CN107304449B - Recombinant nucleic acid fragment RecCR010311 and 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 RecCR010311, 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 receptor material of the invention is a widely used three-series restorer 'Yuehui 9113'. By using the method, the rice blast resistance genome fragment can be introduced to improve the rice blast resistance of the rice blast resistance genome fragment on the premise of keeping the original characteristics of 'Yuehui 9113'. 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 RecCR010311 and 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 Pi1 and Pik cluster alleles are located in the long-arm proximal terminal region of rice chromosome 11 (Hua et al, therapeutic and Applied genetics.2012,125: 1047-1055; Li et al, Molecular Breeding.2007,20: 179-188; Alok et al, Functional & Integrated genetics.2012, 12: 215-228; Yuan et al, therapeutic and Applied genetics.2011,122: 1017-1028).

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 RecCR010311, 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 comprises the sequence 1192-1450bp 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 'Yuehui 9113' 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 1-1192bp of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the region of 1450-1559bp 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 1-1192bp region of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the 1193-and 1449bp 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 1193-and 1449-bp region of the sequence shown in SEQ ID NO.1 and a reverse primer for specifically recognizing the nucleotide sequence in the 1450-and 1559-bp region of the sequence shown in SEQ ID NO. 1;

(III) a forward primer that specifically recognizes a sequence comprising nucleotide 1192 of the sequence shown in SEQ ID No.1 and a reverse primer that specifically recognizes a sequence comprising nucleotide 1450 of the sequence shown in SEQ ID No. 1;

(IV) a forward primer which specifically recognizes a sequence comprising the 1192 nd nucleotide of the sequence shown in SEQ ID NO.1 and a reverse primer which specifically recognizes a nucleotide sequence in the 1450-1559bp region of the sequence shown in SEQ ID NO. 1;

(V) a forward primer which specifically recognizes the nucleotide sequence in the 1-1192bp region of the sequence shown in SEQ ID NO.1 and a reverse primer which specifically recognizes the sequence containing the 1450 th nucleotide of the sequence shown in SEQ ID NO. 1.

Specifically, the present invention provides primer pairs, 5'-ACTCACCGTTTAGTAGTTTGGG-3', and 5'-GTGTTTCATCATTTCTGTAGCC-3', for amplifying the sequence shown in SEQ ID NO. 1.

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

5’-TTTTCCTATGCGGGCGATGC-3’，

5’-CTACAATCTTGGCTATTTCTGGC-3’，

5’-TTAGTAAACGTGGAGGAGCG-3’，

5’-TGAGCTGGAGCTTGACTTT-3’，

5’-GTAGCGTGGGCATCTTATT-3’，

5’-GAGCGGATTGTTGTGACAGATT-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 the 'Yuehui 9113' as a recurrent parent and 'Hua3418B' as a donor parent, backcrossing the obtained hybrid with the recurrent parent 'Yuehui 9113', and 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 Pi31, Pi2ID01, and Pi2ID 05.

Specifically, the breeding method comprises the following steps: 1) hybridizing the recurrent parent and a donor plant, backcrossing the obtained hybrid with the recurrent parent to obtain a backcross generation, screening single-sided homologous recombination fragments of the RICE blast resistant genome fragments by using a positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05, and carrying out background selection on the RICE blast resistant genome fragments by using a RICE whole genome breeding chip, such as RICE 6K; 2) selecting a recombinant single plant with better background recovery (the background recovery value of the generation exceeds 75 percent) to carry out backcross with recurrent parents again to obtain a second backcross generation, detecting the second backcross generation by using a forward selection marker Pi31, selecting the recombinant single plant containing a RICE blast resistant genome segment, and then carrying out background selection on the recombinant single plant by using a RICE whole genome breeding chip, such as RICE 6K; 3) selecting a recombinant single plant with a restored background (the background restoration value of the generation exceeds 87.5 percent) and recurrent parents for backcross again to obtain three backcross generations, screening homologous recombination fragments on the other side of a RICE blast resistant genome fragment by using a positive selection marker Pi31 and negative 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 resistant genome recombinant nucleic acid segment and is homozygous and has good background recovery (the background recovery value exceeds 99%).

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

a primer pair for amplifying the molecular marker Pi31, comprising:

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

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

a primer pair for amplifying the molecular marker Pi2ID01, comprising:

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

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

a primer pair for amplifying the molecular marker Pi2ID05, comprising:

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

reverse primer: 5'-TGGGATTCATTGGGTGAGTAT-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 'Yuehui 9113' 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 fragment with the rice blast resistance.

The improved receptor material of the invention is a widely used three-series restorer 'Yuehui 9113'. By using the method, the rice blast resistance genome fragment can be introduced to improve the rice blast resistance of the rice blast resistance genome fragment on the premise of keeping the original characteristics of 'Yuehui 9113'. 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 CR010311 RICE RICE60K in example 1 of the present invention; wherein, the boxes indicated by the abscissa number sequentially represent 12 chromosomes of the 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 'Yuehui 9113', the black line represents the genotype of the donor parent 'Hua3418B', and the white line represents the section with the same genotype of the two parents, namely no polymorphism. In the figure, the black line of chromosome 6 shows that the segment is the introduced rice blast resistant genome recombinant nucleic acid fragment RecCR 010311.

