CN106480060A - Recombinant nucleic acid fragment RecCR01BC06 and its detection method - Google Patents

Abstract

This application provides recombinant nucleic acid fragment and its detection method.Present invention also provides the selection of the rice plant containing recombinant nucleic acid fragment, carries out foreground selection and Foreground selection using molecular labeling to plant of recombinating, obtains the rice plant containing recombinant nucleic acid fragment.

Description

Recombinant nucleic acid fragment RecCR01BC06 and its detection method

Technical field

The application is related to full-length genome selection and use technology.Specifically, the application relates to the use of Full-length genome selection and use technology seed selection contains the rice plant of recombinant nucleic acid fragment, and thus And the recombinant nucleic acid fragment of acquisition and its detection method.

Background technology

For a long time, the system of selection of traditional breeding method depends on the evaluation of variable rate technology type, Accepted or rejected according to breeding man personal experience, its maximum shortcoming is that time-consuming, less efficient. The efficiency of selection to be improved, optimal method should directly genotype be selected. With the development of molecular biotechnology, molecular labeling is to realize directly selecting offer to genotype May.In recent years, have started to apply molecular marker-assisted selection method to improve individual target Proterties, being capable of significant shortening the breeding cycle.

Rice blast is one of disease of paddy rice most serious, the paddy rice that the whole world is caused by rice blast every year Production loss accounts for 11%～30%, and the therefore research of rice blast and its resistance is particularly important. As what rice blast was studied progressively gos deep into, many rice blast resistant gene DNA fragmentation phases Continue and be positioned and clone.Wherein, the Pi2 interval of the 6th chromosome of paddy rice is positioned and clones A lot of rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm, Pi50, the interval includes Gene cluster (Qu etc., Genetics.2006,172 of one rice blast resistance gene:1331-1914； Wang etc., Phytopathology.2012,102:779-786；Xiao etc., Mol Breeding. 2012,30:1715-1726；Liu etc., Mol Genet Genomics.2002,267:472-480； Jiang etc., Rice.2012,5:29-35；Zhu etc., Theor Appl Genet.2012,124: 1295-1304；Deng etc., Theor Appl Genet.2006,113:705-713).

Content of the invention

On the one hand, this application provides recombinant nucleic acid fragment, which is selected from：I) include SEQ ID NO: The sequence or its fragment of the nucleotides of sequence 790-2130 position shown in 1 or its variant or its complementary sequence Row；Ii) include SEQ ID NO:The sequence of sequence shown in 1 or its fragment or its variant or which is mutual Complementary series；Iii) include SEQ ID NO:The sequence of the nucleotides of sequence 770-1283 position shown in 2 or Its fragment or its variant or its complementary series；Or iv) include SEQ ID NO:Sequence shown in 2 Sequence or its fragment or its variant or its complementary series；And the combination of above fragment.Real one Apply in scheme, the recombinant nucleic acid fragment is genome recombination nucleic acid fragment.

Additionally, this application provides the primer of the detection recombinant nucleic acid fragment, which is selected from： (I) specific recognition SEQ ID NO:The forward direction of the sequence of the nucleotides of sequence 1-790 position shown in 1 Primer and specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 2130-4133 position shown in 1 Reverse primer；(II) the combining of first group of primer pair and second group of primer pair below, which includes (a) first group of primer pair：Specific recognition SEQ ID NO:The core of sequence 1-790 position shown in 1 The forward primer of the sequence of thuja acid and specific recognition SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 791-2129 position nucleotides；(b) second group of primer pair：Specificity Identification SEQ ID NO:The forward primer of the sequence of the nucleotides of sequence 791-2129 position shown in 1 With specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 2130-4133 position shown in 1 Reverse primer；(III) specific recognition includes SEQ ID NO:Sequence 790-791 shown in 1 The forward primer of the sequence of position nucleotides and specific recognition include SEQ ID NO:Sequence shown in 1 Arrange the reverse primer of the sequence of 2129-2130 position nucleotides；(IV) specific recognition includes SEQ ID NO:The forward primer of the sequence of the nucleotides of sequence 790-791 position shown in 1 and special Property identification SEQ ID NO:Reversely the drawing of the sequence of the nucleotides of sequence 2130-4133 position shown in 1 Thing；(V) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-790 position shown in 1 Forward primer and specific recognition include SEQ ID NO:Sequence 2129-2130 position shown in 1 The reverse primer of the sequence of nucleotides；And/or optionally, (VI) specific recognition SEQ ID NO: The forward primer of the sequence of the nucleotides of sequence 1-770 position shown in 2 and specific recognition SEQ ID NO:The reverse primer of the sequence of the nucleotides of sequence 1283-2244 position shown in 2；(VII) below Three groups of primer pairs are combined with the 4th group of primer pair, and which includes (c) the 3rd group of primer pair：Specifically Property identification SEQ ID NO:The forward primer of the sequence of the nucleotides of sequence 1-770 position shown in 2 and Specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 771-1282 position shown in 2 anti- To primer；(d) the 4th group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of the sequence of 771-1282 position nucleotides and specific recognition SEQ ID NO:2 institutes Show the reverse primer of the sequence of sequence 1283-2244 position nucleotides；(VIII) specific recognition Comprising SEQ ID NO:The forward primer of the sequence of the nucleotides of sequence 770-771 position shown in 2 and Specific recognition includes SEQ ID NO:The sequence of the nucleotides of sequence 1282-1283 position shown in 2 Reverse primer；(IX) specific recognition includes SEQ ID NO:Sequence 770-771 shown in 2 The forward primer of the sequence of position nucleotides and specific recognition SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 1283-2244 position nucleotides；(X) specific recognition SEQ ID NO:2 The forward primer of the sequence of shown sequence 1-770 position nucleotides and specific recognition include SEQ ID NO:The reverse primer of the sequence of the nucleotides of sequence 1282-1283 position shown in 2.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-CTCCATACCACATCGGTGACTCT-3 ', and 5 '-CCTCATTGT GGGCTGTGTTCA-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 1 For, for example, 5 '-TGCATTCGTCTAGCATGTGT-3 ', 5 '-GATCTTGTCCGGT GCTAGCA-3 ', 5 '-GTTGGCACACCACGTAAGCT-3 ', 5 '-ATACCTCCA GGCTCTAGTCA-3 ', 5 '-GATGAGAGGGATTCCTACCT-3 ', 5 '-AGTC ACCTCAGTTGTTAGTG-3 ', 5 '-AGATGGGGTGAACATTGAGGA-3 ', 5 '-CAGGAGGTCTGGTTCTGGTT-3 ', and 5 '-CCTCATTGTGGGCTGTGTT CA-3’.

