CN107988213A - Rice genome recombinant nucleic acid fragment RecCR020428 and its detection method - Google Patents

Abstract

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

Description

Rice genome recombinant nucleic acid fragment RecCR020428 and its detection method

Technical field

This application involves full-length genome selection and use technology.Specifically, this application involves educated using full-length genome selection Kind technology selection and breeding have the function of the rice plant of the recombinant nucleic acid fragment of Brown Planthopper Resistance, and the recombinant nucleic acid obtained therefrom Fragment and its detection method.

Background technology

Brown paddy plant hopper, scientific name Nilaparvata lugensBelong to Homoptera, Delphacidae.Brown paddy plant hopper is monophagy evil Worm, only feeding rice, have the features such as happiness temperature, cold tolerance is weak, growth cycle is short, migrate at a distance, fulminant and wildness. Brown paddy plant hopper is typical sucking pest, is made a living with feeding bast and xylem sap, and food ingestion is big, breeding is fast, once greatly Outburst, can causing damage area, No kernels or seeds are gathered, as in a year of scarcity.Further, it is also possible to propagate Virus Diseases of Rice (such as：Grass-like bushy stunt and tingia stunt Disease), serious harm is also resulted in Rice Production.

Since the 1980s, identified more than the 30 brown planthopper resistant sites from wild rice and cultivated rice.By In the complexity of brown paddy plant hopper phenotypic evaluation, cause just to clone in recent years the Individual genes such as Bph14, Bph26 and Bph3 (Du etc., PNAS.2009,106(52):22163-22168；Tamura etc., Sci Rep.2014,4:5872；Liu et al., Nature Biotechnology.2015,33:301-305).In addition, the continuous utilization with brown planthopper resistant kind in production, brown paddy plant hopper The adaptability of kind is gradually strengthened.Some resistant varieties utilized extensively in production, which are just gradually lost, resists brown paddy plant hopper Property (Deen etc., Rice Genet Newsl.2010,25:70-72).

At present, brown paddy plant hopper prevention still relies upon chemical pesticide, not only increases production cost, pollutes environment, and promotes brown fly The lice resistance to the action of a drug strengthens.It can be seen from the above that the cultivation demand of brown planthopper resistant new varieties is very urgent.

The content of the invention

On the one hand, this application provides rice genome recombinant nucleic acid fragment, it includes following recombinant nucleic acid fragment, or It is made of following recombinant nucleic acid fragment：

- the first recombinant nucleic acid fragment, it is selected from：I) SEQ ID NO are included:1 and SEQ ID NO:Sequence shown in 2 or its piece Section or its variation or its complementary series；And/or

- the second recombinant nucleic acid fragment, it is selected from：Ii SEQ ID NO) are included:Sequence the 1054th to 1078 shown in 3 and The sequence of the nucleotide of 1778 to 1798 or its fragment or its variation or its complementary series；Iii SEQ ID NO) are included:3 institutes Show the sequence or its fragment or its variation or its complementary series of the 1065th to 1786 nucleotide of sequence；Iv SEQ ID) are included NO:The sequence or its fragment of sequence the 1054th to 1078 shown in 3 and the nucleotide of the 1778th to 1798 or its variation or its Complementary series, and SEQ ID NO:Sequence the 7th to 29 shown in 3, the 192nd to 216, the 727th to 751 and the 1868th to At least one of 1889 nucleotide sequence or its fragment or its variation or its complementary series；Or v) include SEQ ID NO:3 Shown sequence or its fragment or its variation or its complementary series.

In one embodiment, the rice genome recombinant nucleic acid fragment that the application provides is included selected from the first restructuring i) Nucleic acid fragment, or by being formed selected from the first recombinant nucleic acid fragment i).

In another embodiment, the application provide rice genome recombinant nucleic acid fragment include is selected from ii), iii), Iv), v) in any sequence the second recombinant nucleic acid fragment, or by selected from ii), iii), iv), v) in any sequence second Recombinant nucleic acid fragment forms.

In yet another embodiment, the rice genome recombinant nucleic acid fragment that the application provides is included selected from the first weight i) Group nucleic acid fragment and be selected from ii), iii), iv), v) in any sequence the second recombinant nucleic acid fragment combination, or by selected from I) the first recombinant nucleic acid fragment and be selected from ii), iii), iv), v) in any sequence the second recombinant nucleic acid fragment combination group Into.

In addition, this application provides the primer for detecting the recombinant nucleic acid fragment, wherein the primer includes：

The primer of the first recombinant nucleic acid fragment of-detection, it is selected from：(I) specific recognition SEQ ID NO:1 and SEQ ID NO:The primer of sequence shown in 2；And/or

The primer of the second recombinant nucleic acid fragment of-detection, it is selected from：(II) specific recognition SEQ ID NO:Sequence shown in 3 The primer of the sequence of 1054th to 1078 nucleotide, and specific recognition SEQ ID NO:Sequence the 1778th to 1798 shown in 3 The primer of the sequence of position nucleotide；(III) specific recognition SEQ ID NO:Sequence the 1065th to 1786 shown in 3 nucleotide The primer of sequence；(IV) specific recognition SEQ ID NO:The primer of the sequence of sequence the 1054th to 1078 shown in 3 nucleotide, With specific recognition SEQ ID NO:The primer of the sequence of sequence the 1778th to 1798 shown in 3 nucleotide, and specificity are known Other SEQ ID NO:Sequence the 7th to 29 shown in 3, the 192nd to 216, the 727th to 751 and the 1868th to 1889 nucleosides The primer of at least one of acid sequence；Or (V) specific recognition SEQ ID NO:The primer of sequence shown in 3.

