CN106480056A - Recombinant nucleic acid fragment RecCR01BC02 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, using molecular marker, foreground selection and Foreground selection are carried out to restructuring plant, obtain the rice plant containing recombinant nucleic acid fragment.

Description

Recombinant nucleic acid fragment RecCR01BC02 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 selection-breeding contains the rice plant of recombinant nucleic acid fragment, and thus And the recombinant nucleic acid fragment obtaining 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 marker is for realizing directly selecting offer to genotype May.In recent years, have started to apply molecular marker-assisted selection method to improve individual target Character, being capable of significant shortening the breeding cycle.

Rice blast is one of disease of Oryza sativa L. most serious, the Oryza sativa L. that the whole world is caused by rice blast every year Production loss accounts for 11%～30%, and the research of therefore rice blast and its resistance is particularly important. Progressively go deep into study to rice blast, many rice blast resistant gene DNA fragmentation phases Continue and be positioned and clone.Wherein, the Pi2 interval of Oryza sativa L. the 6th chromosome is positioned and clones A lot of rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm, Pi50, this interval comprises 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, it is selected from：I) comprise SEQ ID NO: The sequence of the nucleotide of sequence 775-917 position shown in 1 or its fragment or its variant or its complementary series； Ii) comprise SEQ ID NO:The sequence of sequence shown in 1 or its fragment or its variant or its complementary sequence Row；Iii) comprise SEQ ID NO:The sequence of the nucleotide of sequence 941-1254 position shown in 2 or its piece Section or its variant or its complementary series；Or iv) comprise SEQ ID NO:The sequence of sequence shown in 2 Or its fragment or its variant or its complementary series；And the combination of above fragment.

Additionally, this application provides detecting the primer of described recombinant nucleic acid fragment, it is selected from： (I) specific recognition SEQ ID NO:The forward direction of the sequence of the nucleotide of sequence 1-775 position shown in 1 Primer and specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 917-1517 position shown in 1 Reverse primer；(II) the combining of first group of primer pair and second group of primer pair below, it comprises (a) first group of primer pair：Specific recognition SEQ ID NO:The core of sequence 1-775 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 776-916 position nucleotide；(b) second group of primer pair：Specificity Identification SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 776-916 position shown in 1 and Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 917-1517 position shown in 1 anti- To primer；(III) specific recognition comprises SEQ ID NO:The core of sequence 775-776 position shown in 1 The forward primer of the sequence of thuja acid and specific recognition comprise SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 916-917 position nucleotide；(IV) specific recognition comprises SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 775-776 position shown in 1 and specific recognition SEQ ID NO:The reverse primer of the sequence of the nucleotide of sequence 917-1517 position shown in 1；(V) Specific recognition SEQ ID NO:The forward direction of the sequence of the nucleotide of sequence 1-775 position shown in 1 is drawn Thing and specific recognition comprise SEQ ID NO:The sequence of the nucleotide of sequence 916-917 position shown in 1 The reverse primer of row；And/or optionally, (VI) specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of sequence of row 1-941 position nucleotide and specific recognition SEQ ID NO:Shown in 2 The reverse primer of the sequence of sequence 1254-2103 position nucleotide；(VII) the 3rd group of primer below To with the combining of the 4th group of primer pair, it comprises (c) the 3rd group of primer pair：Specific recognition SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 1-941 position shown in 2 and specific recognition SEQ ID NO:The reverse primer of the sequence of the nucleotide of sequence 942-1253 position shown in 2；(d) 4th group of primer pair：Specific recognition SEQ ID NO:The core of sequence 942-1253 position shown in 2 The forward primer of the sequence of thuja acid and specific recognition SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 1254-2103 position nucleotide；(VIII) specific recognition comprises SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 941-942 position shown in 2 and specificity are known Do not comprise SEQ ID NO:Reversely the drawing of the sequence of the nucleotide of sequence 1253-1254 position shown in 2 Thing；(IX) specific recognition comprises SEQ ID NO:The nucleotide of sequence 941-942 position shown in 2 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 1254-2103 shown in 2 The reverse primer of the sequence of position nucleotide；(X) specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of sequence of 1-941 position nucleotide and specific recognition comprise SEQ ID NO:2 The reverse primer of the sequence of shown sequence 1253-1254 position nucleotide.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-ACCTGAGCGATGGTGGTCTTGA-3 ', and 5 '-CGAGTCGTTC TTGGCGTACTTGA-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 1 For, for example, 5 '-AGTTGCTGATACCGAGAGGT-3 ', 5 '-GGCGCTAATCA GGGTACAGT-3 ', and 5 '-TGTGCCACCATGCATCTCGA-3 '.