In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2 For, for example, 5 '-ACGGGCTCAAGTGAACGAGT-3 ', and 5 '-CGTCTTGGTA TCTCTCAAGGCAT-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 2 For, for example, 5 '-GGGAGAAAGGGGATCGATCT-3 ', 5 '-ACGCCGAGAA GAAAACTCCA-3 ', 5 '-TGAACGCTGCCGGTGAGAGT-3 ', 5 '-CCTG ACCTACCACCAACACA-3 ', 5 '-GTGCAGTCTTTGGCGAAGGT-3 ', With 5 '-CGTCTTGGTATCTCTCAAGGCAT-3 '.

On the other hand, this application provides seed selection contains the side of the rice plant of recombinant nucleic acid fragment Method, it include using without genes of interest group fragment paddy rice recipient plant parent as recurrent parent, Which is hybridized with the paddy rice donor plant containing genes of interest group fragment, then will be obtained Cenospecies be returned with recurrent parent, then the step of obtained backcrossing kind is carried out selfing, Wherein foreground selection and Foreground selection are carried out to plant of recombinating using molecular labeling.For example, described Recombinant nucleic acid fragment is as previously mentioned.

In the above-mentioned methods, Pi31, Pi2ID02 are selected from for the molecular labeling of the foreground selection With one or more in Pi2S91；And/or institute is carried out using paddy rice full-length genome breeding chip State Foreground selection.

In one embodiment, the seed selection that the application is provided contains blast resisting recombinant nucleic acid fragment Rice plant method, which comprises the following steps：1) recurrent parent is carried out with donor plant Hybridization, obtained cenospecies and recurrent parent are returned, and obtain first backcross generation, are utilized Favorable selection mark Pi31 and negative itemsets mark Pi2ID02, Pi2S91 carry out anti-rice blast to which The unilateral homologous recombination fragment screening of ospc gene group fragment, and utilize paddy rice full-length genome breeding core Piece, such as RICE6K, carry out Foreground selection to which；2) background is selected to reply preferably restructuring single Strain (this generation background recovery value is more than 75%) is returned again with recurrent parent, obtains backcrossing In two generations, which is detected using favorable selection mark Pi31, select to contain blast resistant gene The restructuring individual plant of group fragment, then using paddy rice full-length genome breeding chip, such as RICE6K, Foreground selection is carried out to which；3) restructuring individual plant (this generation background recovery value for selecting background to reply More than 87.5%) it is returned with recurrent parent again, third backcross generation is obtained, using positive choosing Selecting mark Pi31 and negative indicia Pi2ID02, Pi2S91 carries out blast resistant gene pack to which The opposite side homologous recombination fragment screening of section, and paddy rice full-length genome breeding chip is utilized, for example RICE60K, carries out Foreground selection to which；And 4) select introgressed segment little, and background is replied Good restructuring individual plant (background recovery value is more than 93.75%), by the restructuring individual plant selfing that chooses once, Selfed seed is obtained, which is detected using favorable selection mark Pi31, and utilizes the full base of paddy rice Because of group breeding chip, such as RICE60K, Foreground selection is carried out to which, final acquisition contains anti-rice Seasonal febrile diseases genome recombination nucleic acid fragment homozygosis and the water of background reply (background recovery value is more than 99%) Rice plants.

In another embodiment, adopt when carrying out foreground selection using molecular labeling to plant of recombinating Amplimer, including：The primer pair of Pi31 is marked for amplifier molecule, and wherein forward direction is drawn Thing is 5 '-ATCCAAACCCGTTGTTGCAC-3 ', and reverse primer is 5 '-CGGCAATT GCCACGATGATA-3.The primer pair of Pi2ID02 is marked for amplifier molecule, wherein just It is 5 '-AAGTAATAATCCCCGCTGTTGT-3 ' to primer, reverse primer is 5’-TTTCAAGCGAAGGAGGATGT-3’.And Pi2S91 is marked for amplifier molecule Primer pair, wherein forward primer be 5 '-AAGGTGAAGGAGATGATGGC-3 ', instead It is 5 '-GTGCACCCACTCATCAAGAC-3 ' to primer.

Another aspect, this application provides the method for detection recombinant nucleic acid fragment, which includes basis Foregoing recombinant nucleic acid fragment designs specifically primer, is entered with testing gene group as template Performing PCR reacts, and the step of analysing amplified product.Specifically, for example, the primer is for example front Described.Selectively, using the analysing amplified product of Sanger PCR sequencing PCR.