In one embodiment, the primer pair for the first recombinant nucleic acid fragment of amplification, such as expanding SEQ ID NO:The primer pair of sequence shown in 1 is, for example, 5 '-GCACGATCTTGAACAGGTAGTCG-3 ', and 5 '- TTGATGGTACTGGTGCAAGGGAT-3’；Expand SEQ ID NO:The primer pair of sequence shown in 2 is, for example, 5 '- CCGAAGAAGAAGTTCCCATAAA-3 ', and 5 '-GCTGTACCAAACATACCCATAC-3 '.For the first recombinant nucleic acid to be sequenced The primer of fragment, for example, for SEQ ID NO to be sequenced:The primer of sequence shown in 1 is, for example, 5 '- GCACGATCTTGAACAGGTAGTCG-3’；For SEQ ID NO to be sequenced:The primer of sequence shown in 2 is, such as 5 '- GCTGTACCAAACATACCCATAC-3’

In another embodiment, the primer pair for the second recombinant nucleic acid fragment of amplification, such as expanding SEQ ID NO:The primer pair of sequence shown in 3 is, such as 5 '-GGAACGAAGTTAGCAGTAGTAGCA-3 ', and 5 '- CGAGACAGTTTTGAGATGGGATAG-3’.For the primer of the second recombinant nucleic acid fragment to be sequenced, such as SEQ ID to be sequenced NO:The primer of 3 the 7th to 29 and the 192nd to 216 shown sequence is, for example, 5 '-GGAACGAAGTTAGCAGTAGTAGCA- 3’；For SEQ ID NO to be sequenced:The primer of 3 the 727th to 751 and the 1054th to 1078 shown sequence is, for example, 5 '- GTTGCCCACAGTTTCCTCAC-3’；And for SEQ ID NO to be sequenced:3 the 1778th to 1798 and the 1868th to 1889 The primer of shown sequence is, for example, 5 '-CGAGACAGTTTTGAGATGGGATAG-3 '.

On the other hand, the method for the rice plant of the recombinant nucleic acid fragment, wherein institute are contained this application provides selection and breeding Stating recombinant nucleic acid fragment has the function of Brown Planthopper Resistance, and the described method comprises the following steps：1) by recurrent parent rice ' in ' magnificent 130B ' is hybridized kind extensive 629 ', and obtained cenospecies is returned with recurrent parent, is returned with donor rice A generation is handed over, marks BphC03ID03 and negative itemsets mark BphC03S20, BphC03S88 to resist it using favorable selection The unilateral homologous recombination fragment screening of brown paddy plant hopper genomic fragment, and background is carried out to it using rice full-length genome breeding chip Selection；2) select background to reply preferable restructuring single plant to be returned again with recurrent parent, obtain second backcross generation, utilize forward direction Selected marker BphC03ID03 is detected it, selects the restructuring single plant containing brown planthopper resistant gene pack section, then utilizes Rice full-length genome breeding chip carries out Foreground selection to it；3) the restructuring single plant and recurrent parent that selection background has been replied are another It is secondary to be returned, obtain third backcross generation, using favorable selection mark BphC03ID03 and negative itemsets mark BphC03S20, BphC03S88 carries out it opposite side homologous recombination fragment screening of brown planthopper resistant gene pack section, and utilizes rice full genome Group breeding chip carries out Foreground selection to it；And 4) select introgressed segment small, and the restructuring single plant that background has been replied, it will choose Restructuring individual plant selfing once, obtain selfed seed, it be detected using favorable selection mark BphC03ID03, and utilizes water Rice full-length genome breeding chip carries out Foreground selection to it, final to obtain the recombinant nucleic acid fragment of group containing homozygous gene and background reply Rice plant.

In one embodiment, the amplimer used when carrying out foreground selection to restructuring plant using molecular labeling, bag The primer pair for amplifier molecule mark BphC03ID03 is included, wherein forward primer is 5 '-GCAAGAATCCGACGCCATAA- 3 ', reverse primer 5 '-CTCTGCTCCTTGCTCTAATCCTCT-3 '；Primer for amplifier molecule mark BphC03S20 Right, wherein forward primer is 5 '-TGGCAGCATTTTGTTGTAG-3 ', 5 '-TTCCAGCCGTGTCTATTT- of reverse primer 3’；And the primer pair of amplifier molecule mark BphC03S88, wherein forward primer is 5 '-GTGATGCATGCTTTACCACC- 3 ', reverse primer 5 '-ATACCGTAAACTTTGCACGC-3 '.

Another aspect, this application provides the method for detecting the recombinant nucleic acid fragment, it includes being carried using the application The primer of confession, PCR reactions are carried out by template of testing gene group, and the step of analyze PCR product.Specifically, the method with Sample to be tested genomic DNA is template, PCR amplification is carried out using foregoing amplimer, then using foregoing sequencing primer to obtaining The amplified production obtained is sequenced, if sequencing result and SEQ ID NO:1-2 and/or SEQ ID NO:Sequence shown in 3 or its area Section is consistent or complementary, then contains SEQ ID NO in sample to be tested:1-2 and/or SEQ ID NO:The homologous recombination of sequence shown in 3 Nucleic acid fragment.

Determined by detection and contain SEQ ID NO in sample to be tested:1-2 and/or SEQ ID NO:Sequence shown in 3 or its The recombinant nucleic acid fragment of section, you can determine to include the recombinant nucleic acid fragment with Brown Planthopper Resistance in sample to be tested.

In addition, present invention also provides the kit of detection recombinant nucleic acid fragment, it includes primer as the aforementioned.

