CN106480051A - Recombinant nucleic acid fragment RecCR010113 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 RecCR010113 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:1901-1914； 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 180-377 position shown in 1 or its fragment or its variant or its complementary sequence Row；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 528-774 position shown in 2 or Its fragment or its variant or its complementary series；Or iv) comprise 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, described recombinant nucleic acid fragment is genome recombination nucleic acid fragment.

Additionally, this application provides detecting the primer of described recombinant nucleic acid fragment, it is selected from：(I) special Opposite sex identification SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 1-180 position shown in 1 and Specific recognition SEQ ID NO:Reversely the drawing of the sequence of the nucleotide of sequence 377-782 position shown in 1 Thing；(II) the combining of first group of primer pair and second group of primer pair below, it comprises (a) first Group primer pair：Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-180 position shown in 1 The forward primer of row and specific recognition SEQ ID NO:The nucleoside of sequence 181-376 position shown in 1 The reverse primer of the sequence of acid；(b) second group of primer pair：Specific recognition SEQ ID NO: The forward primer of sequence of the nucleotide of sequence 181-376 position shown in 1 and specific recognition SEQ ID NO:The reverse primer of the sequence of the nucleotide of sequence 377-782 position shown in 1；(III) special Property identification comprise SEQ ID NO:The forward direction of the sequence of the nucleotide of sequence 180-181 position shown in 1 Primer and specific recognition comprise SEQ ID NO:The nucleotide of sequence 376-377 position shown in 1 The reverse primer of sequence；(IV) specific recognition comprises SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 180-181 position nucleotide and specific recognition SEQ ID NO:Shown in 1 The reverse primer of the sequence of sequence 377-782 position nucleotide；(V) specific recognition SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 1-180 position shown in 1 and specific recognition bag The NO of ID containing SEQ:The reverse primer of the sequence of the nucleotide of sequence 376-377 position shown in 1；With / or optionally, (VI) specific recognition SEQ ID NO:The nucleotide of sequence 1-528 position shown in 2 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 774-1363 position shown in 2 The reverse primer of the sequence of nucleotide；(VII) the 3rd group of primer pair and the 4th group of primer pair below Combination, it comprises (c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 Arrange the forward primer of sequence and the specific recognition SEQ ID NO of 1-528 position nucleotide:2 institutes Show the reverse primer of the sequence of sequence 529-773 position nucleotide；(d) the 4th group of primer pair： Specific recognition SEQ ID NO:The forward direction of the sequence of the nucleotide of sequence 529-773 position shown in 2 Primer and specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 774-1363 position shown in 2 The reverse primer of row；(VIII) specific recognition comprises SEQ ID NO:Sequence shown in 2 The forward primer of sequence of 528-529 position nucleotide and specific recognition comprise SEQ ID NO:2 The reverse primer of the sequence of shown sequence 773-774 position nucleotide；(IX) specific recognition bag The NO of ID containing SEQ:The forward primer of sequence of the nucleotide of sequence 528-529 position shown in 2 and spy Opposite sex identification SEQ ID NO:The sequence of the nucleotide of sequence 774-1363 position shown in 2 reverse Primer；(X) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-528 position shown in 2 The forward primer of row and specific recognition comprise SEQ ID NO:Sequence 773-774 position shown in 2 The reverse primer of the sequence of nucleotide.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-CCAGCTTCTAGTTTGTTCCCTGTA-3 ', and 5 '-TGCTTAGTT AGGTCCTGTTCGTTT-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 1 is drawn Thing is, for example, 5 '-CCAGCTTCTAGTTTGTTCCCTGTA-3 ', and 5 '-TGCTTA GTTAGGTCCTGTTCGTTT-3’.