Specifically, in the method for the detection recombinant nucleic acid fragment that the application is provided, for expanding And detection SEQ ID NO:The primer combination of sequence shown in 1 is as follows：Amplimer, including forward direction Primer：5 '-CTCCATACCACATCGGTGACTCT-3 ', and reverse primer： 5’-CCTCATTGTGGGCTGTGTTCA-3’；Sequencing primer, including reverse primer： 5 '-TGCATTCGTCTAGCATGTGT-3 ', reverse primer：5’-GATCTTGTCCG GTGCTAGCA-3 ', forward primer：5 '-GTTGGCACACCACGTAAGCT-3 ', Forward primer：5 '-ATACCTCCAGGCTCTAGTCA-3 ', forward primer：5’-GAT GAGAGGGATTCCTACCT-3 ', reverse primer：5’-AGTCACCTCAGTTGTT AGTG-3 ', forward primer：5 '-AGATGGGGTGAACATTGAGGA-3 ', positive Primer：5 '-CAGGAGGTCTGGTTCTGGTT-3 ', and reverse primer：5’-CCTCATTG

TGGGCTGTGTTCA-3’.Methods described with testing sample genomic DNA as template, Enter performing PCR amplification using above-mentioned amplimer, then the expansion using above-mentioned sequencing primer to acquisition Volume increase thing is sequenced, if sequencing result and SEQ ID NO:1 sequence is consistent or complementary, then treat Contain SEQ ID NO in test sample product:Homologous recombination fragment shown in 2.

In addition, in the method for the detection genome recombination nucleic acid fragment that the application is provided, being used for Amplification and detection SEQ ID NO:The primer combination of sequence shown in 2 is as follows：Amplimer, including Forward primer：5 '-ACGGGCTCAAGTGAACGAGT-3 ', and reverse primer： 5’-CGTCTTGGTATCTCTCAAGGCAT-3’；Sequencing primer, including reverse primer： 5 '-GGGAGAAAGGGGATCGATCT-3 ', reverse primer：5’-ACGCCGAGAA GAAAACTCCA-3 ', forward primer：5 '-TGAACGCTGCCGGTGAGAGT-3 ', Forward primer：5 '-CCTGACCTACCACCAACACA-3 ', forward primer： 5 '-GTGCAGTCTTTGGCGAAGGT-3 ', and reverse primer：5’-CGTCTTGGTA

TCTCTCAAGGCAT-3’.Methods described with testing sample genomic DNA as template, Enter performing PCR amplification using above-mentioned amplimer, then the expansion using above-mentioned sequencing primer to acquisition Volume increase thing is sequenced, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, then treat Contain SEQ ID NO in test sample product:Homologous recombination fragment shown in 2.

Determined containing SEQ ID NO in testing sample by detection:1 and/or SEQ ID NO: The recombinant nucleic acid fragment of sequence shown in 2, you can determine in testing sample and contain resistant gene Recombinant nucleic acid fragment.

Additionally, present invention also provides the kit of detection recombinant nucleic acid fragment, which is included as front The primer that states.

Further, present invention also provides screening the rice plant containing recombinant nucleic acid fragment or Whether the method for seed, it are included containing as previously mentioned in the genome for detect rice plant to be measured Recombinant nucleic acid fragment the step of.In one embodiment, examined using foregoing primer Survey whether containing foregoing recombinant nucleic acid fragment in the genome of rice plant to be measured.Another In one embodiment, the method using foregoing detection recombinant nucleic acid fragment is to be measured to detect Whether foregoing recombinant nucleic acid fragment is contained in the genome of rice plant.In another enforcement In scheme, whether detected using foregoing kit in the genome of rice plant to be measured Containing foregoing recombinant nucleic acid fragment.

It yet still another aspect, this application provides containing the application by what methods described screening was obtained The rice plant of disclosed recombinant nucleic acid fragment or its seed.

What the application was provided contains blast resisting base based on the seed selection of full-length genome selection and use technology Method because organizing the rice plant of recombinant nucleic acid fragment, with quick, accurate, stable advantage. Only pass through the transformation of five generations, you can only by target gene group fragment importing acceptor material, and simultaneously Realize the reply of background.The acceptor material of the application improvement is for ' deep 95B ' is widely used Sterile line ' the matching used maintainer of deep 95A '.Using said method, can retain ' deeply On the premise of 95B ' cytoplasmic skeleton, only by rice blast resistance segments into cells karyogene In group, and other sites on nuclear genome are not changed.Further, by least one Generation hybridization, generation backcrossing can obtain stable containing rice blast resistance fragment ' deep 95A ', Again increasing substantially for cenospecies rice blast resistance is realized by combo.Meanwhile, the application is provided Genome recombination fragment be closely related with rice blast resistance, which can be applied to as Resistance resource The cultivation of his kind.

Description of the drawings

Fig. 1 is that CR01BC06 paddy rice RICE60K full-length genome is educated in the embodiment of the present application 1 Plant chip detection result；Wherein, the indicated square frame of abscissa numeral represents 12 dyes of paddy rice successively Colour solid, ordinate numeral are the physical location [with megabasse (Mb) as unit] on rice genome, Grey lines represent receptor parent, and ' deep 95B ' genotype, black lines represent donor parents ' China 3418B ' genotype, white line represent two parent genotypes unanimously i.e. nothing polymorphism section. At No. 6 chromosome black round dot of in figure, lines display block is the blast resistant gene for importing Group recombinant nucleic acid fragment RecCR01BC06.

Fig. 2A and Fig. 2 B is RecCR01BC06 upstream homologous recombination in the embodiment of the present application 2 Sequencing fragment comparison result；Asterisk shown in figure represents identical base, in figure in comparison result CR01BC06 is the new lines for obtaining, and T001 is that ' deep 95B ', R002 are confession to receptor parent ' magnificent 3418B ' is donor parents to body parent.