Further, present invention also provides screening the rice plant containing recombinant nucleic acid fragment or seed method, its The step of whether containing foregoing recombinant nucleic acid fragment in genome including detecting rice plant to be measured.

In one embodiment, detected using foregoing primer and whether contained in the genome of rice plant to be measured Foregoing recombinant nucleic acid fragment.In another embodiment, using the method for foregoing detection recombinant nucleic acid fragment To detect in the genome of rice plant to be measured whether contain foregoing recombinant nucleic acid fragment.In yet another embodiment, Detected using foregoing kit in the genome of rice plant to be measured and whether contain foregoing recombinant nucleic acid piece Section.

It yet still another aspect, this application provides what is screened by the method to contain recombinant nucleic acid disclosed in the present application The rice plant of fragment or its seed.

The selection and breeding based on full-length genome selection and use technology that the application provides contain brown planthopper resistant gene group recombinant nucleic acid The method of the rice plant of fragment, has advantage that is quick, accurate, stablizing.Only pass through the transformation of five generations, you can only by target base Because pack section imports acceptor material, and the reply of background is realized at the same time.The application improvement acceptor material for ' middle kind extensive 629 ', It is high yield, high-quality, strong excellent wide spectrum restorer.Using the above method, can retain ' in the case of middle extensive 629 ' original advantage of kind Increase substantially its Brown Planthopper Resistance.Meanwhile the recombinant nucleic acid fragment that the application provides is closely related with Brown Planthopper Resistance, can make It is applied to the cultivation of other kinds for Resistance resource.

Brief description of the drawings

Fig. 1 is CR020428 rice RICE60K full-length genome breeding chip testing results in the embodiment of the present application 1；Wherein, Square frame indicated by abscissa numeral represents 12 chromosomes of rice successively, and ordinate numeral is the physical location on rice genome [with megabasse (Mb) for unit], grey lines represent receptor parent, and ' the middle extensive 629 ' genotype of kind, black lines represent donor parent ' magnificent 130B ' genotype, it is consistent i.e. without polymorphism section that white line represents two parent genotypes for this.No. 3 chromosome is black in figure Section shown in colo(u)r streak bar is the brown planthopper resistant gene group recombinant nucleic acid fragment RecCR020428 imported.

Fig. 2 is qualification result in CR020428 Brown Planthopper Resistances room in the embodiment of the present application 3；Blade shown in figure is successively For：(A) high sense brown paddy plant hopper kind ' coming No. 1 in platform '；(B) new lines CR020428 is improved；(C) original kind ' middle kind extensive 629 '； (D) donor parents ' magnificent 130B '.

Embodiment

Defined below and method is provided preferably to define the application and instruct this area general in the application practice Logical technical staff.Unless otherwise mentioned, term understands according to the common usage of person of ordinary skill in the relevant.

As used herein, " nucleotide sequence " includes the deoxyribonucleotide or ribose core for being related to single-stranded or double-stranded form Thuja acid polymer, and write from left to right with 5 ' to 3 ' directions unless otherwise limitation, nucleotide sequence, including with natural nucleus The known analog (for example, peptide nucleic acid) of thuja acid fundamental property, the analog with as naturally occurring ucleotides side Formula hybridizes with single-chain nucleic acid.

In some embodiments, the nucleotide sequence of the application can be changed, is replaced with carrying out conserved amino acid Change.In certain embodiments, the nucleotide sequence of the application can not changed according to unifacial leaf codon preference The replacement of amino acid sequence, such as the codon of monocotyledon preference can be used to replace password of the coding with amino acid sequence Son, without changing the encoded amino acid sequence of the nucleotide sequence.

Specifically, this application involves to SEQ ID NO:1-2 and/or SEQ ID NO:3 further optimize the nucleosides of gained Acid sequence.The more details of this method are described in Murray etc. (1989) Nucleic Acids Res.17:477-498.Optimization Nucleotide sequence can be used for improving the expression of brown planthopper resistant gene group recombinant nucleic acid fragment in rice.

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

The application further relates to include SEQ ID NO:1-2 and/or SEQ ID NO:The sequence of specific site in sequence shown in 3 Or its fragment or its variation or its complementary series, for example, including SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2 or its piece Section or its variation or its complementary series (including upstream homologous recombination area section boundary SNP/Indel sites).Alternatively, include SEQ ID NO:The sequence or its fragment of sequence the 1054th to 1078 shown in 3 and the 1778th to 1798 nucleotide or its variation or its Complementary series (includes downstream homologous recombination area section boundary SNP/Indel sites)；Include SEQ ID NO:Sequence shown in 3 The sequence or its fragment of 1065 to 1786 nucleotide or its variation or its complementary series (comprising downstream homologous recombination section and its Border SNP/Indel sites)；Include SEQ ID NO:Sequence the 1054th to 1078 shown in 3 and the 1778th to 1798 nucleosides The sequence or its fragment or its variation or its complementary series of acid, and it is any of following or a variety of：SEQ ID NO:Shown in 3 The sequence or its fragment of the 7th to 29, the 192nd to 216, the 727th to 751 and the 1868th to 1889 nucleotide of sequence or Its variation or its complementary series (include downstream homologous recombination area section boundary SNP/Indel sites, and are respectively derived from donor The SNP/Indel sites of fragment and/or receptor fragments).

According to fragment or section comprising above-mentioned specific site, corresponding SEQ ID NO can be specifically identified:1- 2 and/or SEQ ID NO:Sequence shown in 3.Further, by identifying containing SEQ ID NO:1-2 and/or SEQ ID NO: The recombinant nucleic acid fragment of sequence shown in 3, you can determine to include the recombinant nucleic acid piece with Brown Planthopper Resistance in sample to be tested Section.