In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2 For, for example, 5 '-TCAGCTTGAAAGAGCGAAGTGGT-3 ', and 5 '-ACTATTTG CGTCCTACTCCCTCCG-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 2 Primer is, for example, 5 '-CATGTCCCTCAACGGTTAGA-3 ', 5 '-CATAACA CGAACGGAAACCA-3 ', 5 '-GTGCATTGTTCCACAGGAGA-3 ', and 5’-GAAGAGAGGGACAAAGGAGA-3’.

On the other hand, this application provides selection-breeding contains the side of the rice plant of recombinant nucleic acid fragment Method, it includes using the Oryza sativa L. recipient plant parent without genes of interest group fragment as recurrent parent, It is hybridized with the Oryza sativa L. donor plant containing genes of interest fragment, then will be obtained Cenospecies are returned with recurrent parent, then the step that obtained backcrossing kind is carried out selfing, Wherein using molecular marker, foreground selection and the back of the body are carried out respectively to obtained backcrossing kind and selfed seed Scape selects.For example, described recombinant nucleic acid fragment is as previously mentioned.

In the above-mentioned methods, the molecular marker for described foreground selection is selected from Pi31, Pi2ID02 One or more of with Pi2S91；And/or carry out institute using Oryza sativa L. full-length genome breeding chip State Foreground selection.

In one embodiment, the selection-breeding that the application provides contains blast resisting recombinant nucleic acid fragment Rice plant method, it 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, utilize Favorable selection labelling Pi31 and negative itemsets labelling Pi2ID02, Pi2S91 carry out anti-rice blast to it The unilateral homologous recombination fragment screening of ospc gene group fragment, and utilize Oryza sativa L. full-length genome breeding core Piece, such as RICE6K, carry out Foreground selection to it；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 Secondary, using favorable selection labelling Pi31, it is detected, select to contain blast resistant gene The restructuring individual plant of group fragment, then using Oryza sativa L. full-length genome breeding chip, such as RICE6K, Foreground selection is carried out to it；3) select restructuring individual plant (this generation background recovery value that background is replied More than 87.5%) it is returned again with recurrent parent, obtain third backcross generation, using positive choosing Select labelling Pi31 and negative indicia Pi2ID02, Pi2S91 and blast resistant gene pack is carried out to it The opposite side homologous recombination fragment screening of section, and utilize Oryza sativa L. full-length genome breeding chip, for example RICE60K, carries out Foreground selection to it；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 chosen individual plant selfing once, Obtain selfed seed, using favorable selection labelling Pi31, it is detected, and utilize the full base of Oryza sativa L. Because organizing breeding chip, such as RICE60K, Foreground selection is carried out to it, final acquisition contains anti-rice The Oryza sativa L. of pestilence genome recombination fragment homozygosis and background reply (background recovery value is more than 99%) is planted Strain.

In another embodiment, using molecular marker, restructuring plant is carried out adopting during foreground selection Amplimer, including：For the primer pair of amplifier molecule labelling Pi31, wherein forward direction is drawn Thing is 5 '-ATCCAAACCCGTTGTTGCAC-3 ', and reverse primer is 5 '-CGGCAATT

GCCACGATGATA-3’；For the primer pair of amplifier molecule labelling Pi2ID02, its Middle forward primer is 5 '-AAGTAATAATCCCCGCTGTTGT-3 ', and reverse primer is 5’-TTTCAAGCGAAGGAGGATGT-3’；And it is used for amplifier molecule labelling Pi2S91 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, it includes basis Foregoing recombinant nucleic acid fragment design specifically primer, is entered with testing gene group for template Performing PCR reacts, and the step of analysing amplified product.Specifically, for example, described primer is for example front Described.Selectively, using the analysing amplified product of Sanger sequencing.