FIG. 2 shows the sequencing alignment result of the homologous recombination fragment downstream of RecCR010311 in example 2 of the present invention; the asterisks shown in the figure represent the same bases in the alignment, in the figure, CR010311 is the new strain obtained, T005 is the acceptor parent 'Yuehui 9113', and R002 is the donor parent 'Hua 3418B'.

FIG. 3 is a structural diagram of a homologous recombination fragment downstream of RecCR010311 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 'Yuehui 9113'. The gray segment is derived from the genome segment of Yuehui 9113, the black segment is derived from the genome segment of Hua 3418B, the white segment is the homologous recombination segment, and the abscissa is the fragment length in base pair number (bp).

FIG. 4 shows the results of indoor identification of resistance to CR010311 rice blast 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) original variety 'Yuehui 9113'; (C) improving a new strain CR 010311; (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 sequence complementary thereto, for example a sequence comprising nucleotides 1192-1450 of the sequence indicated by SEQ ID NO.1 or a fragment or variant thereof or a sequence complementary thereto. 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, the rice 'yuehui 9113' was used as a recurrent parent, and the rice 'hua 3418B' 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 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 marker Pi31 that is closely linked to the target genomic fragment, the negative selection marker is marker Pi2ID01 that is upstream of the target fragment, and marker Pi2ID05 that is downstream of the target fragment.

In the embodiment of the present invention, 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 pattern as 'hua 3418B', and Pi2ID01 or Pi2ID05 detects the same band pattern as 'yuehui 9113'; the criteria for judging bilateral or bilateral homologous recombination were that Pi31 detected the same band pattern as ` Hua 3418B ` and Pi2ID01 and Pi2ID05 detected the same band pattern as ` Yuehui 9113 `.

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 'yuehui 9113' and rice 'hua 3418B'.

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

In the process of breeding the recombinant plants, the molecular markers are used for carrying out prospect selection on the recombinant plants, and the adopted prospect selection molecular markers are screened. The 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 ` Yuehui 9113 ` were screened by PCR, and finally, the foreground selection molecular markers having polymorphisms and high amplification efficiency in both materials, i.e., the positive selection marker Pi31 and the negative selection markers Pi2ID01 and Pi2ID05, were selected. The specific primer information for PCR amplification of the above molecular markers is shown in Table 1.

TABLE 1 Foreground selection of molecular marker primer information

The genome segment of the gene in the rice 'Hua 3418B' is introduced into the rice 'Yuehui 9113', and the specific process is as follows:

"Yuehui 9113" as recurrent parent and "Hua3418B" as donor parent, backcrossing the hybrid with recurrent parent "Yuehui 9113" to obtain BC1F1 seed, selecting recombinant individuals using positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05 after seedling raising, selecting 11 individuals that are homologously recombined on the side of the target genomic DNA fragment, i.e., Pi31 detected the same banding pattern as "Hua3418B" and Pi2ID01 or Pi2ID05 detected the same banding pattern as "Yuehui 9113", and background selecting them using RICE genome-wide breeding chip RICE6K (CN102747138A) (Yu et al, Plant Biotechnology journal.2014,12: 28-37).

Comparing the chip results in 11 screened single-side homologous recombination single plants, selecting the recombinant single plant with the best background recovery (the background recovery value of the generation exceeds 75%), carrying out backcross again with a recurrent parent 'Yuehui 9113' to obtain BC2F1 seeds, detecting the seeds by using a positive selection marker Pi31 after seedling raising, selecting the recombinant single plant containing a target genome segment, namely detecting the same banding pattern as 'Hua3418B' by using a Pi31, and carrying out background selection on the recombinant single plant by using a RICE whole genome breeding chip RICE 6K.

And (3) selecting a single plant with better background recovery (the generation background recovery value exceeds 87.5%), carrying out backcross again with a recurrent parent 'Yuehui 9113' to obtain BC3F1 seeds, screening homologous recombination fragments on the other side of a target genome fragment on the harvested seeds by using a positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05 after seedling culture, and obtaining 5 single plants recombined on two 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 'Yuehui 9113'.

The 7 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%).

The selected individuals are selfed once to obtain BC3F2, after seedling raising, the positive selection marker Pi31 is used for detecting the plants, the individuals containing target genome fragments, namely Pi31 detects the same banding pattern as 'Hua 3418B', and the RICE whole genome breeding chip RICE60K is used for carrying out background selection on the individuals.

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 010311. 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 single strain of the ` Yuehui 9113 ` introduced fragment was subjected to sequencing of homologous recombination fragments flanking the target genomic fragment. The rice blast resistant genomic recombinant nucleic acid fragment contained in CR010311 was designated RecCR 010311.

