CN107304426B - Recombinant nucleic acid fragment RecCR010161, detection primer and application thereof - Google Patents

Abstract

The invention relates to molecular biology, and particularly discloses a rice blast-resistant recombinant nucleic acid fragment RecCR010161, and a detection primer and application thereof. The invention also provides a method for breeding rice plants containing the rice blast resistant genome recombinant nucleic acid segments based on the whole genome selective breeding technology, which introduces target genome segments into receptor materials and simultaneously realizes the reversion of the background. The improved acceptor material is Luxiang 618B which is a maintenance line matched with an odor type sterile line Luxiang 618A with low-middle 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, the rice blast resistance of the hybrid seeds is greatly improved through matching. The genome recombinant nucleic acid fragment provided by the invention is closely related to rice blast resistance, and can be used as a resistance resource to be applied to the cultivation of other varieties.

Description

Recombinant nucleic acid fragment RecCR010161, detection primer and application thereof

Technical Field

The invention relates to molecular biology, in particular to a rice blast resistant recombinant nucleic acid fragment.

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 Pil and Pik cluster alleles are located in the long-arm proximal region of the 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).

In order to improve the stability of breeding, shorten the breeding process and time, and effectively utilize rice resistance resources, it is necessary to study rice blast resistance gene recombinant fragments generated during cross breeding of rice, so as to provide an effective means for efficiently and stably realizing rice resistance breeding.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a rice blast-resistant recombinant nucleic acid fragment RecCR010161, and a detection primer and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a recombinant nucleic acid fragment, wherein the nucleotide sequence thereof comprises the sequence of 331-478bp shown in SEQ ID NO.1 or a fragment thereof, or a variant thereof, or a complementary sequence thereof.

Preferably, the nucleotide sequence comprises the sequence shown in SEQ ID NO.1 or a fragment thereof, or a variant thereof, or a complementary sequence thereof. The sequence shown in SEQ ID NO.1 is from a recombinant plant produced by exchanging the genomic regions of 'Luxiang 618B' and 'Hua 3418B'.

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 invention 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.

In a second aspect, the invention provides primers for amplifying the recombinant nucleic acid fragments.

The primers include all primers that can be designed by those skilled in the art for the target of the amplification.

When the amplification target is the sequence shown in SEQ ID NO.1, the primer may be selected from:

(I) a forward primer for specifically recognizing the nucleotide sequence in the region of 1-331bp of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the region of 478-766bp 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 for specifically recognizing the nucleotide sequence in the 1-331bp region of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the 332-477bp region of the sequence shown in SEQ ID NO. 1;

(b) a second set of primer pairs: a forward primer for specifically recognizing the nucleotide sequence in the 332-477bp region of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the 478-766bp region of the sequence shown in SEQ ID NO. 1;

(III) a forward primer specifically recognizing a sequence comprising nucleotide 331 of the sequence shown in SEQ ID NO.1 and a reverse primer specifically recognizing a sequence comprising nucleotide 478 of the sequence shown in SEQ ID NO. 1;

(IV) a forward primer which specifically recognizes a sequence comprising the 331 st nucleotide of the sequence shown in SEQ ID NO.1 and a reverse primer which specifically recognizes a nucleotide sequence in the 478-and 766bp region of the sequence shown in SEQ ID NO. 1;

(V) a forward primer which specifically recognizes the nucleotide sequence in the 1 st to 331bp region of the sequence shown in SEQ ID NO.1 and a reverse primer which specifically recognizes a sequence comprising the 478 nd nucleotide of the sequence shown in SEQ ID NO. 1.

Specifically, the primer pair for amplifying the sequence shown in SEQ ID NO.1 provided by the invention is as follows: 5'-TGCCGAACGGTGAATAATGTAA-3' the flow of the air in the air conditioner,

and 5'-GCCTTGATCTAGGAGGGAATGT-3'.

And the sequencing primer for detecting the sequence shown by SEQ ID NO.1 is as follows:

5’-TGCCGAACGGTGAATAATGTAA-3’，

and 5'-GCCTTGATCTAGGAGGGAATGT-3'.