Specifically, in the method for detection recombinant nucleic acid fragment that the application provides, for expanding And detection SEQ ID NO:The primer combination of sequence shown in 1 is as follows：Amplimer, including positive Primer：5 '-ACCTGAGCGATGGTGGTCTTGA-3 ', and reverse primer： 5’-CGAGTCGTTCTTGGCGTACTTGA-3’；Sequencing primer, including forward primer： 5 '-AGTTGCTGATACCGAGAGGT-3 ', forward primer：5’-GGCGCTAATCA GGGTACAGT-3 ', and forward primer：5’-TGTGCCACCATGCATCTCGA-3’. Methods described, with testing sample genomic DNA as template, enters performing PCR using above-mentioned amplimer Amplification, is then sequenced to the amplified production obtaining using above-mentioned sequencing primer, if sequencing knot Fruit and SEQ ID NO:1 sequence is consistent or complementary, then contain SEQ ID NO in testing sample:2 Shown homologous recombination fragment.

In addition, the application provide detection recombinant nucleic acid fragment method in, for amplification and Detection SEQ ID NO:The primer combination of sequence shown in 2 is as follows：Amplimer, draws including forward direction Thing：5 '-TCAGCTTGAAAGAGCGAAGTGGT-3 ', and reverse primer： 5’-ACTATTTGCGTCCTACTCCCTCCG-3’；Sequencing primer, including forward primer： 5 '-CATGTCCCTCAACGGTTAGA-3 ', forward primer：5’-CATAACACGAA CGGAAACCA-3 ', forward primer：5 '-GTGCATTGTTCCACAGGAGA-3 ', And forward primer：5’-GAAGAGAGGGACAAAGGAGA-3’.Methods described is to treat Test sample product genomic DNA is template, enters performing PCR amplification using above-mentioned amplimer, then Using above-mentioned sequencing primer, the amplified production obtaining is sequenced, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, then contain SEQ ID NO in testing sample:Homology weight shown in 2 Group fragment.

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

Additionally, present invention also provides the test kit of detection recombinant nucleic acid fragment, it includes as front The primer stated.

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

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

What the application provided contains blast resisting base based on the selection-breeding of full-length genome selection and use technology Method because organizing the rice plant of recombinant nucleic acid fragment, has quick, accurate, stable advantage. Only pass through the transformation of five generations, you can only target gene group fragment is imported acceptor material, and simultaneously Realize the reply of background.The acceptor material of the application improvement is that ' 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 do not change other sites on nuclear genome.Further, by least one Generation hybridization, generation backcrossing can obtain stable containing rice blast resistance fragment ' deep 95A ', Again increasing substantially of cenospecies rice blast resistance is realized by combo.Meanwhile, the application provides Genome recombination nucleic acid fragment be closely related with rice blast resistance, can apply as Resistance resource Cultivation in other kinds.

Brief description

Fig. 1 is that in the embodiment of the present application 1, CR01BC02 Oryza sativa L. RICE60K full-length genome is educated Plant chip detection result；Wherein, square frame indicated by abscissa numeral represents 12 dyes of Oryza sativa L. successively Colour solid, vertical coordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome, Lycoperdon polymorphum Vitt lines represent receptor parent, and ' deep 95B ' genotype, black lines represent donor parents ' China 3418B ' genotype, white line represents the consistent i.e. no polymorphism section of two parent genotypes. At No. 6 chromosome black round dot of in figure, lines display block is the blast resistant gene importing Group recombinant nucleic acid fragment RecCR01BC02.

Fig. 2 is RecCR01BC02 upstream homologous recombination sequencing fragment in the embodiment of the present application 2 Comparison result；Asterisk shown in figure represents identical base, in figure CR01BC02 in comparison result For the new lines obtaining, T001 is that ' deep 95B ', R002 are donor parents ' China to receptor parent 3418B ' is donor parents.

Fig. 3 is RecCR01BC02 downstream homologous recombination sequencing fragment in the embodiment of the present application 2 Comparison result.