The result of the RICE whole genome breeding chip RICE60K test preliminarily confirmed that RecCR010311 is located upstream of SNP marker R0610434052 AG.

Meanwhile, Miseq sequencing technology is used for whole genome sequencing of three samples of 'Yuehui 9113', 'Hua3418B' and CR 010311. 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 downstream of RecCR010311 is positioned in the interval of 10430262bp to 10433740bp on the 6 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 'Yuehui 9113' and a donor parent 'Hua3418B' are used as controls, amplification primers are designed for homologous recombination fragments at the downstream of RecCR010311, 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 to ensure that an amplification product shows a single bright band in agarose gel electrophoresis detection. Wherein the reaction conditions of the downstream homologous recombination fragment amplification primers are as follows: 94 ℃ for 2 min; 10sec at 98 ℃, 30sec at 61 ℃, 120sec at 68 ℃ for 37 cycles; 1min at 20 ℃. Thus, a pair of amplification primers is finally selected for the amplification of the downstream homologous recombination fragment.

In addition, the amplification product is used as a template, sequencing is carried out by a Sanger sequencing method, and finally 6 sequencing primers are screened out for sequencing downstream homologous recombination fragments 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 the homologous recombination fragment downstream of RecCR010311 is 1559bp (SEQ ID NO: 1). 1-1192bp is the genome segment of donor 'Hua3418B', and compared with acceptor 'Yuehui 9113', there are 2 SNPs and 1 Indel. The 257bp segment of 1193-1449bp is a homologous recombination segment. 1450 and 1559bp are acceptor 'Yuehui 9113' genome fragments, and compared with donor 'Hua3418B', 5 SNPs exist.

FIG. 3 is a diagram of the structure of a homologous recombination fragment downstream of RecCR 010311. The upper base is SNP or InDel mark of donor 'Hua 3418B', and the lower base is SNP or InDel mark of acceptor 'Yuehui 9113'. The gray segment is derived from the genome segment of Yuehui 9113', the black segment is derived from the genome segment of ' Hua 3418B ', 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 after introduction of genomic fragment against Rice blast in Yuehui 9113

In order to identify the resistance effect, the new strain CR010311, the recurrent parent Yuehui 9113', the rice blast disease-resistant variety flos Pruni mume No. 4 (as a positive control) and the rice blast susceptible variety Lijiang Xinjiang new-ball black grain (as a negative control) are planted indoors, and the new strain CR010311, the recurrent parent Yuehui 9113', the rice blast disease-resistant variety flos Pruni mume (as a negative control) are cultured to 3-4 leaves and then identified by adopting the following method:

13-1209-1 strain separated from rice blast disease samples of Guangdong disease nursery in 2013 is selected as an inoculation strain. The strain is preserved at-20 deg.C by sorghum grain method, and the preserved sorghum grains are taken out to potato glucose medium (PDA) plate for activation (PDA: peeled potato)200g of potato, 20g of glucose, 15g of agar powder and distilled water to a constant volume of 1L), culturing for 5 days at 28 ℃ by illumination, taking a fresh hypha block with the diameter of 5mm, transferring the hypha block into a sorghum grain culture medium (500 g of sorghum grains are added with 1.5L of distilled water, filtering out liquid after boiling, taking out sorghum grains, putting the sorghum grains into a 250ml triangular flask, 100 ml/flask, performing moist heat sterilization for 20 minutes), culturing for 10 grains/flask, inoculating bacteria for 2 days, shaking the sorghum grains out every day, and culturing in the dark 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.

Mixed conidium suspension is sprayed to inoculate CR010311, Yuehui 9113', flos Pruni mume No. 4 and Lijiang Xinjiang new ball black grain, and the inoculation is 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 'Yuehui 9113' as a recurrent parent and 'Hua3418B' as a donor parent to carry out hybridization, hybridizing the recurrent parent with a donor plant, carrying out backcross on the obtained hybrid and the recurrent parent to obtain a backcross first generation, carrying out single-side homologous recombination fragment screening on a rice blast resistant genome fragment by utilizing a positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05, and carrying out background selection on the rice blast resistant genome fragment by utilizing a rice whole genome breeding chip;

2) selecting a recombinant single plant with a background recovery value of more than 75 percent to carry out backcross with recurrent parents again to obtain a second backcross generation, detecting the second backcross generation by using a forward selection marker Pi31, selecting the recombinant single plant containing a rice blast resistant genome segment, and then carrying out background selection on the recombinant single plant by using a rice whole genome breeding chip;

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, screening homologous recombination fragments on the other side of the rice blast resistant genome fragment by using a positive selection marker Pi31 and negative selection markers Pi2ID01 and Pi2ID05, and carrying out background selection on the recombinant single plant by using 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 Pi31, 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;

a primer pair for amplifying the molecular marker Pi31, comprising:

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

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

a primer pair for amplifying the molecular marker Pi2ID01, comprising:

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

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

a primer pair for amplifying the molecular marker Pi2ID05, comprising:

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

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

the RICE whole genome breeding chip is RICE 60K.

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