In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2 For, for example, 5 '-GACGAACCTGCAAAGCCTAAAGA-3 ', and 5 '-GAGTCAG CTAACGGGCACATACC-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 2 Primer is, for example, 5 '-GACGAACCTGCAAAGCCTAAAGA-3 ', and 5 '-GAG TCAGCTAACGGGCACATACC-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 group fragment, then will be obtained Cenospecies be 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.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, Pi2ID01 One or more of with Pi2ID05；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 resistant gene group recombinant nuclear The method of the rice plant of acid fragment, it comprises the following steps：1) recurrent parent is planted with donor Thing is hybridized, and obtained cenospecies and recurrent parent are returned, and obtains first backcross generation, Using favorable selection labelling Pi31 and negative itemsets labelling Pi2ID01, Pi2ID05, it is carried out The unilateral homologous recombination fragment screening of blast resistant gene group fragment, and utilize Oryza sativa L. full-length genome Breeding chip, such as RICE6K, carry out Foreground selection to it；2) background is selected to reply preferably Restructuring individual plant (this generation background recovery value is more than 75%) is returned again with recurrent parent, obtains Obtain second backcross generation, using favorable selection labelling Pi31, it is detected, select to contain anti-rice blast The restructuring individual plant of ospc gene group fragment, then utilizes Oryza sativa L. full-length genome breeding chip, for example RICE6K, carries out Foreground selection to it；3) select restructuring individual plant (this generation that background is replied Background recovery value is more than 87.5%) it is returned again with recurrent parent, obtain third backcross generation, Using favorable selection labelling Pi31 and negative indicia Pi2ID01, Pi2ID05, anti-rice is carried out to it The opposite side homologous recombination fragment screening of pestilence genomic fragment, and educated using Oryza sativa L. full-length genome Plant chip, such as RICE60K, Foreground selection is carried out to it；And 4) select introgressed segment little, And the restructuring individual plant (background recovery value is more than 93.75%) 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, such as RICE60K, Foreground selection is carried out to it, Obtain the recombinant nucleic acid fragment homozygosis of group containing blast resistant gene eventually and background replys (background recovery value More than 99%) rice plant.

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 '-CGGCAA TTGCCACGATGATA-3’；For the primer pair of amplifier molecule labelling Pi2ID01, wherein Forward primer is 5 '-CGTAAACTTGTTAGGTGGGTG-3 ', and reverse primer is 5’-AAAATATGAGGAACTGGGCA-3’；And it is used for amplifier molecule labelling Pi2ID05 Primer pair, wherein forward primer be 5 '-CCTTATCACAGCCACATAGAGC-3 ', Reverse primer is 5 '-TGGGATTCATTGGGTGAGTAT-3 '.

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 analyzing pcr amplification product.Specifically, for example, described draw Thing is as previously mentioned.Selectively, analyze pcr amplification product using 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 '-CCAGCTTCTAGTTTGTTCCCTGTA-3 ', and reverse primer： 5’-TGCTTAGTTAGGTCCTGTTCGTTT-3’；Sequencing primer, including forward primer： 5 '-CCAGCTTCTAGTTTGTTCCCTGTA-3 ', and reverse primer：5’-TGCTTA GTTAGGTCCTGTTCGTTT-3’.Methods described is with testing sample genomic DNA as mould Plate, enters performing PCR amplification using above-mentioned amplimer, then utilizes above-mentioned sequencing primer to acquisition Amplified production be sequenced, if sequencing result and SEQ ID NO:1 sequence is consistent or complementary, Then contain SEQ ID NO in testing sample:Homologous recombination fragment shown in 1.

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 '-GACGAACCTGCAAAGCCTAAAGA-3 ', and reverse primer： 5’-GAGTCAGCTAACGGGCACATACC-3’；Sequencing primer, including forward primer： 5 '-GACGAACCTGCAAAGCCTAAAGA-3 ', and reverse primer：5’-GAGTCA GCTAACGGGCACATACC-3’.Methods described is with testing sample genomic DNA as mould Plate, enters performing PCR amplification using above-mentioned amplimer, then utilizes above-mentioned sequencing primer to acquisition Amplified production be sequenced, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, Then contain SEQ ID NO in testing sample:Homologous recombination fragment shown in 2.

Determined by detection and in testing sample, contain SEQ ID NO:1 and/or SEQ ID NO:2 The recombinant nucleic acid fragment of shown sequence, 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 application improvement acceptor material be ' flourishing age B ', be quality early rice not Educate is that ' maintainer of flourishing age A ' has the good outstanding feature of meter matter.Using said method, ' in the case of the original advantage of flourishing age B ', its rice blast resistance can be increased substantially retaining. Further, can be obtained by least first cross, generation backcrossing and stable contain rice blast Resistance fragments ' flourishing age A ', more significantly carrying of cenospecies rice blast resistance is realized by combo High.Meanwhile, the recombinant nucleic acid fragment that the application provides is closely related with rice blast resistance, can make It is applied to the cultivation of other kinds for Resistance resource.