Fig. 3 is RecCR01BC06 downstream homologous recombination sequencing fragment in the embodiment of the present application 2 Comparison result.

Fig. 4 is the knot of RecCR01BC06 both sides homologous recombination fragment in the embodiment of the present application 2 Composition；Wherein, (A) is upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination piece Segment structure figure, top base are donor ' the SNP or InDel mark of magnificent 3418B ', lower section Base is acceptor ' the SNP or InDel mark of deep 95B '.Grey section be from ' deep 95B ' genomic segment, black section be from ' magnificent 3418B ' genomic segment, white Section is homologous recombination section, and abscissa is fragment length, with base pairs (bp) as unit.

Fig. 5 is qualification result in CR01BC06 rice blast resistance room in the embodiment of the present application 3； Shown in figure, blade is followed successively by：(A) rice blast susceptible variety Lijiang xintuanheigu；(B) original kind ' deep 95B '；(C) new lines CR01BC06 are improved；(D) rice blast disease-resistant variety paddy plum No. 4.

Specific embodiment

Defined below and method is provided in order to preferably to define the application and middle finger is put into practice in the application Lead those of ordinary skill in the art.Unless otherwise mentioned, term is according to association area ordinary skill people The common usage of member understands.

As used herein, " nucleotide sequence " includes to be related to the deoxyribose of single-stranded or double-stranded form Nucleotides or ribonucleotide polymer, and unless otherwise restriction, nucleotide sequence with 5 ' extremely 3 ' directions are write from left to right, including the known analog (example with natural nucleotide fundamental property Such as, peptide nucleic acid), the analog with naturally occurring nucleotides similar mode and single-stranded core Acid hybridization.

In some embodiments, the nucleotide sequence of the application can be changed, to enter Row conserved amino acid replacement.In certain embodiments, can be according to unifacial leaf codon preference Property is not changed the replacement of amino acid sequence to the nucleotide sequence of the application, for example, can use Codon of the coding with amino acid sequence replaced by the codon of monocotyledon preference, and does not change Become the amino acid sequence coded by the nucleotide sequence.

Specifically, the application is related to SEQ ID NO:1 or SEQ ID NO:2 is excellent further Change the nucleotide sequence of gained.The more details of the method are described in Murray etc. (1989) Nucleic Acids Res.17:477-498.Optimize nucleotide sequence to can be used to improve blast resisting base Because of the expression in paddy rice.

In some embodiments, the application further relates to SEQ ID NO:1 or SEQ ID NO:2 The variant of shown sequence.In general, the variant of specific nucleotide sequence will be with the specific nucleosides Acid sequence have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%th, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% Or 99.9% or higher sequence iden, or more complementary series.Such variant sequence thereof Interpolation, disappearance or replacement including one or more nucleic acid, corresponding such that it is able to cause The interpolation of amino acid residue, remove or replace.By alignment programs known in the art Sequence iden is determined including hybridization technique.The nucleotide sequence variants of embodiment and the application The difference of sequence may be as few as 1-15 nucleotides, as little as 1-10 (such as 6-10), As little as 5, as little as 4,3,2 or even 1 nucleotides.

The application is further related to comprising SEQ ID NO:1 or SEQ ID NO:Special in sequence shown in 2 The fragment of anchor point or its variant or its complementary series, for example, comprising SEQ ID NO:Shown in 1 The sequence or its fragment of the 790-2130 position nucleotides of sequence or its variant or its complementary series, or Person includes SEQ ID NO:The sequence or its fragment of the 770-1283 position nucleotides of sequence shown in 2 Or its variant or its complementary series.According to the fragment comprising above-mentioned specific site, being capable of specificity Identify corresponding SEQ ID NO:1 or SEQ ID NO:Sequence shown in 2.Further, By identifying containing SEQ ID NO:1 or SEQ ID NO:The recombinant nucleic acid of sequence shown in 2 Fragment, you can determine comprising the recombinant nucleic acid fragment containing resistant gene in testing sample.

As used herein, " paddy rice " is any rice plant including can be all with rice breeding Plant variety.As used herein, " plant " or " plant ", including whole plant, plant cell, Plant cell tissue cultures that plant organ, plant protoplast, plant therefrom can regenerate, Complete plant cell in plant callus, vegetation bed and plant or plant part, the plant Part for example embryo, pollen, ovule, seed, leaf, flower, branch, fruit, cane, root, the tip of a root, Flower pesticide etc..

Go for any rice varieties for needing seed selection in the present processes.That is, (i.e. Comprehensive Traits are preferable, it is contemplated that have and send out for the improved seeds that certain beneficial traits can be lacked by any The kind of exhibition future) it is used as recurrent parent.With another with beneficial traits lacking in this receptor Kind is used as donor parents, and the beneficial traits for being provided are preferably dominant Dominant gene. In the embodiment of the application, using paddy rice, ' deep 95B ' is adopted as recurrent parent ' magnificent 3418B ' is used as donor to be proven to have the paddy rice of good rice blast resistance.

In the selection of restructuring plant provided herein, using molecular labeling to restructuring Plant carries out foreground selection.The reliability of foreground selection is depended primarily between mark and target gene Chain tightness degree, is the accuracy rate for improving selection, general while with adjacent two in both sides Mark is tracked selecting to target gene.

In the embodiment of the application, the foreground selection mark of employing includes that favorable selection is marked With negative itemsets mark, wherein, favorable selection mark be away from target gene group fragment (containing anti-rice Seasonal febrile diseases gene) screening in the range of upstream and downstream 50kb (in paddy rice its genetic distance be 0.2cM) Polymorphism molecular labeling.Negative itemsets mark be away from target gene group fragment upstream and downstream The polymorphism molecular labeling screened in the range of 500kb (its genetic distance is 2cM in paddy rice).? In specific embodiment, the positive foreground selection mark of optimized Select to use is and target gene Group fragment close linkage mark Pi31, negative itemsets mark be positioned at target fragment upstream about The mark Pi2ID02 of 230kb, and the mark Pi2S91 positioned at target fragment downstream about 270kb.