Fig. 4 is the knot of RecCR01BC02 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 is donor ' SNP the or InDel labelling of magnificent 3418B ', lower section Base is receptor ' SNP the or InDel labelling of deep 95B '.Lycoperdon polymorphum Vitt section is from ' deep 95B ' genomic segment, black section is 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 CR01BC02 rice blast resistance interior qualification result 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 CR01BC02 are improved；(D) rice blast disease-resistant variety paddy prunus mume (sieb.) sieb.et zucc. No. 4.

Specific embodiment

There is provided defined below and method in order to preferably to define the application and to put into practice middle finger 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 being related to the deoxyribose of single-stranded or double-stranded form Nucleotide or ribonucleotide polymer, and unless otherwise restriction, nucleotide sequence is with 5 ' extremely 3 ' directions are write from left to right, including the known analog (example with natural nucleotide fundamental property As peptide nucleic acid(PNA)), described analog with naturally occurring nucleotide 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 aminoacid sequence to the nucleotide sequence of the application, for example, can use The codon with amino acid sequence for the coding replaced by the codon of monocotyledon preference, and do not change Become the aminoacid sequence coded by this 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 for improving blast resisting base Because of the expression in Oryza sativa L..

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 this specific nucleoside Acid sequence has 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 seriess.Such variant sequence thereof Including the interpolation of one or more nucleic acid, disappearance or replacement, corresponding such that it is able to lead to The interpolation of amino acid residue, remove or replace.By alignment programs known in the art Determine sequence iden including hybridization technique.The nucleotide sequence variants of embodiment and the application The difference of sequence may be as few as 1-15 nucleotide, as little as 1-10 (such as 6-10), As little as 5, as little as 4,3,2 or even 1 nucleotide.

The application further relates to comprise 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, comprise SEQ ID NO:Shown in 1 The sequence of 775-917 position nucleotide of sequence or its fragment or its variant or its complementary series, or Person comprises SEQ ID NO:The sequence of 941-1254 position nucleotide of sequence shown in 2 or its fragment 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 the recombinant nucleic acid fragment comprising in testing sample containing resistant gene.

As used herein, " Oryza sativa L. " is any rice plant include 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 can therefrom regenerate, Complete plant cell in plant callus, vegetation bed and plant or plant part, described 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 needing selection-breeding in the present processes.That is, Can (i.e. Comprehensive Traits be preferably sent out it is contemplated that having by any improved seeds lacking certain beneficial traits The kind of exhibition future) it is used as recurrent parent.With another, there are beneficial traits lacking in this receptor Kind is as donor parents, and the beneficial traits being provided are preferably dominant Dominant gene. In the embodiment of the application, using Oryza sativa L., ' deep 95B ', as recurrent parent, adopts ' magnificent 3418B ' is as donor to be proven to have the Oryza sativa L. of good rice blast resistance.

In the selection of restructuring plant provided herein, using molecular marker to restructuring Plant carries out foreground selection.The reliability of foreground selection depends primarily between labelling and target gene Chain tightness degree, for improving the accuracy rate of selection, typically uses two that both sides are adjacent simultaneously Labelling is tracked to target gene selecting.

In the embodiment of the application, the foreground selection labelling of employing includes favorable selection labelling With negative itemsets labelling, wherein, favorable selection labelling be away from target gene group fragment (containing anti-rice Pestilence gene) screening in the range of upstream and downstream 50kb (in Oryza sativa L. its genetic distance be 0.2cM) Pleiomorphism molecular marker.Negative itemsets labelling is away from target gene group fragment upstream and downstream The pleiomorphism molecular marker of screening in the range of 500kb (its genetic distance is 2cM in Oryza sativa L.).? In specific embodiments, the positive foreground selection labelling of optimized Select to use is and target gene The labelling Pi31 of group fragment close linkage, negative itemsets labelling is in target fragment upstream about The labelling Pi2ID02 of 230kb, and it is located at the labelling Pi2S91 of target fragment downstream about 270kb.