Brief description

Fig. 1 is CR010113 Oryza sativa L. RICE60K full-length genome breeding in the embodiment of the present application 1 Chip detection result；Wherein, square frame indicated by abscissa numeral represents 12 dyeing of Oryza sativa L. successively Body, vertical coordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome, Lycoperdon polymorphum Vitt lines represent receptor parent, and ' flourishing age B ' genotype, black lines represent donor parents ' China 2048B ' 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 RecCR010113.

Fig. 2 is RecCR010113 upstream homologous recombination sequencing fragment ratio in the embodiment of the present application 2 To result；Asterisk shown in figure represents identical base in comparison result, and in figure CR010113 is The new lines obtaining, T003 is that ' flourishing age B ', R006 are donor parents ' magnificent 2048B ' to receptor parent For donor parents.

Fig. 3 is RecCR010113 downstream homologous recombination sequencing fragment ratio in the embodiment of the present application 2 To result.

Fig. 4 is the structure of RecCR010113 both sides homologous recombination fragment in the embodiment of the present application 2 Figure；Wherein, (A) is upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment Structure chart, top base is donor ' SNP the or InDel labelling of magnificent 2048B ', lower section alkali Base is receptor ' SNP the or InDel labelling of flourishing age B '.Lycoperdon polymorphum Vitt section is from ' the flourishing age B ' genomic segment, black section is from ' magnificent 2048B ' genomic segment, white area Section is homologous recombination section, and abscissa is fragment length, with base pairs (bp) as unit.

Fig. 5 is CR010113 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 ' flourishing age B '；(C) new lines CR010113 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:Specific in sequence shown in 2 The fragment in site or its variant or its complementary series, for example, comprise SEQ ID NO:Sequence shown in 1 The sequence of 180-377 position nucleotide of row or its fragment or its variant or its complementary series, or Comprise SEQ ID NO:The sequence of 528-774 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, can specifically reflect Make corresponding SEQ ID NO:1 or SEQ ID NO:Sequence shown in 2.Further, pass through Identify containing SEQ ID NO:1 or SEQ ID NO:The recombinant nucleic acid fragment of sequence shown in 2, Can determine that 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., ' flourishing age B ', as recurrent parent, adopts ' magnificent 2048B ' 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 Pi2ID01 of 360kb, and it is located at the labelling of target fragment downstream about 450kb Pi2ID05.

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 2048B ' Identical banding pattern, and Pi2ID01 or Pi2ID05 detection and ' the identical banding pattern of flourishing age B '；Both sides Or the criterion of bilateral homologous recombination be Pi31 detection with ' the identical banding pattern of magnificent 2048B ', Pi2ID01 with Pi2ID05 detection and ' the identical banding pattern of flourishing age B '.

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. ' flourishing age B ' and Oryza sativa L. ' magnificent 2048B '.

' magnificent 2048B ' 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 2048B ' and ' Sheng Generation B ' in polymorphism, finally pick out and there is in two parts of materials polymorphism, amplification efficiency High foreground selection molecular marker, is favorable selection labelling Pi31 and negative itemsets labelling respectively Pi2ID01、Pi2ID05.

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 2048B ', forementioned gene cluster is located imports to Oryza sativa L. ' in flourishing age B ', detailed process is as follows：

So that ' as recurrent parent, ' magnificent 2048B ' is hybridized flourishing age B ' for donor parents, will Obtained cenospecies and recurrent parent are returned, and obtain BC₁F₁Seed, using just after nursery Carry out individual plant choosing of recombinating to selected marker Pi31 and negative itemsets labelling Pi2ID01, Pi2ID05 Select, filter out 11 individual plants in target gene group fragment side homologous recombination, be i.e. Pi31 inspection Go out and ' the identical banding pattern of magnificent 2048B ', and Pi2ID01 or Pi2ID05 detection and ' flourishing age B ' Identical banding pattern, and right using Oryza sativa L. full-length genome breeding chip RICE6K (CN102747138A) It carries out Foreground selection (Yu etc., Plant Biotechnology Journal.2014,12:28-37).

Comparable chip result in the unilateral homologous recombination individual plant of 11 filtering out, selects background to return Multiple best restructuring individual plant (this generation background recovery value is more than 75%) is so as to ' contain with recurrent parent Generation B ' it is returned again, obtain 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 2048B ', 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 ' flourishing age, B ' was returned again recurrent parent, obtained BC₃F₁Seed, utilizes after nursery Favorable selection labelling Pi31 and negative indicia Pi2ID01, Pi2ID05 are carried out to the seed harvesting The screening of target gene group fragment opposite side homologous recombination fragment, obtains 9 in target fragment two Stress the individual plant organized, that is, Pi31 detection with ' the identical banding pattern of magnificent 2048B ', and Pi2ID01 with Pi2ID05 detection and ' the identical banding pattern of flourishing age B '.