In the embodiment of the application, homologous recombination is carried out using above-mentioned foreground selection mark During detection, the criterion of side or unilateral homologous recombination is Pi31 detection and ' magnificent 3418B ' Identical banding pattern, and Pi2ID02 or Pi2S91 detection and ' the identical banding pattern of deep 95B '；Both sides or The criterion of bilateral homologous recombination be Pi31 detection with ' the identical banding pattern of magnificent 3418B ', and Pi2ID02 and Pi2S91 detection and ' the identical banding pattern of deep 95B '.

In this application, it is possible to use any available chip carries out provided herein Breeding method in Foreground selection.In preferred embodiments, the applicant can be adopted Paddy rice full-length genome breeding chip disclosed in Chinese patent application CN102747138A RICE6K, or the paddy rice full genome disclosed in PCT international application WO/2014/121419 Group breeding chip RICE60K.Full content in this two parts of application documents is integrally incorporated to be made herein It is reference.

Following examples are merely to illustrate and the purpose of unrestricted the application scope.If not referring in particular to Bright, embodiment is all according to conventional laboratory conditions, such as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J&Russell DW,Molecular cloning:a laboratory manual, 2001) condition, or according to manufacturer's specification suggestion.

Rice plant material information used in this application all can be found in rice in China kind and its Pedigree database (http://www.ricedata.cn/variety/index.htm).

The rice genome physical location being previously mentioned in the application is all with reference to the fine genome of paddy rice Japan MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).

Embodiment 1Seed selection imports the restructuring plant of blast resistant gene group fragment

Material used in the present embodiment is paddy rice ' deep 95B ' and paddy rice ' magnificent 3418B '.

' magnificent 3418B ' is with good rice blast resistance, and speculates possibly No. 6 for paddy rice Rice blast resistance of the gene cluster region that Pi2, Pi9 and Pigm of chromosome is located to the material Serve key effect.

In the Breeding Process of restructuring plant, prospect choosing is carried out to plant of recombinating using molecular labeling Select, the foreground selection molecular labeling to being adopted is screened.With reference to the fine gene of paddy rice Japan Group MSU/TIGR annotates the 6.1st edition, the 6th chromosome 9 of download, and 559,000 to 10,990,000 DNA sequence dna.The SSR site in above-mentioned sequence is scanned using SSRLocator software. Using 3.0 software of Primer Premier to the SSR site design primer for searching out, design altogether and draw Thing 162 pairs.By the method for PCR, above-mentioned primer pair is screened in ' magnificent 3418B ' and ' depth Polymorphism in 95B ', finally picks out with polymorphism, amplification efficiency in two parts of materials High foreground selection molecular labeling, is favorable selection mark Pi31 and negative itemsets mark respectively Pi2ID02、Pi2S91.The concrete primer information for above-mentioned molecular labeling being expanded for PCR is shown in Table 1.

1 foreground selection molecular labeling primer information of table

By paddy rice, ' genomic fragment that forementioned gene cluster is located in magnificent 3418B ' imports to paddy rice ' in deep 95B ', detailed process is as follows：

So that ' as recurrent parent, ' magnificent 3418B ' is hybridized deep 95B ' for donor parents, will ' deep 95B ' is returned obtained cenospecies, obtains BC with recurrent parent₁F₁Seed, Carried out using favorable selection mark Pi31 and negative itemsets mark Pi2ID02, Pi2S91 after nursery Restructuring Single-plant selection, filters out 9 individual plants in target gene group fragment side homologous recombination, That is Pi31 detection with ' the identical banding pattern of magnificent 3418B ', and Pi2ID02 or Pi2S91 detection with ' deep The identical banding pattern of 95B ', and utilize paddy rice full-length genome breeding chip RICE6K (CN102747138A) carries out Foreground selection (Yu etc., Plant Biotechnology to which Journal.2014,12:28-37).

Comparable chip result in the 9 unilateral homologous recombination individual plants for filtering out, selects background to return Multiple best restructuring individual plant (this generation background recovery value is more than 75%) so as to ' deep with recurrent parent 95B ' is returned again, obtains BC₂F₁Seed, marks Pi31 using favorable selection after nursery Which is detected, select the restructuring individual plant containing target gene group fragment, i.e. Pi31 detection with ' the identical banding pattern of magnificent 3418B ', is entered to which using paddy rice full-length genome breeding chip RICE6K Row Foreground selection.

Select background to reply preferable individual plant (this generation background recovery value is more than 87.5%) so as to ' deep 95B ' is returned again recurrent parent, obtains BC₃F₁Seed, utilizes after nursery Favorable selection mark Pi31 and negative indicia Pi2ID02, Pi2S91 carry out mesh to the seed for harvesting The screening of mark genomic fragment opposite side homologous recombination fragment, obtains 5 in target fragment both sides The detection of the individual plant of restructuring, i.e. Pi31 and ' the identical banding pattern of magnificent 3418B ', and Pi2ID02 and Pi2S91 Detection and ' the identical banding pattern of deep 95B '.

Using paddy rice full-length genome breeding chip RICE60K (WO/2014/121419) to above-mentioned 5 Individual bilateral exchanges individual plant and carries out background and target fragment selection (Chen etc., Molecular Plant. 2014,7:541-553), it is less that importing target fragment is screened, and the target list that background is replied One (this generation background recovery value is more than 93.75%) of strain.