Further, the invention also provides a kit containing the primer.

In a third aspect, the invention provides an application of the recombinant nucleic acid fragment in rice blast resistance rice breeding.

For example, the fragment is introduced into other rice plants to obtain rice plants having resistance to rice blast.

In a fourth aspect, the invention provides a method of screening for rice plants containing the recombinant nucleic acid fragment.

Designing a specific primer according to the recombined nucleic acid fragment, carrying out PCR reaction by taking the genome to be detected as a template, and analyzing the PCR amplification product. Specifically, the primers are as described above. Alternatively, PCR amplification products were analyzed using Sanger sequencing.

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.

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

Preferably, the aforementioned primer or the aforementioned kit can be used to detect whether the genome of the sample to be tested contains the recombinant nucleic acid fragment according to claim 1.

It is understood that the rice plants or seeds thereof containing the recombinant nucleic acid fragments disclosed in the present invention screened by the method also belong to the scope of the present invention.

In a fifth aspect, the invention provides a breeding method of rice plants containing the recombinant nucleic acid fragment, which specifically comprises the following steps: hybridizing 'Luxiang 618B' as a recurrent parent and 'Hua3418B' as a donor parent, backcrossing the obtained hybrid with 'Luxiang 618B' as a recurrent parent, and then selfing the obtained backcrossed seed to obtain a rice plant containing the recombinant nucleic acid fragment of claim 1;

wherein, the hybrid, the backcross and the inbred need to respectively use the molecular marker and the rice whole genome breeding chip to carry out foreground selection and background selection.

The molecular markers are one or more of PiC11ID17, PiC11S122 and PiC11S 166.

Specifically, the breeding method comprises the following steps: 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.

Wherein, the amplification primers adopted when the molecular marker is used for foreground selection are as follows:

a primer pair for amplifying a molecular marker PiC11ID17, comprising:

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

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

a primer pair for amplifying a molecular marker PiC11S122, comprising:

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

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

a primer pair for amplifying a molecular marker PiC11S166, comprising:

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

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

The invention has the beneficial effects that:

the invention obtains and provides a recombinant nucleic acid fragment of a rice blast resistance gene through the whole genome selective breeding technology, and the recombinant nucleic acid fragment provided by the invention is closely related to the resistance of rice blast and can be used as a resistance resource to be applied to the cultivation of other varieties.

The invention provides a method for breeding rice plants containing rice blast resistant genome recombinant nucleic acid fragments based on a whole genome selective breeding technology, which has the advantages of rapidness, accuracy and stability, and only through five generations of transformation, only target genome fragments can be introduced into a receptor material, and the background recovery is realized at the same time.

On the basis, the invention takes 'Luxiang 618B' as a recurrent parent and 'Hua3418B' as a donor parent to carry out hybridization, backcross and selfing to obtain the recombinant plant with rice blast resistance and obtain the recombinant nucleic acid segment with the rice blast resistance.

The improved acceptor material is Luxiang 618B which is a maintenance line matched with an odor type sterile line Luxiang 618A with low-middle 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, the rice blast resistance of the hybrid seeds is greatly improved through matching. The genome recombinant nucleic acid fragment provided by the invention is closely related to rice blast resistance, and can be used as a resistance resource to be applied to the cultivation of other varieties.

Drawings

FIG. 1 shows the results of the chip detection of the whole genome breeding of CR010161 RICE RICE60K in example 1 of the present invention; 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 Luxiang 618B', and the white line represents the same genotype of the two parents, namely no polymorphic segment. The black line of chromosome 11 in the figure shows that the segment is the introduced rice blast resistant genomic recombinant nucleic acid fragment RecCR 010161.

FIG. 2 shows the sequencing alignment of upstream homologous recombination fragments of RecCR010161 in example 2 of the present invention; the asterisks shown in the figure represent the same base in the alignment, in the figure, CR010161 is the new line obtained, T004 is the acceptor parent Luxiang 618B ', and R002 is the donor parent Luzhou 618B'.

