other side of the target genome fragment of the harvested seeds by using a positive selective marker Pi31 and negative markers Pi2ID01 and Pi2ID05 after seedling raising, and obtaining 6 individuals recombined on both sides of the target fragment, namely Pi31 detects the same banding pattern as 'Hua 3418B', and Pi2ID01 and Pi2ID05 detect the same banding pattern as 'Luxiang 618B'.

The 6 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 Pi31 was used to detect it, and individuals containing the target genomic fragment, namely Pi31, were selected to have the same band type as that of 'Hua3418B', and were background-selected using the RICE whole genome breeding chip RICE 60K.

Finally, one strain which is homozygous for the target fragment and has a background reversion (the background reversion value exceeds 99%) is obtained and named as CR 010215. 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 homozygous individual for the "Luxiang 618B" introduced fragment was sequenced for homologous recombination fragments flanking the genomic fragment of interest. The rice blast resistant genomic recombinant nucleic acid fragment contained in CR010215 was designated RecCR 010215.

As preliminarily determined by the detection result of a RICE whole genome breeding chip RICE60K, RecCR010215 is positioned between two SNP markers R0610197319GT and F0610684610 TC.

Meanwhile, three samples of 'Luxiang 618B', 'Hua3418B' and CR010215 were subjected to whole genome sequencing using Miseq sequencing technology. Library construction was performed using TruSeq Nano DNA LT Kit (illumina) Kit, quantification was performed using LibraryQuantification 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 RecCR010215 is positioned in the interval from 10200150bp to 10200974bp of chromosome 6, and the downstream homologous recombination fragment is positioned in the interval from 10682107bp to 10682859 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 acceptor parent Luxiang 618B 'and the donor parent Hua3418B' as controls, amplification primers are respectively designed for upstream and downstream homologous recombination fragments of RecCR010215, 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 the optimal amplification condition, so that the 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 ℃,30 sec at 61 ℃, 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 ℃,30 sec 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 2 sequencing primers are finally screened out for sequencing upstream homologous recombination fragments and downstream homologous recombination fragments respectively 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 homologous recombination fragment upstream of RecCR010215 is 825bp (SEQ ID NO: 1). 1-361bp is the genome segment of the acceptor 'Luxiang 618B', and compared with the donor 'Hua3418B', 8 SNPs exist. The 97bp segment of 362-458bp is a homologous recombination segment. 459-825bp is the donor 'Hua3418B' genome fragment, and compared with 'Luxiang 618B', there are 5 SNPs and 1 Indel.

The sequencing length of the homologous recombination fragment downstream of RecCR010215 is 764bp (SEQ ID NO: 2). 1-289bp is the genome segment of donor 'Hua3418B', and there are 9 SNPs compared with 'Luxiang 618B'. The 107bp segment of 290-396bp is a homologous recombination segment. 397-764bp is the genome segment of the acceptor 'Luxiang 618B', and there are 5 SNPs compared with the donor 'Hua3418B'.

FIG. 4 shows the structure of homologous recombination fragments flanking RecCR 010215. Wherein (A) is the structure diagram of an upstream homologous recombination fragment; (B) is a structure diagram of a downstream homologous recombination fragment. The top base is the SNP or InDel marker of the donor 'Hua 3418B', and the bottom base is the SNP or InDel marker of the acceptor 'Luxiang 618B'. The grey segment is derived from 'Luxiang 618B' genome segment, the black segment is derived from 'Hua3418B' 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 of Luxiang 618B' after introduction of genome fragment resistant to rice blast

In order to identify the resistance effect, the new strain CR010215, the recurrent parent 'Luxiang 618B', the rice blast disease-resistant variety flos Pruni mume No. 4 (as a positive control) and the rice blast susceptible variety Lijiang new group black valley (as a negative control) are planted indoors, and the new strain is cultured to 3-4 leaves and then identified by the following method:

6 rice blast strains of 6102-1, 21K14-1, 21K02-1 and 4105-1 of Hubei disease nursery, 1209-1 of Guangdong disease nursery and 8111-1 of Fujian disease nursery which are separated from rice blast leaves and disease necks of Sichuan disease nursery in 2013 are selected as inoculation strains. The strain is preserved at-20 ℃ by adopting a sorghum grain method, the preserved sorghum grains are taken out to a potato glucose culture medium (PDA) for plate activation before use (the PDA is 200g of peeled potatoes, 20g of glucose and 15g of agar powder, the constant volume of distilled water is 1L), fresh mycelium blocks with the diameter of 5mm are taken out after illumination culture at 28 ℃ for 5 days and transferred to 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 taken out and put into a 250ml triangular flask, 100 ml/flask, moist heat sterilization is carried out for 20 minutes), 10 sorghum grains are taken out, the sorghum grains are shaken every day after inoculation for 2 days, and dark culture at 28 ℃ is carried out until the mycelium is full of the sorghum grains. Then theSpreading 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.

CR010215, 'luxiang 618B', oryzanol 4 and lijiang new ball black grain were inoculated with mixed conidia suspension spray, 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 the first recombinant nucleic acid fragment and/or the second recombinant nucleic acid fragment of:

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

i) a sequence comprising nucleotides 361-459 of the sequence shown in SEQ ID NO. 1 or a complementary sequence thereof, or

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

-a second recombinant nucleic acid fragment selected from:

iii) a sequence comprising nucleotide sequence 289-397 shown in SEQ ID NO 2 or a complementary sequence thereof, or

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

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

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

(I) a forward primer which specifically recognizes the sequence of nucleotides 1 to 361 shown in the sequence SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 459-825 shown in the sequence 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 361 of the sequence shown in SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 362-458 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 362-458 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 459-825 of the sequence shown by SEQ ID NO. 1;

(III) a forward primer which specifically recognizes the sequence shown by SEQ ID NO. 1 and comprises the 361 st and 362 st nucleotides of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence shown by SEQ ID NO. 1 and comprises the 458 st and 459 st nucleotides of the sequence shown by SEQ ID NO. 1;

(IV) a forward primer specifically recognizing the sequence containing the 361 st and 362 st nucleotides of the sequence shown by SEQ ID NO. 1 and a reverse primer specifically recognizing the 459 st and 825 st nucleotides of the sequence shown by SEQ ID NO. 1 in the sequence shown by SEQ ID NO. 1; or

(V) a forward primer which specifically recognizes the sequence of nucleotides 1 to 361 of the sequence shown by SEQ ID NO. 1 and a reverse primer which specifically recognizes the sequence of nucleotides 458 and 459 of the sequence shown by SEQ ID NO. 1 in the sequence shown by SEQ ID NO. 1;

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

(VI) a forward primer specifically recognizing the sequence of nucleotides 1 to 289 of the sequence shown in SEQ ID NO. 2 and a reverse primer specifically recognizing the sequence of nucleotides 397-764 of the sequence shown in SEQ ID NO. 2;

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

(c) Third set of primer pairs: a forward primer which specifically recognizes the sequence of nucleotides 1 to 289 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes the sequence of nucleotides 290 to 396 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 nucleotides 290-;

(VIII) a forward primer which specifically recognizes a sequence comprising nucleotides 289 and 290 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 396 and 397 of the sequence shown by SEQ ID NO:2 in the sequence shown by SEQ ID NO: 2;

(IX) a forward primer which specifically recognizes a sequence comprising nucleotides 289 and 290 of the sequence shown by SEQ ID NO. 2 and a reverse primer which specifically recognizes a sequence comprising nucleotides 397 and 764 of the sequence shown by SEQ ID NO. 2 in the sequence shown by SEQ ID NO. 2; or

(X) a forward primer which specifically recognizes the sequence of nucleotides 1 to 289 of the sequence shown by SEQ ID NO:2 and a reverse primer which specifically recognizes the sequence containing nucleotides 396 and 397 of the sequence shown by SEQ ID NO:2 among the sequences shown by SEQ ID NO: 2.

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

(I) primer for amplifying and sequencing sequence shown as SEQ ID NO. 1

5’-TCACGACGGTTGACGGGACTTA-3’，

5'-TCGGCTGCTTATGAAATGGAAA-3', respectively; and/or

(II) primers for amplifying and sequencing the sequence shown in SEQ ID NO. 2

5’-ACGCAGTCAAGCGAGCCGTTCT-3’，

5’-TTCTTCAACCGCAATGCCAGAT-3’。

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

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

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

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

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

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

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