By the individual plant selfing that chooses once, BC is obtained₃F₂, marked using favorable selection after nursery Pi31 detected to which, selects the individual plant containing target gene group fragment, i.e. Pi31 detection with ' the identical banding pattern of magnificent 3418B ', is entered to which using paddy rice full-length genome breeding chip RICE60K Row Foreground selection.

Final acquisition target fragment homozygosis, and background replys the strain of (background recovery value is more than 99%) It is one, is named as CR01BC06.Chip detection result is shown in Fig. 1.

Embodiment 2The determination of homologous recombination fragment after importing rice blast resistance gene group fragment

In order to determine the rice blast resistance gene group clip size of importing, to ' deep 95B ' is imported The homozygosis individual plant of fragment has carried out the sequencing of target gene group fragment both sides homologous recombination fragment.Will Blast resistant gene group recombinant nucleic acid fragment contained by CR01BC06 is named as RecCR01BC06.

Primarily determined that by paddy rice full-length genome breeding chip RICE60K testing result, RecCR01BC06 is located at two SNP marker R0610372586AG and R0610424502CT Between.

Meanwhile, using Miseq sequencing technologies to ' deep 95B ', ' magnificent 3418B ' and CR01BC06 Three samples carry out genome sequencing.Using TruSeq Nano DNA LT Kit (illumina) Kit carries out library foundation, using Library Quantification Kit Universal (KAPA Biosystems) kit carries out quantitation, using MiSeq V2Reagent Kit (illumina) kit carries out sequencing reaction.Entered using the desk-top sequenator of Miseq (illumina) Row detection.Concrete steps and method are referring to each kit and sequenator operation instructions.

According to aforementioned SNP chip and Miseq sequencing result, RecCR01BC06 upstream is same Source recombinant fragment Primary Location is interval in 10375077bp to the 10379192bp of the 6th chromosome, It is interval that downstream homologous recombination fragment is positioned at 10418697bp to 10420932bp.

On this basis, the 6.1st edition is annotated with reference to the fine genome MSU/TIGR of paddy rice Japan, Download respective segments DNA sequence dna.Expanded using 5.0 Software for Design of Primer Premier and survey Sequence primer, design requirement is for long 22nt of primer or so, G/C content 40-60% and without mispairing.

With receptor parent, ' ' magnificent 3418B ' is as control, right for deep 95B ' and donor parents RecCR01BC06 upstream and downstream homologous recombination fragment separately designs amplimer, using LA Taq (TAKARA) is expanded, and finds optimal amplification condition using two-step method or three-step approach, really Protect amplified production single bright band is shown as in agarose gel electrophoresis detection.Wherein determine Upstream homologous recombination fragment amplification primer reaction condition be：94℃3min；98 DEG C of 10sec, 68 DEG C of 5min, 35 circulations；72℃10min；25℃1min.Downstream homologous recombination fragment Amplimer reaction condition is：94℃3min；98 DEG C of 10sec, 68 DEG C of 5min, 35 Circulation；72℃10min；25℃1min.Thus, finally filter out two pairs of amplimers to divide Not Yong Yu upstream and downstream homologous recombination fragment amplification.

In addition, with amplified production as template, being sequenced using Sanger PCR sequencing PCR, according to reality Border is sequenced effect, finally filters out 9 and 6 sequencing primers are respectively used to upstream and downstream together The sequencing of source recombinant fragment.Specific amplimer and sequencing primer sequence are shown in Table 2, sequencing knot Fruit sees Fig. 2 and Fig. 3.

RecCR01BC06 upstream homologous recombination sequencing fragment length is 4133bp (SEQ ID NO: 1).1-790bp is that ' genomic segment of deep 95B ', with donor ' magnificent 3418B ' ratio for acceptor Relatively, there are 3 SNP.This 1339bp section of 791-2129bp is homologous recombination section. 2130-4133bp is that ' magnificent 3418B ' genomic fragment, with ' deep 95B ' compares donor, deposits In 4 SNP.

RecCR01BC06 downstream homologous recombination sequencing fragment length is 2244bp (SEQ ID NO: 2).1-770bp is that deep 95B ' compares donor ' genomic segment of magnificent 3418B ', with ', There are 11 SNP.This 512bp section of 771-1282bp is homologous recombination section. 1283-2244bp is that ' genomic segment of deep 95B ', with donor ' magnificent 3418B ' ratio for acceptor Relatively, there are 31 SNP, 3 Indel.

Fig. 4 is the structure chart of RecCR01BC06 both sides homologous recombination fragment.Wherein, (A) For upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment structure figure.Top alkali Base is that ' the SNP or InDel mark of magnificent 3418B ', lower section base is acceptor ' deep 95B ' to donor SNP or InDel mark.Grey section be from ' deep 95B ' genomic segment, black Color section be from ' magnificent 3418B ' genomic segment, white section be homologous recombination section. Abscissa is fragment length, with base pairs (bp) as unit.