As used herein, " rice " is any rice plant and including can be with all plant varieties of rice breeding.Such as It is used herein, " plant " or " plant ", including whole plant, plant cell, plant organ, plant protoplast, plant can be with Therefrom complete plant in regenerated plant cell tissue cultures, plant callus, vegetation bed and plant or plant part Cell, the plant part is such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, cane, root, the tip of a root, flower pesticide.

It can be adapted for the rice varieties of any required selection and breeding in the present processes.That is, it can be lacked any The improved seeds (i.e. Comprehensive Traits are preferable, it is contemplated that promising kind) of certain few beneficial traits are used as recurrent parent.With Another kind with beneficial traits lacking in this receptor is as donor parents, and the beneficial traits provided are preferably aobvious Property Dominant gene.In the embodiment of the application, using rice ' middle kind extensive 629 ' is used as recurrent parent, using by Confirm that ' magnificent 130B ' is used as donor to the rice with good Brown Planthopper Resistance.

In the selection of restructuring plant provided herein, prospect choosing is carried out to restructuring plant using molecular labeling Select.The reliability of foreground selection depends primarily upon tightness degree chain between mark and target gene, to improve the accurate of selection Rate is general that target gene is selected into line trace with two adjacent marks of both sides at the same time.

In the embodiment of the application, the foreground selection mark of use includes favorable selection mark and negative itemsets mark Note.In a particular embodiment, the positive foreground selection mark of optimized Select to use is closely connected with target gene group fragment The mark BphC03ID03 of lock, negative itemsets mark are the mark BphC03S20 positioned at target fragment upstream, and positioned at target The mark BphC03S88 in fragment downstream.

In the embodiment of the application, using above-mentioned foreground selection mark carry out homologous recombination detection when, side or The criterion of unilateral homologous recombination be BphC03ID03 detections with ' the identical banding pattern of magnificent 130B ', and BphC03S20 or BphC03S88 is detected and ' middle extensive 629 ' identical banding pattern of kind；The criterion of both sides or bilateral homologous recombination is BphC03ID03 inspections Go out and ' the identical banding pattern of magnificent 130B ', and BphC03S20 and BphC03S88 detections and ' middle extensive 629 ' identical banding pattern of kind.

In this application, the available chip of any type can be used to carry out in breeding method provided herein Foreground selection.In preferred embodiments, the applicant can be used disclosed in Chinese patent application CN102747138A Rice full-length genome breeding chip RICE6K, or the full base of rice disclosed in PCT international applications WO/2014/121419 Because of a group breeding chip RICE60K.Full content in this two parts of application documents is incorporated herein by reference.

Following embodiments are merely to illustrate and the purpose of unrestricted the application scope.Unless otherwise specified, embodiment is pressed More solito experiment condition, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.

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

The rice genome physical location being previously mentioned in the application is with reference to rice Nipponbare genome MSU/TIGR annotations 6.1st edition (http://rice.plantbiology.msu.edu/).

Embodiment 1Selection and breeding import the restructuring plant of brown planthopper resistant gene pack section

The material used in the present embodiment is rice ' middle kind extensive 629 ' and rice ' magnificent 130B '.

Rice ' magnificent 130B ' has good Brown Planthopper Resistance, thus it is speculated that be probably No. 3 chromosome QBph3 (Hu etc., Molecular Breeding.2015,35:And Bph14 (Du etc., PNAS.2009,106 (52) 3):Where 22163-22168) Gene cluster region key effect has been risen to the Brown Planthopper Resistance of the material.

In the Breeding Process of restructuring plant, foreground selection is carried out to restructuring plant using molecular labeling, to used Foreground selection molecular labeling is screened.Used molecular labeling is derived partly from website http:// Www.gramene.org/, partially self design.Design method is, with reference to rice Nipponbare genome MSU/TIGR annotations the 6.1 editions, download aforementioned zones segment DNA sequence.The SSR sites in above-mentioned sequence are scanned using SSRLocator softwares.Profit Primer is designed to the SSR sites searched out with 3.0 softwares of Primer Premier, it is right to design primer 128 altogether.Pass through the side of PCR Method, screens above-mentioned primer pair in ' magnificent 130B ' and ' polymorphism in middle kind extensive 629 ', finally pick out has in two parts of materials The high foreground selection molecular labeling of polymorphism, amplification efficiency, is favorable selection mark BphC03ID03 and negative itemsets mark respectively Remember BphC03S20, BphC03S88.Specific primer information for the above-mentioned molecular labeling of PCR amplification is shown in Table 1.

1 foreground selection molecular labeling primer information of table

By rice, ' genomic fragment in magnificent 130B ' where forementioned gene imported into rice ' in middle kind extensive 629 ', specifically Process is as follows：

Hybridized with ' middle kind extensive 629 ' for recurrent parent, ' magnificent 130B ' for donor parents, by obtained cenospecies with ' middle kind extensive 629 ' is returned recurrent parent, obtains BC₁F₁Seed, marks BphC03ID03 using favorable selection after nursery and bears Restructuring Single-plant selection is carried out to selected marker BphC03S20, BphC03S88, filters out 7 in target gene group DNA fragmentation one The single plant of side homologous recombination, i.e. BphC03ID03 detect and ' the identical banding pattern of magnificent 130B ', and BphC03S20 or BphC03S88 is examined Go out with ' middle extensive 629 ' identical banding pattern of kind, and using rice full-length genome breeding chip RICE6K (CN102747138A) to its into Row Foreground selection (Yu etc., Plant Biotechnology Journal.2014,12:28-37).