FIG. 3 is a diagram showing the structure of the upstream homologous recombination fragment of RecCR010161 in example 2 of the present invention; wherein, 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. 4 shows the results of indoor identification of resistance to CR010161 blast of rice in example 3 of the present invention; 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 010161; (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 invention and to guide those of ordinary skill in the art in the practice of the present invention. 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 invention to make conservative amino acid substitutions. In certain embodiments, substitutions that do not alter the amino acid sequence of the nucleotide sequence of the present invention 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 invention relates to further optimizing the resulting nucleotide sequence for SEQ ID NO. 1. 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 invention also relates to variants of the sequence shown in SEQ ID NO. 1. 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 invention 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 invention also relates to a sequence comprising the specified position in the sequence indicated by SEQ ID NO.1 or a fragment or variant thereof or a complement thereof, for example, a sequence comprising nucleotides 331-478 of the sequence indicated by SEQ ID NO.1 or a fragment or variant thereof or a complement thereof. Based on the fragment containing the specific site, the corresponding sequence shown in SEQ ID NO.1 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.

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 of the present invention is suitable for any rice variety needing selective 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 an embodiment of the present invention, rice 'Luxiang 618B' is used as a recurrent parent, and rice 'Hua3418B' that has been confirmed to have good resistance to rice blast is used as a donor.

In the breeding method of the recombinant plant provided by the invention, 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 invention, 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 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 embodiment of the present invention, when the detection of homologous recombination is performed using the above-mentioned foreground selection marker, the criteria for judging one-sided or one-sided homologous recombination are that Pic11ID17 detects the same banding pattern as ` Hua 3418B `, and Pic11S122 or Pic11S166 detects the same banding pattern as ` Luxiang 618B `; the criteria for the bilateral or bilateral homologous recombination were PiC11ID17 detecting the same banding pattern as ` Hua3418B ` and PiC11S122 and PiC11S166 detecting the same banding pattern as ` Luxiang 618B `.

In the present invention, any available chip can be used for background selection in the breeding method provided by the present invention. In a preferred embodiment, the RICE whole genome breeding chip RICE6K disclosed by the present inventors in Chinese patent application CN102747138A, or the RICE whole genome breeding chip RICE60K disclosed in PCT international application WO/2014/121419, may 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 invention. 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 invention can be seen in Chinese rice varieties and genealogical databases thereof (http:// www.ricedata.cn/variety/index. htm).

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

Example 1 selection of recombinant plants into which genomic fragments resistant to Rice blast were introduced

The materials used in this example were rice 'luxiang 618B' and rice 'hua 3418B'.

Rice 'Hua 3418B' has good rice blast resistance, and presumably the region where the Pil and Pik cluster alleles of chromosome 11 are located plays 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 11 th chromosome 27,155,000 to 28,495,000DNA 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 polymorphism of the primer pair in 'Hua3418B' and 'Luxiang 618B' is screened by a PCR method, and finally, the foreground selective molecular markers which have polymorphism and high amplification efficiency in two materials are selected, wherein the foreground selective molecular markers are respectively a positive selective marker Pic11ID17, a negative selective marker Pic11S122 and a negative selective marker Pic11S 166. 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 '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 BC₁F₁Seed, after seedling raising, utilizing positive selection marker Pic11ID17 and negative selection markers Pic11S122 and Pic11S166 to make recombination individual plant selection, screening 9 plants with identical target genome DNA fragment sideThe recombinant individual, i.e., Pic11ID17, detected the same banding pattern as ` Hua 3418B ` and Pic11S122 or Pic11S166 detected the same banding pattern as ` Luxiang 618B ` and was 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 the 9 screened unilateral homologous recombinant individuals, selecting the recombinant individual with the best background recovery (the background recovery value of the generation exceeds 75%), backcrossing the recombinant individual with the recurrent parent 'Luxiang 618B' 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 same banding pattern as 'Hua 3418B' is detected, and the RICE whole genome breeding chip RICE6K is used for carrying out background selection on the recombinant single plant.