2 blast resistant gene group recombinant nucleic acid fragment amplification of table and sequencing primer information

Embodiment 3' deep 95B ' imports the Resistance Identification after blast resistant gene group fragment

In order to identify resistance effect, new lines CR01BC06 to the application seed selection, samsara parent This ' deep 95B ', rice blast disease-resistant variety paddy plum No. 4 (as positive control), and rice blast Susceptible variety Lijiang xintuanheigu (as negative control) carries out indoor plantation, is cultivated to 3-4 Adopt after the leaf phase and identified with the following method：

Bacterial strain select be 2013, enshi bacterial strain 13-21K02,13-21K14 and 13-2104, Jinggangshan County, Jiangxi Province bacterial strain 13-5121, Luzhou, Sichuan bacterial strain 13-6102, Fujian bacterial strain 13-8101,13-8108, totally 7 plants of rice blast bacterial strains are used as inoculating strain..Bacterial strain adopts Chinese sorghum The sorghum grain of preservation is taken out to potato dextrose medium using front by -20 DEG C of preservations of grain method (PDA) flat board activation (PDA：Peeled potatoes 200g, glucose 20g, agar powder 15g, Distilled water is settled to 1L), 28 DEG C of illumination cultivation take diameter 5mm fresh mycelia block after 5 days turns (sorghum grain 500g addition 1.5L distilled water, elimination after boiling to boiling are connected in sorghum grain culture medium Liquid, sorghum grain is pulled out loading 250ml triangular flask, 100ml/ bottle, moist heat sterilization 20 minutes), 10 pieces/bottle, connect bacterium and daily shake sorghum grain scattered after 2 days, and 28 DEG C of dark culturing cover with height to mycelia Fine strain of millet grain.Then sorghum grain is spread out on sterile gauze, aseptic damp gauze is covered, at 25 DEG C, RH >=95%, cultivates under 12h illumination condition 4-5 days and produces to a large amount of spores, (contained with sterilized water 0.02% polysorbas20) lower spore is washed, wait spore amount combined inoculation bacterial strain, adjustment concentration to 5 × 10⁵ Individual/ml.

With mixing conidial suspension spray inoculation CR01BC06, ' deep 95B ', Gu Mei 4 Number and Lijiang xintuanheigu, be inoculated with three repetition.Transparent cover on inoculation back cover, 28 DEG C of dark 24h is cultivated, then 16h illumination cultivation was investigated after 5 days.

Investigation standard is 0 grade (high anti-, HR)：Without symptom；1 grade (anti-, R)：Very little brown Color scab；2 grades (in resist, MR)：Diameter is about the brown scab of 1mm；3 grades (MS, in Sense)：Directly it is about the band circle scab of 2-3mm, central canescence, edge brown；4 grades (sense, S)：It is about the oval scab of 1-3cm, central canescence, edge brown；5 grades (high sense, HS)：Long and wide big ellipse scab, scab fusion are in flakes, withered to blade.Wherein 0-2 Level is disease-resistant, and 3-5 level is susceptible.Inoculation the results are shown in Table 3 and Fig. 5.

Table 3 is inoculated with the Resistant expression after rice blast fungus

Although, above with a general description of the specific embodiments the application has been made in detail Most description, but on the basis of the application, it can be made some modifications or improvements, this is to this It is obvious for skilled person.Therefore, on the basis without departing from the application spirit Upper these modifications or improvements, belong to this application claims scope.

Claims (10)

1. recombinant nucleic acid fragment, which is selected from：

I) include SEQ ID NO:The sequence of the nucleotides of sequence 790-2130 position shown in 1 or its piece Section or its variant or its complementary series；

Ii) include SEQ ID NO:The sequence of sequence shown in 1 or its fragment or its variant or which is mutual Complementary series；

Iii) include SEQ ID NO:The sequence of the nucleotides of sequence 770-1283 position shown in 2 or its piece Section or its variant or its complementary series；

Iv) include SEQ ID NO:The sequence of sequence shown in 2 or its fragment or its variant or which is mutual Complementary series；And

The combination of above fragment.

2. test right requires the primer of fragment described in 1, and wherein described primer is selected from：

(I) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-790 position shown in 1 Forward primer and specific recognition SEQ ID NO:The nucleotides of sequence 2130-4133 position shown in 1 The reverse primer of sequence；

(II) the combining of first group of primer pair and second group of primer pair below, which includes

(a) first group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of the sequence of 1-790 position nucleotides and specific recognition SEQ ID NO:Sequence shown in 1 Arrange the reverse primer of the sequence of 791-2129 position nucleotides；With

(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of the sequence of 791-2129 position nucleotides and specific recognition SEQ ID NO:1 institute Show the reverse primer of the sequence of sequence 2130-4133 position nucleotides；

(III) specific recognition includes SEQ ID NO:The nucleotides of sequence 790-791 position shown in 1 The forward primer of sequence and specific recognition include SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 2129-2130 position nucleotides；

(IV) specific recognition includes SEQ ID NO:The nucleotides of sequence 790-791 position shown in 1 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 2130-4133 shown in 1 The reverse primer of the sequence of position nucleotides；

(V) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-790 position shown in 1 Forward primer and specific recognition include SEQ ID NO:Sequence 2129-2130 position shown in 1 The reverse primer of the sequence of nucleotides；And/or optionally,

(VI) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-770 position shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleotides of sequence 1283-2244 position shown in 2 The reverse primer of sequence；

(VII) the combining of the 3rd group of primer pair and the 4th group of primer pair below, which includes

(c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of the sequence of 1-770 position nucleotides and specific recognition SEQ ID NO:Sequence shown in 2 Arrange the reverse primer of the sequence of 771-1282 position nucleotides；With

(d) the 4th group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of the sequence of 771-1282 position nucleotides and specific recognition SEQ ID NO:2 institutes Show the reverse primer of the sequence of sequence 1283-2244 position nucleotides；

(VIII) specific recognition includes SEQ ID NO:The nucleosides of sequence 770-771 position shown in 2 The forward primer of the sequence of acid and specific recognition include SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 1282-1283 position nucleotides；

(IX) specific recognition includes SEQ ID NO:The nucleotides of sequence 770-771 position shown in 2 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 1283-2244 shown in 2 The reverse primer of the sequence of position nucleotides；

(X) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-770 position shown in 2 Forward primer and specific recognition include SEQ ID NO:Sequence 1282-1283 position shown in 2 The reverse primer of the sequence of nucleotides.