Comparable chip is as a result, selection background replys best restructuring list in the 7 unilateral homologous recombination single plants filtered out Strain (this generation background recovery value is more than 75%), making it, ' middle kind extensive 629 ' is returned again, obtains BC with recurrent parent₂F₁Kind Son, is detected it using favorable selection mark BphC03ID03 after nursery, selects the restructuring containing target gene group fragment Single plant, i.e. BphC03ID03 detect with ' the identical banding pattern of magnificent 130B ', using rice full-length genome breeding chip RICE6K to its into Row Foreground selection.

Select background to reply preferable single plant (this generation background recovery value is more than 87.5%), make its with recurrent parent ' in Kind extensive 629 ' is returned again, obtains BC₃F₁Seed, utilizes favorable selection mark BphC03ID03 and negative sense mark after nursery Remember that BphC03S20, BphC03S88 carry out the seed of harvest the screening of target gene group fragment opposite side homologous recombination fragment, Obtain 11 single plants in the restructuring of target fragment both sides, i.e. BphC03ID03 detect with ' the identical banding pattern of magnificent 130B ', and BphC03S20 and BphC03S88 detections and ' middle extensive 629 ' identical banding pattern of kind.

Single plant is exchanged to above-mentioned 9 bilaterals using rice full-length genome breeding chip RICE60K (WO/2014/121419) Carry out background and target fragment selection (Chen etc., Molecular Plant.2014,7:541-553), importing target is screened Fragment is smaller, and the target single plant one that background has been replied (this generation background recovery value is more than 93.75%).

By the individual plant selfing chosen once, BC is obtained₃F₂, after nursery using favorable selection mark BphC03ID03 to its into Row detection, selects the single plant containing target gene group fragment, i.e. BphC03ID03 is detected and ' the identical banding pattern of magnificent 130B ', utilizes water Rice full-length genome breeding chip RICE60K carries out Foreground selection to it.

It is final to obtain target fragment homozygosis, and background replys the strain one of (background recovery value is more than 99%), is named as CR020428.Chip testing result is shown in Fig. 1.

Embodiment 2Homologous recombination fragment determines after importing resistance gene of brown planthopper pack section

In order to determine the resistance gene of brown planthopper group clip size imported, to ' homozygosis of the middle extensive 629 ' introgressed segment of kind is single Strain has carried out the sequencing of target gene group fragment both sides homologous recombination fragment.By the brown planthopper resistant gene group weight contained by CR020428 Group nucleic acid fragment is named as RecCR020428.

Primarily determine that RecCR020428 upstreams are homologous heavy by rice full-length genome breeding chip RICE60K testing results Pack section is between mark R0335402302GT and F0335657380GA, and downstream homologous recombination fragment is positioned at mark Between F0335749323TC and R0335762925CT.

Meanwhile carried out using Miseq sequencing technologies tri- samples of 130B ' and CR020428 magnificent to ' middle kind extensive 629 ', ' complete Gene order-checking.Library foundation is carried out using TruSeq Nano DNA LT Kit (illumina) kit, uses Library Quantification Kit-Universal (KAPA Biosystems) kit is quantified, and uses MiSeq V2Reagent Kit (illumina) kit carries out sequencing reaction.Examined using the desk-top sequenators of Miseq (illumina) Survey.Specific steps and method are referring to each kit and sequenator operation instructions.

It is according to foregoing SNP chip and Miseq sequencing results, RecCR020428 upstreams homologous recombination fragment is further true 35420807bp to the 35467580bp sections of the 3rd chromosome are scheduled on, downstream homologous recombination fragment is positioned at the 3rd chromosome 35758124bp to 35762279bp sections.

On this basis, the 6.1st edition is annotated with reference to rice Nipponbare genome MSU/TIGR, downloads respective segments DNA sequences Row.Using the amplification of 5.0 Software for Design of Primer Premier and sequencing primer, design requirement contains for long 22nt of primer or so, GC Measure 40-60% and no mispairing.

With receptor parent, ' ' magnificent 130B ' is control, homologous to RecCR020428 upstream and downstream for middle kind extensive 629 ' and donor parents Recombinant fragment designs amplimer, is expanded using high-fidelity enzyme KOD FX Neo (TOYOBO), uses two-step method or three steps Method finds optimal amplification condition, it is ensured that amplified production is shown as single bright band in agarose gel electrophoresis detection.Design 26 pairs filter out the amplification that 2 pairs of primers are used for upstream homologous recombination fragment, its reaction condition is respectively 1) BphCC03X13K5：94 ℃2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 60sec, 37 circulations；20℃1min；2)BphCC03X13K23：94℃ 2min；98 DEG C of 10sec, 59 DEG C of 30sec, 68 DEG C of 240sec, 37 circulations；20℃1min.It is same for downstream to filter out 1 pair of primer The amplification of source recombinant fragment, its reaction condition are：94℃2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 150sec, 37 Circulation；20℃1min.

In addition, using amplified production as template, it is sequenced using Sanger PCR sequencing PCRs, upstream homologous recombination fragment is set altogether 116 sequencing primers are counted, effect is sequenced according to actual, picks out 2 sequencings for being used for SNP or Indel sites.It is homologous to downstream Recombinant fragment designs 4 sequencing primers altogether, and effect is sequenced according to actual, picks out 3 sequencings for being used for SNP or Indel sites. Specific amplimer and sequencing primer sequence are shown in Table 2.