In the embodiment of the application, carry out homologous recombination using above-mentioned foreground selection labelling 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 with 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 Oryza sativa L. full-length genome breeding chip disclosed in Chinese patent application CN102747138A RICE6K, or the Oryza sativa L. 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 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 description suggestion.

Rice plant material information used in this application all can be found in rice in China kind and its Pedigree data base (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 Oryza sativa L. Japan MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).

Embodiment 1Selection-breeding imports the restructuring plant of blast resistant gene group fragment

Used in the present embodiment, material is Oryza sativa L. ' deep 95B ' and Oryza sativa L. ' magnificent 3418B '.

' magnificent 3418B ' has good rice blast resistance to Oryza sativa L., and speculates possibly No. 6 The rice blast resistance to this material for the gene cluster region that Pi2, Pi9 and Pigm of chromosome is located Serve pivotal role.

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

Table 1 foreground selection molecular labeling primer information

By Oryza sativa L., ' genomic fragment that in magnificent 3418B ', forementioned gene cluster is located imports to Oryza sativa L. ' 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, and obtains BC for obtained cenospecies and recurrent parent₁F₁Seed, Carried out using favorable selection labelling Pi31 and negative itemsets labelling Pi2ID02, Pi2S91 after nursery Restructuring Single-plant selection, filters out 9 individual plants in target gene group fragment side homologous recombination, I.e. Pi31 detection and ' the identical banding pattern of magnificent 3418B ', and Pi2ID02 or Pi2S91 detection and ' depth The identical banding pattern of 95B ', and utilize Oryza sativa L. full-length genome breeding chip RICE6K (CN102747138A) carries out Foreground selection (Yu etc., Plant Biotechnology to it Journal.2014,12:28-37).

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

Select background 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 labelling Pi31 and negative indicia Pi2ID02, Pi2S91 carry out mesh to the seed harvesting The screening of mark genomic fragment opposite side homologous recombination fragment, obtains 5 in target fragment both sides The individual plant of restructuring, i.e. Pi31 detection and ' the identical banding pattern of magnificent 3418B ', and Pi2ID02 and Pi2S91 Detection and ' the identical banding pattern of deep 95B '.

Using Oryza sativa L. 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), screen importing target fragment less, 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 chosen once, obtain BC₃F₂, after nursery, utilize favorable selection labelling Pi31 detects to it, select the individual plant containing target gene group fragment, that is, Pi31 detection with ' the identical banding pattern of magnificent 3418B ', is entered to it using Oryza sativa L. 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, be named as CR01BC02.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 ' imports 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 CR01BC02 is named as RecCR01BC02.

Primarily determined that by Oryza sativa L. full-length genome breeding chip RICE60K testing result, RecCR01BC02 is located at two SNP marker F0610285497TG and F0610565956CA Between.

Meanwhile, using Miseq sequencing technologies to ' deep 95B ', ' magnificent 3418B ' and CR01BC02 Three samples carry out genome sequencing.Using TruSeq Nano DNA LT Kit (illumina) Test kit carries out library foundation, using Library Quantification Kit Universal (KAPA Biosystems) test kit carries out quantitation, using MiSeq V2Reagent Kit (illumina) test kit carries out sequencing reaction.Entered using the desk-top sequenator of Miseq (illumina) Row detection.Concrete steps and method are referring to each test kit and sequenator operation instructions.

According to aforementioned SNP chip and Miseq sequencing result, RecCR01BC02 upstream is same Source recombinant fragment Primary Location is interval in 10285572bp to the 10287075bp of the 6th chromosome, It is interval that downstream homologous recombination fragment is positioned 10563830bp to 10565938bp.

On this basis, annotate the 6.1st edition with reference to the fine genome MSU/TIGR of Oryza sativa L. Japan, Download respective segments DNA sequence.Using the amplification of Primer Premier 5.0 software design and survey Sequence primer, design requirement be the long 22nt of primer about, G/C content 40-60% and do not have mispairing.

With receptor parent, ' ' magnificent 3418B ' is for comparison, right for deep 95B ' and donor parents RecCR01BC02 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 and be shown as single bright band in agarose gel electrophoresiies 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 sequencing, according to reality Border sequencing effect, finally filters out 3 and 4 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.