3. test right requires the primer of fragment described in 1, and wherein described primer is selected from：

(I) SEQ ID NO is expanded:The primer pair of sequence shown in 1

5 '-CTCCATACCACATCGGTGACTCT-3 ',

5’-CCTCATTGTGGGCTGTGTTCA-3’；And

(II) SEQ ID NO is sequenced:The primer of sequence shown in 1

5 '-TGCATTCGTCTAGCATGTGT-3 ',

5 '-GATCTTGTCCGGTGCTAGCA-3 ',

5 '-GTTGGCACACCACGTAAGCT-3 ',

5 '-ATACCTCCAGGCTCTAGTCA-3 ',

5 '-GATGAGAGGGATTCCTACCT-3 ',

5 '-AGTCACCTCAGTTGTTAGTG-3 ',

5 '-AGATGGGGTGAACATTGAGGA-3 ',

5 '-CAGGAGGTCTGGTTCTGGTT-3 ',

5’-CCTCATTGTGGGCTGTGTTCA-3’；And/or optionally,

(III) SEQ ID NO is expanded:The primer pair of sequence shown in 2

5 '-ACGGGCTCAAGTGAACGAGT-3 ',

5’-CGTCTTGGTATCTCTCAAGGCAT-3’；And

(IV) SEQ ID NO is sequenced:The primer of sequence shown in 2

5 '-GGGAGAAAGGGGATCGATCT-3 ',

5 '-ACGCCGAGAAGAAAACTCCA-3 ',

5 '-TGAACGCTGCCGGTGAGAGT-3 ',

5 '-CCTGACCTACCACCAACACA-3 ',

5 '-GTGCAGTCTTTGGCGAAGGT-3 ',

5’-CGTCTTGGTATCTCTCAAGGCAT-3’.

4. the method that seed selection contains the rice plant of the recombinant nucleic acid fragment described in claim 1, Which includes, using the paddy rice recipient plant parent without genes of interest group fragment as recurrent parent, to incite somebody to action Which is hybridized with the paddy rice donor plant containing genes of interest group fragment, then will be obtained Cenospecies is returned with recurrent parent, then the step of obtained backcrossing kind is carried out selfing, Wherein foreground selection and Foreground selection are carried out to plant of recombinating using molecular labeling.

5. method as claimed in claim 4, is wherein used for the molecular labeling of the foreground selection One or more in Pi31, Pi2ID02 and Pi2S91；And/or

Optionally, the Foreground selection is carried out using paddy rice full-length genome breeding chip.

6. the method as described in claim 4 or 5, wherein described recombinant nucleic acid fragment contains anti- Rice blast ospc gene, and the method comprising the steps of：

1) recurrent parent is hybridized with donor plant, by obtained cenospecies and samsara parent Originally it is returned, first backcross generation is obtained, using favorable selection mark Pi31 and negative itemsets mark Pi2ID02, Pi2S91 carry out the unilateral homologous recombination fragment sieve of blast resistant gene group fragment to which Choosing, and Foreground selection is carried out to which using paddy rice full-length genome breeding chip；

2) select background to reply preferably restructuring individual plant to be returned with recurrent parent again, obtain Second backcross generation, is detected to which using favorable selection mark Pi31, selects to contain blast resisting The restructuring individual plant of genomic fragment, is then carried on the back to which using paddy rice full-length genome breeding chip Scape is selected；

3) select the restructuring individual plant that background is replied to be returned with recurrent parent again, obtain Third backcross generation, using favorable selection mark Pi31 and negative indicia Pi2ID02, Pi2S91 to which The opposite side homologous recombination fragment screening of blast resistant gene group fragment is carried out, and complete using paddy rice Genomic breeding chip carries out Foreground selection to which；And

4) select introgressed segment little, and the restructuring individual plant that background is replied, by the restructuring list that chooses Strain selfing once, obtains selfed seed, which is detected using favorable selection mark Pi31, and Foreground selection is carried out to which using paddy rice full-length genome breeding chip, finally obtain containing the anti-rice of homozygosis The rice plant that seasonal febrile diseases genome recombination nucleic acid fragment and background are replied.

7. the method as any one of claim 4 to 6, wherein using molecular labeling pair The amplimer that restructuring plant carries out adopting during foreground selection is as follows：

The primer pair of amplifier molecule mark Pi31, which includes：

Forward primer：5 '-ATCCAAACCCGTTGTTGCAC-3 ',

Reverse primer：5’-CGGCAATTGCCACGATGATA-3’；

The primer pair of amplifier molecule mark Pi2ID02, which includes：

Forward primer：5 '-AAGTAATAATCCCCGCTGTTGT-3 ',

Reverse primer：5’-TTTCAAGCGAAGGAGGATGT-3’；And

The primer pair of amplifier molecule mark Pi2S91, which includes：

Forward primer：5 '-AAGGTGAAGGAGATGATGGC-3 ',

Reverse primer：5’-GTGCACCCACTCATCAAGAC-3’.

8. the method that test right requires the recombinant nucleic acid fragment described in 1, which includes to adopt right The primer described in 2 or 3 is required, performing PCR reaction is entered as template with testing gene group, and is analyzed The step of PCR primer.

9. test right requires the kit of the recombinant nucleic acid fragment described in 1, and which includes that right will Seek the primer described in 2 or 3.

10. the rice plant containing the recombinant nucleic acid fragment described in claim 1 or seed are screened Method, whether which is included in the genome for detect rice plant to be measured or seed containing has the right will The step of seeking the recombinant nucleic acid fragment described in 1；

Preferably, using the primer described in Claims 2 or 3, or claim 8 is adopted Described method, or detected using the kit described in claim 9.