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

RecCR020428 upstreams homologous recombination fragment includes characteristic sequence SEQ ID NO:1 and SEQ ID NO:2.SEQ ID NO:1 is ' characteristic sequence of middle kind extensive 629 ', ' compared with magnificent 130B ', there are 1 Indel with donor from acceptor.SEQ ID NO:2 are ' characteristic sequence of magnificent 130B ', ' compared with middle kind extensive 629 ', there are 1 SNP with acceptor from donor.

RecCR020428 downstreams homologous recombination fragment length is 1892bp (SEQ ID NO:3).1-1065bp is donor ' genomic segment of magnificent 130B ', ' compared with middle kind extensive 629 ', there are 3 SNP, 1 Indel with acceptor.1066-1785bp this One 720bp sections are homologous recombination section.1786-1892bp is acceptor ' the middle extensive 629 ' genomic fragment of kind, with donor ' China 130B ' compares, and there are 1 SNP, 1 Indel.

CR020428, ' middle kind extensive 629 ' and ' magnificent 130B ' SNP or Indel Site discrepancies are shown in Table 3." position " is in table：On The position of trip homologous recombination fragment SNP or Indel Site discrepancy is respectively relative to SEQ ID NO：1 or SEQ ID NO：For 2, The position of downstream homologous recombination fragment SNP or Indel Site discrepancy is relative to SEQ ID NO：For 3.

Table 3CR020428, ' middle kind extensive 629 ' and ' magnificent 130B ' SNP or Indel Site discrepancies

Embodiment 3' middle kind extensive 629 ' imports the Resistance Identification after brown planthopper resistant gene pack section

In order to identify resistance effect, ' middle kind is extensive 629 ', supplies by new lines CR020428, receptor parent to the application selection and breeding Body parent ' magnificent 130B ' (as positive control), and high sense brown paddy plant hopper kind ' coming No. 1 in platform ' (as negative control) into Resistance Identification in row brown paddy plant hopper room, identification method are as follows.

Each part material is soaked seed indoors, vernalization, then sowing is in the plastic basin of grid line is pulled, every part of material point 3 rows sow 45 plants altogether, and when two one heart stages of leaf, totally 30 plants of consistent plant of healthy growing ways are used to connect worm for 10 plants of often row reservation.Worm Source is come the brown paddy plant hopper that is collected since field, and captive breeding indoors.2-3 ages nymph is taken to plant to be identified, every plant There are 5-10 nymphs.Start recording respectively identifies the resistance class of seedling when ' coming No. 1 in platform ' dead seedling 95%, takes 30 young plants Resistance class average value, as the resistance class of the material, its resistance level is evaluated according to resistance class.Wherein, resistance Grade is divided into 10 grades：0 or 1 grade, blade is not aggrieved or first leaf blade tip turns to be yellow；2 grades, first leaf 1/2 turns to be yellow or blade tip Shrinkage；3 grades, first leaf turns to be yellow or withers；4 grades, second leaf portion distribution is yellow or lobus cardiacus blade tip turns to be yellow；5 grades, second leaf Jaundice, shrinkage or withered, the blue or green volume of lobus cardiacus；6 grades, lobus cardiacus curling, lobus cardiacus tip burn on leaf；7 grades, lobus cardiacus curling is withered, and plant is not It is dead；8 grades, lobus cardiacus is withered, and is somewhat lodged；9 grades, whole strain lodging.According to above-mentioned resistance class, 0-0.9 grades are determined as highly resistance, 1.0-2.9 grades are anti-, and 3.0-5.9 grades are moderate resistance, and 6.0-6.9 grades are middle sense, and 7.0-7.9 grades are sense, and 8.0-9.0 grades are high sense.

Resistance Identification is the result is shown in Fig. 2 in brown paddy plant hopper room, wherein ' coming No. 1 in platform ' is felt to be high, original kind ' middle kind extensive 629 ' For middle sense, improvement new lines CR020141 is anti-, and ' magnificent 2048B ' is highly resistance to donor parents.

Although above having made detailed description to the application with a general description of the specific embodiments, On the basis of the application, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, the these modifications or improvements on the basis of without departing from the application spirit, belong to this application claims scope.

Sequence table

<110>Chinese subset rolls into a ball Co., Ltd

<120>Rice genome recombinant nucleic acid fragment RecCR020428 and its detection method

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213>Rice (Oryza sativa)

<400> 1

atttttattg gggaagagag agaga 25

<210> 2

<211> 25

<212> DNA

<213>Rice (Oryza sativa)

<400> 2

cctgcttgac ccaattatat gggtg 25

<210> 3

<211> 1892

<212> DNA

<213>Rice (Oryza sativa)

<220>

<221>From ' magnificent 130B' genomic segments

<222> (1)..(1065)

<220>

<221>Homologous recombination section

<222> (1066)..(1785)

<220>

<221>From ' the middle extensive 629' genomic segments of kind

<222> (1786)..(1892)