RecCR01BC02 upstream homologous recombination sequencing fragment length is 1517bp (SEQ ID NO: 1).1-775bp is that ' genomic segment of deep 95B ', with donor ' magnificent 3418B ' ratio for receptor Relatively, there are 15 SNP, 2 Indel.This 141bp section of 776-916bp is homology weight Group section.917-1517bp is that ' magnificent 3418B ' genomic fragment, with ' deep 95B ' for donor Relatively, there are 5 SNP, 1 Indel.

RecCR01BC02 downstream homologous recombination sequencing fragment length is 2103bp (SEQ ID NO: 2).1-941bp is that deep 95B ' compares donor ' genomic segment of magnificent 3418B ', with ', There are 16 SNP, 4 Indel.This 312bp section of 942-1253bp is homologous recombination Section.1254-2103bp is that ' genomic segment of deep 95B ', with donor ' magnificent 3418B ' for receptor Relatively, there are 15 SNP, 2 Indel.

Fig. 4 is the structure chart of RecCR01BC02 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 ' SNP the or InDel labelling of magnificent 3418B ', lower section base is receptor ' deep 95B ' to donor SNP or InDel labelling.Lycoperdon polymorphum Vitt section be from ' deep 95B ' genomic segment, black Zone section is from ' magnificent 3418B ' genomic segment, white section is homologous recombination section. Abscissa is fragment length, with base pairs (bp) as unit.

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

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

In order to identify resistance effect, to new lines CR01BC02 of the application selection-breeding, samsara parent This ' deep 95B ', rice blast disease-resistant variety paddy prunus mume (sieb.) sieb.et zucc. 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 as inoculating strain..Bacterial strain adopts Sorghum vulgare Pers. 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 the fresh mycelia block of diameter 5mm to turn after 5 days Be connected in sorghum grain culture medium (sorghum grain 500g add 1.5L distilled water, boil to boiling after filter off 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, 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, covers aseptic damp gauze, at 25 DEG C, RH >=95%, culture under 12h illumination condition produces for 4-5 days to a large amount of spores, (is contained with sterilized water 0.02% polysorbas20) wash lower spore, wait spore amount combined inoculation bacterial strain, adjustment concentration to 5 × 10⁵ Individual/ml.

With mixing conidial suspension spray inoculation CR01BC02, ' deep 95B ', Gu Mei 4 Number and Lijiang xintuanheigu, inoculation three repetition.Transparent cover on inoculation back cover, 28 DEG C of dark Cultivate 24h, then 16h illumination cultivation was investigated after 5 days.

Investigation standard is 0 grade (high anti-, HR)：There is no symptom；1 grade (anti-, R)：Very little brown Color scab；2 grades (in anti-, 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 oval greatly scab, scab merges 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.

The Resistant expression after rice blast fungus inoculated by table 3

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, it is selected from：

I) comprise SEQ ID NO:The sequence of the nucleotide of sequence 775-917 position shown in 1 or its fragment Or its variant or its complementary series；

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

Iii) comprise SEQ ID NO:The sequence of the nucleotide of sequence 941-1254 position shown in 2 or its piece Section or its variant or its complementary series；

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

The combination of above fragment.

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

(I) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-775 position shown in 1 Forward primer and specific recognition SEQ ID NO:The nucleotide of sequence 917-1517 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, it comprises

(a) first group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 1-775 position nucleotide and specific recognition SEQ ID NO:Sequence shown in 1 Arrange the reverse primer of the sequence of 776-916 position nucleotide；With

(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 776-916 position nucleotide and specific recognition SEQ ID NO:Shown in 1 The reverse primer of the sequence of sequence 917-1517 position nucleotide；

(III) specific recognition comprises SEQ ID NO:The nucleotide of sequence 775-776 position shown in 1 The forward primer of sequence and specific recognition comprise SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 916-917 position nucleotide；

(IV) specific recognition comprises SEQ ID NO:The nucleotide of sequence 775-776 position shown in 1 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 917-1517 shown in 1 The reverse primer of the sequence of position nucleotide；