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 from the harvested seeds by using a positive selection marker PiC11ID17 and negative markers PiC11S122 and PiC11S166 after seedling raising, and obtaining 8 individuals recombined on both sides of the target fragment, namely, the individuals with the same banding patterns as those of 'Hua3418B' are detected by PiC11ID17, and the individuals with the same banding patterns as those of 'Luxiang 618B' are detected by PiC11S122 and PiC11S 166.

The 8 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 forward selection marker Pic11ID17 was used to detect it, and the single plant containing the target genome fragment, i.e., Pic11ID17, was selected to detect the same banding pattern as that of 'Hua 3418B', and was subjected to background selection using 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 010161. The chip detection results are shown in FIG. 1.

Example 2 determination 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 CR010161 was designated RecCR 010161.

The RecCR010161 is located at the downstream of SNP marker R1127353173AG as preliminarily determined by the detection result of RICE whole genome breeding chip RICE 60K.

Meanwhile, three samples of 'Luxiang 618B', 'Hua3418B' and CR010161 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 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 homologous recombination fragment at the upstream of RecCR010161 is positioned in the interval of 27353218bp to 27354367bp on the 11 th chromosome.

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.

An acceptor parent Luxiang 618B 'and a donor parent Hua3418B' are used as controls, amplification primers are designed for homologous recombination fragments upstream of RecCR010161, 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 ℃, 30sec at 60 ℃, 60sec at 68 ℃, 37 cycles; 1min at 20 ℃. Thus, a pair of amplification primers is finally selected for amplification of the upstream homologous recombination fragment.

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 the upstream 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 FIG. 2.

The sequencing length of homologous recombination fragment upstream of RecCR010161 is 766bp (SEQ ID NO: 1). 1-331bp is the recipient 'Luxiang 618B' genome segment, compared with the donor 'Hua3418B', there are 2 SNPs, 2 indels. The 146bp segment of 332-477bp is a homologous recombination segment. 478-766bp is a donor genome fragment of 'Hua3418B', and compared with an acceptor genome fragment of 'Luxiang 618B', 4 SNPs exist.

FIG. 3 is a diagram of the structure of a homologous recombination fragment upstream of RecCR 010161. 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 3 identification of resistance of Luxiang 618B' after introduction of genomic fragment against Rice blast

In order to identify the resistance effect, the new strain CR010161, 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:

selecting M15 separated from Yichang rice blast disease leaves in 2015Bb-1-1, M15Bb-1-2, M15Bb-2-1, M15Bb-3-1, M15Bb-4-1, M15Bb-5-1 and M15Bb-6-1, wherein 7 rice blast strains are used 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. 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.

CR010161, '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. 4.

TABLE 3 resistance Performance after inoculation with Pyricularia oryzae

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

Claims (1)

1. The breeding method of rice plants with rice blast resistance is characterized by comprising the following steps:

1) taking 'Luxiang 618B' as a recurrent parent and 'Hua3418B' as a donor parent for hybridization, hybridizing the recurrent parent with a donor plant, backcrossing the obtained hybrid with the recurrent parent to obtain a backcross generation, screening single-side homologous recombination fragments of rice blast resistant genome fragments by using a positive selection marker Pic11ID17 and negative selection markers Pic11S122 and Pic11S166, and performing background selection on the rice blast resistant genome fragments by using a rice whole genome breeding chip;

2) selecting a recombinant single plant with a background recovery value of over 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 by using a rice whole genome breeding chip;

3) carrying out backcross again on the selected recombinant single plant with the background recovery value of more than 87.5 percent and recurrent parents to obtain three backcross generations, carrying out screening on homologous recombination fragments on the other side of the rice blast resistant genome fragment by utilizing a positive selection marker Pic11ID17 and negative markers Pic11S122 and Pic11S166, and carrying out background selection on the rice blast resistant genome fragment by utilizing a rice whole genome breeding chip;

4) selecting a recombinant single plant with a small introduced segment and a background recovery value of more than 93.75 percent, 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 which is homozygous and contains rice blast resistant genome recombinant nucleic acid segments and has a background recovery value of more than 99 percent;

the primer pair for amplifying the marker Pic11ID17 is:

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

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

the primer pair for amplifying the 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 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', respectively;

the RICE whole genome breeding chip is RICE 60K.

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