<400> 3

gtggcggtca cggaggcgac ggggcggcag gcggcgtcgt tcgtgctcgg ctgcgtggcc 60

acgctcaccg tcatgctgct cttccagtac caggcgccgc cggactacgg ccgcgccgcc 120

aggtcgccgg tgcagttctc cacctccaga gaccagctgc tgctgcactg cggcggcaat 180

ggaacggcgc cgccgccgcc ggtgatcgca cgtggcggcg aggaggctaa catcaccggc 240

aagcctccga cgactgctac tgctgtcgcc gaggagcagc cgccaaccaa gcctcctgcg 300

acctctactg catcttcacc aactcatcat attccagcaa ccagtacaga tcttgaagaa 360

gaggttagtt ctcattaatc tcccggctta attaattttc acttctttaa tttcttatgc 420

gtcatctggt atcagtaatc tgctaaattt gtgccgtctt gaattcactc tgctgcccac 480

agtttcctca catgttcaaa attaactcaa cagtacaact catcataata aaaacagggg 540

gttcttcaat ttcagtagga cacaactaac cctttttggg tgtctgccaa atgcaccaag 600

aaaatgattt tgtaagatat atgctacata taaccacgaa ttcagaaatc attccatgag 660

acttgatcta ctattttgac ttcgaaaaag agataggcta atcaatttgt tatatattaa 720

tctcgatcct tgattacctt attgtacttg tttcgagtgt acgaaaagtg gcaaagaaag 780

tcctaggatc ttacaccacc tagtgattca cgctaatttg atcatcaatg gcggcagggc 840

ggagagttcc gggggttggc ggcggcggtg gcgcgggcgg cgacggatga ccggacggtg 900

atcatcacgt gcgtgaacca cgcgttcgcg gcgcccgact cgctcctgga catcttcctc 960

gagggcttcc gcgtcggcga cggcacgccg gagctcctcc gccacgtcct cgtcgtcgcc 1020

atggatccca ccgcgctcac ccggtgccgc gccgtccacc cccactgcta cctctacacc 1080

atgcccggcc tcgacgtcga cttcacctcc gagaagttct tcgcctccaa ggactacctc 1140

gagctcgtct ggagcaagct caagctccag cgccgaattc tccagctcgg ctacaacttc 1200

ctcttcacgg taattcagta actgtaaatt gaaattaaca ccgccattga ctgcaaatca 1260

agatgaactg attaattaac caattgcaag atcaggacgt tgacattgtg tggctgagga 1320

atccgttcaa gcacgtggcg gtgtacgcgg acatggcgat ctcgagcgac gtgttcttcg 1380

gtgacccgga caacatcgac aacttcccaa acacgggctt cttctacgtg aagccgagcg 1440

cgaggacgat cgccatgacc aaggagtggc acgaggcgag gagctcgcac ccggggctca 1500

acgagcagcc ggtgttcaac cacatcaaga agaagctggt gaagaagctg aagctcaagg 1560

ttcagtacct ggacacggcc tacatcggcg gattctgcag ctacggcaag gatctgagca 1620

agatctgcac catgcacgcc aactgctgca tcggcctcca atccaagatc agtgatctca 1680

agggtgttct tgctgactgg aagaactaca ccaggttgcc accctgggca aagcccaatg 1740

ccaggtggac tgtgcctggt aaatgcatcc attgattttg attgtcgatc gggcagtatc 1800

tggtgttgta ggctacgtac agtcaggaag atagctttat ttgcacatga aagggcgaat 1860

tttgtttttt ttttggtttg ttgtagattg at 1892

<210> 4

<211> 23

<212> DNA

<213>Artificial sequence

<400> 4

gcacgatctt gaacaggtag tcg 23

<210> 5

<211> 23

<212> DNA

<213>Artificial sequence

<400> 5

ttgatggtac tggtgcaagg gat 23

<210> 6

<211> 22

<212> DNA

<213>Artificial sequence

<400> 6

ccgaagaaga agttcccata aa 22

<210> 7

<211> 22

<212> DNA

<213>Artificial sequence

<400> 7

gctgtaccaa acatacccat ac 22

<210> 8

<211> 24

<212> DNA

<213>Artificial sequence

<400> 8

ggaacgaagt tagcagtagt agca 24

<210> 9

<211> 24

<212> DNA

<213>Artificial sequence

<400> 9

cgagacagtt ttgagatggg atag 24

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<400> 10

gttgcccaca gtttcctcac 20

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence

<400> 11

gcaagaatcc gacgccataa 20

<210> 12

<211> 24

<212> DNA

<213>Artificial sequence

<400> 12

ctctgctcct tgctctaatc ctct 24

<210> 13

<211> 19

<212> DNA

<213>Artificial sequence

<400> 13

tggcagcatt ttgttgtag 19

<210> 14

<211> 18

<212> DNA

<213>Artificial sequence

<400> 14

ttccagccgt gtctattt 18

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence

<400> 15

gtgatgcatg ctttaccacc 20

<210> 16

<211> 20

<212> DNA

<213>Artificial sequence

<400> 16

ataccgtaaa ctttgcacgc 20

Claims (8)

1. rice genome recombinant nucleic acid fragment, it includes：

- the first recombinant nucleic acid fragment, it is selected from：

I) SEQ ID NO are included:1 and SEQ ID NO:Sequence shown in 2 or its fragment or its variation or its complementary series；And/or

- the second recombinant nucleic acid fragment, it is selected from：

Ii SEQ ID NO) are included:The sequence of sequence the 1054th to 1078 shown in 3 and the nucleotide of the 1778th to 1798 or Its fragment or its variation or its complementary series；

Iii SEQ ID NO) are included:The sequence or its fragment of sequence the 1065th to 1786 shown in 3 nucleotide or its variation or its Complementary series；

Iv SEQ ID NO) are included:The sequence of sequence the 1054th to 1078 shown in 3 and the nucleotide of the 1778th to 1798 or Its fragment or its variation or its complementary series, and SEQ ID NO:Sequence the 7th to 29 shown in 3, the 192nd to 216, At least one of 727 to 751 and the 1868th to 1889 nucleotide sequence or its fragment or its variation or its complementary series； Or

V) SEQ ID NO are included:Sequence or its fragment or its variation or its complementary series shown in 3.