(V) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-775 position shown in 1 Forward primer and specific recognition comprise SEQ ID NO:The core of sequence 916-917 position shown in 1 The reverse primer of the sequence of thuja acid；And/or optionally,

(VI) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-941 position shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleotide of sequence 1254-2103 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, it comprises

(c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of sequence of 1-941 position nucleotide and specific recognition SEQ ID NO:Sequence shown in 2 Arrange the reverse primer of the sequence of 942-1253 position nucleotide；With

(d) the 4th group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer of sequence of 942-1253 position nucleotide and specific recognition SEQ ID NO:2 institutes Show the reverse primer of the sequence of sequence 1254-2103 position nucleotide；

(VIII) specific recognition comprises SEQ ID NO:The nucleoside of sequence 941-942 position shown in 2 The forward primer of sequence of acid and specific recognition comprise SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 1253-1254 position nucleotide；

(IX) specific recognition comprises SEQ ID NO:The nucleotide of sequence 941-942 position shown in 2 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 1254-2103 shown in 2 The reverse primer of the sequence of position nucleotide；

(X) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-941 position shown in 2 Forward primer and specific recognition comprise SEQ ID NO:Sequence 1253-1254 position shown in 2 The reverse primer of the sequence of nucleotide.

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

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

5 '-ACCTGAGCGATGGTGGTCTTGA-3 ',

5’-CGAGTCGTTCTTGGCGTACTTGA-3’；And

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

5 '-AGTTGCTGATACCGAGAGGT-3 ',

5 '-GGCGCTAATCAGGGTACAGT-3 ',

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

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

5 '-TCAGCTTGAAAGAGCGAAGTGGT-3 ',

5’-ACTATTTGCGTCCTACTCCCTCCG-3’；And

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

5 '-CATGTCCCTCAACGGTTAGA-3 ',

5 '-CATAACACGAACGGAAACCA-3 ',

5 '-GTGCATTGTTCCACAGGAGA-3 ',

5’-GAAGAGAGGGACAAAGGAGA-3’.

4. the method that selection-breeding contains the rice plant of recombinant nucleic acid fragment described in claim 1, It includes using the Oryza sativa L. recipient plant parent without genes of interest group fragment as recurrent parent, will It is hybridized with the Oryza sativa L. donor plant containing genes of interest group fragment, then will be obtained Cenospecies are returned with recurrent parent, then the step that obtained backcrossing kind is carried out selfing, Wherein using molecular marker, foreground selection and Foreground selection are carried out to restructuring plant.

5. method as claimed in claim 4, is wherein used for the molecular marker of described foreground selection Selected from one or more of Pi31, Pi2ID02 and Pi2S91；And/or

Optionally, carry out described Foreground selection using Oryza sativa L. full-length genome breeding chip.

6. the method as described in claim 4 or 5, wherein said 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, obtained first backcross generation, using favorable selection labelling Pi31 and negative itemsets labelling Pi2ID02, Pi2S91 carry out the unilateral homologous recombination fragment sieve of blast resistant gene group fragment to it Choosing, and using Oryza sativa L. full-length genome breeding chip, Foreground selection is carried out to it；

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

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

4) select introgressed segment little, and the restructuring individual plant that background is replied, by the restructuring chosen list Strain selfing once, is obtained selfed seed, using favorable selection labelling Pi31, it is detected, and Using Oryza sativa L. full-length genome breeding chip, Foreground selection is carried out to it, final acquisition anti-rice containing homozygosis Pestilence genome recombination nucleic acid fragment and the rice plant of background reply.

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

The primer pair of amplifier molecule labelling Pi31, it includes：

Forward primer：5 '-ATCCAAACCCGTTGTTGCAC-3 ',

Reverse primer：5’-CGGCAATTGCCACGATGATA-3’；

The primer pair of amplifier molecule labelling Pi2ID02, it includes：

Forward primer：5 '-AAGTAATAATCCCCGCTGTTGT-3 ',

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

The primer pair of amplifier molecule labelling Pi2S91, it 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, it includes adopting right Require the primer described in 2 or 3, performing PCR reaction is entered for template with testing gene group, and analyzes The step of PCR primer.

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

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

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