Using Oryza sativa L. full-length genome breeding chip RICE60K (WO/2014/121419) to above-mentioned 9 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 2048B ', 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 CR010113.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 ' flourishing age B ' imports The homozygosis individual plant of fragment has carried out the sequencing of target gene fragment both sides homologous recombination fragment.Will Blast resistant gene group recombinant nucleic acid fragment contained by CR010113 is named as RecCR010113.

Primarily determined that by Oryza sativa L. full-length genome breeding chip RICE60K testing result, RecCR010113 is located at two SNP marker F0609583345CA and F0610437629AG Between.

Meanwhile, using Miseq sequencing technologies to ' flourishing age B ', ' magnificent 2048B ' and CR010113 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, RecCR010113 upstream is same 9608906bp to the 9609692bp that source recombinant fragment is positioned at the 6th chromosome is interval, and downstream is same It is interval that source recombinant fragment is positioned 10433889bp to 10435252bp.

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 2048B ' is for comparison, right for flourishing age B ' and donor parents RecCR010113 upstream and downstream homologous recombination fragment separately designs amplimer, is protected using high True enzyme KOD FX Neo (TOYOBO) is expanded, and is found using two-step method or three-step approach Good amplification condition is it is ensured that amplified production is shown as single bright in agarose gel electrophoresiies detection Band.The upstream homologous recombination fragment amplification primer reaction condition of wherein determination is：94℃ 2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 60sec, 37 circulations；20℃ 1min. Downstream homologous recombination fragment amplification primer reaction condition is：94℃ 2min；98 DEG C of 10sec, 61 DEG C 30sec, 68 DEG C of 60sec, 37 circulations；20℃ 1min.Thus, two are finally filtered out right Amplimer is respectively used to the amplification of upstream and downstream homologous recombination fragment.

In addition, with amplified production as template, being sequenced using Sanger sequencing, according to reality Border sequencing effect, finally filters out 2 and 2 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.

RecCR010113 upstream homologous recombination sequencing fragment length is 782bp (SEQ ID NO:1).1-180bp is that ' genomic segment of flourishing age B ', with donor ' magnificent 2048B ' for receptor Relatively, there are 2 SNP, 3 Indel.This 196bp section of 181-376bp is homology Restructuring section.377-782bp is that ' magnificent 2048B ' genomic fragment, with ' flourishing age B ' for donor Relatively, there are 9 SNP.

RecCR010113 downstream homologous recombination sequencing fragment length is 1363bp (SEQ ID NO:2).1-528bp is that ' genomic segment of magnificent 2048B ', with ' flourishing age B ' compares donor Relatively, there are 7 SNP, 1 Indel.This 245bp section of 529-773bp is homology weight Group section.774-1363bp is that ' genomic segment of flourishing age B ', with donor ' magnificent 2048B ' for receptor Relatively, there are 10 SNP, 2 Indel.

Fig. 4 is the structure chart of RecCR010113 both sides homologous recombination fragment.Wherein, (A) is Upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment structure figure.Top base For donor, ' SNP the or InDel labelling of magnificent 2048B ', lower section base is receptor ' flourishing age B ' SNP or InDel labelling.Lycoperdon polymorphum Vitt section be from ' flourishing age B ' genomic segment, black Zone section is from ' magnificent 2048B ' genomic segment, white section is homologous recombination section. Abscissa is fragment length, with base pairs (bp) as unit.

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

Embodiment 3' flourishing age B ' imports the Resistance Identification after blast resistant gene group fragment

In order to identify resistance effect, to new lines CR010113 of the application selection-breeding, recurrent parent ' flourishing age B ', rice blast disease-resistant variety paddy prunus mume (sieb.) sieb.et zucc. No. 4 (as positive control), and rice blast sense Sick kind 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：