2. test right requires the primer of the 1 recombinant nucleic acid fragment, wherein the primer includes：

The primer of the first recombinant nucleic acid fragment of-detection, it is selected from：

(I) specific recognition SEQ ID NO:1 and SEQ ID NO:The primer of sequence shown in 2；And/or

The primer of the second recombinant nucleic acid fragment of-detection, it is selected from：

(II) specific recognition SEQ ID NO:The primer of the sequence of sequence the 1054th to 1078 shown in 3 nucleotide, and specifically Property identification SEQ ID NO:The primer of the sequence of sequence the 1778th to 1798 shown in 3 nucleotide；

(III) specific recognition SEQ ID NO:The primer of the sequence of sequence the 1065th to 1786 shown in 3 nucleotide；

(IV) specific recognition SEQ ID NO:The primer of the sequence of sequence the 1054th to 1078 shown in 3 nucleotide, and specifically Property identification SEQ ID NO:The primer of the sequence of sequence the 1778th to 1798 shown in 3 nucleotide, and specific recognition SEQ ID NO:In sequence the 7th to 29 shown in 3, the 192nd to 216, the 727th to 751 and the 1868th to 1889 nucleotide extremely A kind of few primer of sequence；Or

(V) specific recognition SEQ ID NO:The primer of sequence shown in 3.

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

(I) primer pair of the first recombinant nucleic acid fragment is expanded

5 '-GCACGATCTTGAACAGGTAGTCG-3 ',

5’-TTGATGGTACTGGTGCAAGGGAT-3’；

5 '-CCGAAGAAGAAGTTCCCATAAA-3 ',

5’-GCTGTACCAAACATACCCATAC-3’；And

(II) primer of the first recombinant nucleic acid fragment is sequenced

5’-GCACGATCTTGAACAGGTAGTCG-3’；

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

(III) SEQ ID NO are expanded:The primer pair of sequence shown in 3

5 '-GGAACGAAGTTAGCAGTAGTAGCA-3 ',

5’-CGAGACAGTTTTGAGATGGGATAG-3’；

(IV) SEQ ID NO are sequenced:The primer of sequence shown in 3

5’-GGAACGAAGTTAGCAGTAGTAGCA-3’；

5’-GTTGCCCACAGTTTCCTCAC-3’；

5’-CGAGACAGTTTTGAGATGGGATAG-3’；.

4. selection and breeding contain the method for the rice plant of the recombinant nucleic acid fragment described in claim 1, wherein the recombinant nucleic acid piece Section has the function of Brown Planthopper Resistance, and the described method comprises the following steps：

1) by recurrent parent rice ' middle kind extensive 629 ' with donor rice ', magnificent 130B ' is hybridized, by obtained cenospecies with Recurrent parent is returned, and obtains first backcross generation, marks BphC03ID03 and negative itemsets to mark using favorable selection BphC03S20, BphC03S88 carry out it unilateral homologous recombination fragment screening of brown planthopper resistant gene pack section, and utilize water Rice full-length genome breeding chip carries out Foreground selection to it；

2) select background to reply preferable restructuring single plant to be returned again with recurrent parent, obtain second backcross generation, utilize forward direction Selected marker BphC03ID03 is detected it, selects the restructuring single plant containing brown planthopper resistant gene pack section, then utilizes Rice full-length genome breeding chip carries out Foreground selection to it；

3) the restructuring single plant that selection background has been replied is returned again with recurrent parent, is obtained third backcross generation, is utilized forward direction Selected marker BphC03ID03 and negative itemsets mark BphC03S20, BphC03S88 carry out it brown planthopper resistant gene pack section The screening of opposite side homologous recombination fragment, and Foreground selection is carried out to it using rice full-length genome breeding chip；And

4) select introgressed segment small, and the restructuring single plant that background has been replied, by the restructuring individual plant selfing chosen once, it is selfed Kind, it is detected using favorable selection mark BphC03ID03, and it is carried out using rice full-length genome breeding chip Foreground selection, the final rice plant for obtaining the recombinant nucleic acid fragment of group containing homozygous gene and background and replying.

5. method as claimed in claim 4, wherein the expansion used when carrying out foreground selection to restructuring plant using molecular labeling It is as follows to increase primer：

Amplifier molecule marks the primer pair of BphC03ID03, it includes：

Forward primer：5 '-GCAAGAATCCGACGCCATAA-3 ',

Reverse primer：5’-CTCTGCTCCTTGCTCTAATCCTCT-3’；

Amplifier molecule marks the primer pair of BphC03S20, it includes：

Forward primer：5 '-TGGCAGCATTTTGTTGTAG-3 ',

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

Amplifier molecule marks the primer pair of BphC03S88, it includes：

Forward primer：5 '-GTGATGCATGCTTTACCACC-3 ',

Reverse primer：5’-ATACCGTAAACTTTGCACGC-3’.

6. the method for the recombinant nucleic acid fragment described in test right requirement 1, it is included using the primer described in Claims 2 or 3, The step of PCR reactions being carried out by template of testing gene group, and analyzing PCR product.

7. the kit of the recombinant nucleic acid fragment described in test right requirement 1, it includes the primer described in Claims 2 or 3.

8. screening the method for rice plant or seed containing the recombinant nucleic acid fragment described in claim 1, it includes detection and treats The step of whether surveying in the genome of rice plant or seed containing recombinant nucleic acid fragment described in claim 1；

Preferably, using the primer described in Claims 2 or 3, either using the method described in claim 6 or using power Profit requires the kit described in 7 to be detected.