Choose detached 6102-1 the rice blast disease leaves from Sichuan sick nursery in 2013 and sick neck, lake 21K14-1,21K02-1,4105-1 of northern sick nursery, the 1209-1 of Guangdong sick nursery, Fujian sick nursery 8111-1, totally 6 plants of rice blast bacterial strains are as inoculating strain.Bacterial strain adopts -20 DEG C of sorghum grain method Preserve, using front, the sorghum grain of preservation is taken out to potato dextrose medium (PDA) flat board Activation (PDA：Peeled potatoes 200g, glucose 20g, agar powder 15g, distilled water constant volume To 1L), 28 DEG C of illumination cultivation take the fresh mycelia block of diameter 5mm to be forwarded to sorghum grain after 5 days In culture medium, (sorghum grain 500g adds 1.5L distilled water, boils and filters off liquid to boiling, by height Fine strain of millet grain pulls loading 250ml triangular flask, 100ml/ bottle, moist heat sterilization 20 minutes out), 10 pieces/ Bottle, connects bacterium and daily shakes sorghum grain scattered after 2 days, 28 DEG C of dark culturing cover with sorghum grain to mycelia. Then sorghum grain is spread out on sterile gauze, covers aseptic damp gauze, at 25 DEG C, RH Under >=95%, 12h illumination condition, culture produces, with sterilized water (containing 0.02% for 4-5 days to a large amount of spores Polysorbas20) wash lower spore, wait spore amount combined inoculation bacterial strain, adjustment concentration to 5 × 10⁵Individual/ml.

With mixing conidial suspension spray inoculation CR010113, ' flourishing age B ', Gu Mei No. 4 numbers And Lijiang xintuanheigu, three repetitions of inoculation.Transparent cover on inoculation back cover, 28 DEG C of dark trainings Foster 24h, then 16h illumination cultivation investigate 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 180-377 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 528-774 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 1-180 position shown in 1 Forward primer and specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 377-782 position shown in 1 The reverse primer of row；

(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 1-180 position nucleotide and specific recognition SEQ ID NO:Sequence shown in 1 Arrange the reverse primer of the sequence of 181-376 position nucleotide；With

(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 181-376 position nucleotide and specific recognition SEQ ID NO:Shown in 1 The reverse primer of the sequence of sequence 377-782 position nucleotide；

(III) specific recognition comprises SEQ ID NO:The nucleotide of sequence 180-181 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 376-377 position nucleotide；

(IV) specific recognition comprises SEQ ID NO:The nucleotide of sequence 180-181 position shown in 1 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 377-782 position shown in 1 The reverse primer of the sequence of nucleotide；

(V) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-180 position shown in 1 Forward primer and specific recognition comprise SEQ ID NO:The core of sequence 376-377 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 1-528 position shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleotide of sequence 774-1363 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 1-528 position nucleotide and specific recognition SEQ ID NO:Sequence shown in 2 Arrange the reverse primer of the sequence of 529-773 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 529-773 position nucleotide and specific recognition SEQ ID NO:Shown in 2 The reverse primer of the sequence of sequence 774-1363 position nucleotide；

(VIII) specific recognition comprises SEQ ID NO:The nucleoside of sequence 528-529 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 773-774 position nucleotide；

(IX) specific recognition comprises SEQ ID NO:The nucleotide of sequence 528-529 position shown in 2 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 774-1363 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 1-528 position shown in 2 Forward primer and specific recognition comprise SEQ ID NO:The core of sequence 773-774 position shown in 2 The reverse primer of the sequence of thuja acid.

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

(I) it is used for the SEQ ID NO that expands and be sequenced:The primer of sequence shown in 1

5 '-CCAGCTTCTAGTTTGTTCCCTGTA-3 ',

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

(II) it is used for the SEQ ID NO that expands and be sequenced:The primer pair of sequence shown in 2

5 '-GACGAACCTGCAAAGCCTAAAGA-3 ',

5’-GAGTCAGCTAACGGGCACATACC-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, Pi2ID01 and Pi2ID05；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 Pi2ID01, Pi2ID05 carry out the unilateral homologous recombination fragment of blast resistant gene group fragment to it Screening, 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 Pi2ID01, Pi2ID05 pair It carries out the opposite side homologous recombination fragment screening of blast resistant gene group fragment, and utilizes Oryza sativa L. Full-length genome 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 Pi2ID01, it includes：

Forward primer：5 '-CGTAAACTTGTTAGGTGGGTG-3 ',

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

The primer pair of amplifier molecule labelling Pi2ID05, it includes：

Forward primer：5 '-CCTTATCACAGCCACATAGAGC-3 ',

Reverse primer：5’-TGGGATTCATTGGGTGAGTAT-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 amplified production.

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.