CN106609277A - Recombined nucleic acid fragment RecCR020142 and detection method thereof - Google Patents

Abstract

The invention provides a recombined nucleic acid fragment and a detection method thereof. The invention further provides a breeding method for a rice plant containing the recombined nucleic acid fragment. Foreground selection and background selection are conducted on a recombined plant through molecular markers, and the rice plant containing the recombined nucleic acid fragment is obtained.

Description

Recombinant nucleic acid fragment RecCR020142 and its detection method

Technical field

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

Background technology

Brown paddy plant hopper, scientific name Nilaparvata lugensCategory Homoptera, Delphacidae.It is brown Plant hopper is monophagy insect, only takes food paddy rice, and with happiness temperature, cold tolerance is weak, growth cycle It is short, migrate at a distance, the feature such as fulminant and wildness.Brown paddy plant hopper is typical pierce-suck type evil Worm, is made a living with taking food bast and xylem sap, and food ingestion is big, breeding is fast, once produce shockingly Send out, No kernels or seeds are gathered, as in a year of scarcity can to cause damage area.Further, it is also possible to propagate Virus Diseases of Rice (such as： Grass-like bushy stunt and tingia dwarf wilt), serious harm is also resulted in Rice Production.

It is identified more than 30 from wild rice and cultivated rice since the eighties in 20th century Individual brown planthopper resistant site.Due to 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 etc., Nature Biotechnology.2015,33:301-305).In addition, as brown planthopper resistant kind is in production In continuous utilization, brown paddy plant hopper gradually strengthens the adaptability of kind.Some are wide in production The general resistant variety for utilizing just gradually is losing (Deen etc., the Rice Genet of the resistance to brown paddy plant hopper Newsl.2010,25:70-72)。

At present, brown paddy plant hopper preventing and treating still relies upon chemical pesticide, not only increases production cost, pollutes ring Border, and promote the brown paddy plant hopper resistance to the action of a drug to strengthen.As can be seen here, the cultivation of brown planthopper resistant new varieties Demand is very urgent.

The content of the invention

On the one hand, this application provides recombinant nucleic acid fragment, it is selected from：I) SEQ ID NO are included: The sequence or its fragment of the nucleotides of sequence 534-936 position shown in 1 or its variant or its complementary series； Ii) SEQ ID NO are included:The sequence of sequence shown in 1 or its fragment or its variant or its complementary sequence Row；Iii) SEQ ID NO are included:The sequence or its piece of the nucleotides of sequence 163-288 position shown in 2 Section or its variant or its complementary series；Iv) SEQ ID NO are included:The sequence of sequence shown in 2 or Its fragment or its variant or its complementary series；And the combination of above fragment.In an embodiment In, the recombinant nucleic acid fragment is genome recombination nucleic acid fragment.

Additionally, this application provides detecting the primer of the recombinant nucleic acid fragment, it is selected from：(I) it is special Opposite sex identification SEQ ID NO:The forward primer of the sequence of the nucleotides of sequence 1-534 position shown in 1 and Specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 936-1609 position shown in 1 it is reverse Primer；(II) combination of first group of primer pair and second group of primer pair below, it includes (a) first Group primer pair：Specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-534 positions shown in 1 The forward primer and specific recognition SEQ ID NO of row:The nucleosides of sequence 535-935 positions 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 and specific recognition SEQ of the sequence of the nucleotides of sequence 535-935 positions shown in 1 ID NO:The reverse primer of the sequence of the nucleotides of sequence 936-1609 positions shown in 1；(III) it is special Opposite sex identification includes SEQ ID NO:The sequence of the nucleotides of sequence 534-535 positions shown in 1 is just SEQ ID NO are included to primer and specific recognition:The nucleotides of sequence 935-936 positions shown in 1 Sequence reverse primer；(IV) specific recognition includes SEQ ID NO:Sequence shown in 1 The forward primer and specific recognition SEQ ID NO of the sequence of 534-535 positions nucleotides:Shown in 1 The reverse primer of the sequence of sequence 936-1609 positions nucleotides；(V) specific recognition SEQ ID NO:The forward primer and specific recognition bag of the sequence of the nucleotides of sequence 1-534 positions shown in 1 The NO of ID containing SEQ:The reverse primer of the sequence of the nucleotides of sequence 935-936 positions shown in 1；With / or optionally, (VI) specific recognition SEQ ID NO:The nucleosides of sequence 1-163 positions shown in 2 The forward primer and specific recognition SEQ ID NO of the sequence of acid:Sequence 288-907 shown in 2 The reverse primer of the sequence of position nucleotides；(VII) the 3rd group of primer pair and the 4th group of primer below To combination, it includes (c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Shown in 2 The forward primer and specific recognition SEQ ID NO of the sequence of sequence 1-163 positions nucleotides:2 The reverse primer of the sequence of shown sequence 164-287 positions nucleotides；(d) the 4th group of primer It is right：Specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 164-287 positions shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleotides of sequence 288-907 positions shown in 2 The reverse primer of sequence；(VIII) specific recognition includes SEQ ID NO:Sequence shown in 2 The forward primer and specific recognition of the sequence of 163-164 positions nucleotides includes SEQ ID NO:2 The reverse primer of the sequence of shown sequence 287-288 positions nucleotides；(IX) specific recognition bag The NO of ID containing SEQ:The forward primer of the sequence of the nucleotides of sequence 163-164 positions shown in 2 and spy Opposite sex identification SEQ ID NO:The sequence of the nucleotides of sequence 288-907 positions shown in 2 is reversely drawn Thing；(X) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-163 positions shown in 2 Forward primer and specific recognition include SEQ ID NO:The cores of sequence 287-288 positions shown in 2 The reverse primer of the sequence of thuja acid.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-ACGAGTGTAAAGTGGCAAGC-3 ', 5 '-AACAGCACCCACCA CTATTA-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 1 is, for example, 5’-ACGAGTGTAAAGTGGCAAGC-3’；5’-ACAACGAGGCAACATGG TAA-3’；5’-TTACTCTAAACCGAACAAGC-3’；5’-ATACCTCCCAAG AACGTAGA-3’；With 5 '-TCTACATCACCTCCACCTTC-3 '.

In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2 For, for example, 5 '-GTCCCTGCACCGTCTCCTACACT-3 ', 5 '-ACTCCACAGA CCTCTATCCCATC-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 2, For example, 5 '-GGTCGTTACGCAAGATTGA-3 '；With 5 '-CTCACAAGGCCA CAGGTATG-3’。

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

In the above-mentioned methods, for the foreground selection molecular labeling selected from BphC04ID06, One or more in RM6659 and MS5；And/or entered using paddy rice full-length genome breeding chip The row Foreground selection.

In one embodiment, the seed selection that the application is provided contains brown planthopper resistant gene group recombinant nuclear The method of the rice plant of acid fragment, it is comprised the following steps：1) recurrent parent and donor are planted Thing is hybridized, then resulting cenospecies and recurrent parent are returned, and obtains backcrossing one In generation, using favorable selection BphC04ID06 and negative itemsets mark RM6659, MS5 are marked The unilateral homologous recombination fragment screening of brown planthopper resistant gene pack section is carried out to it, and utilizes paddy rice Full-length genome breeding chip, such as RICE6K carries out Foreground selection to it；2) background is selected to return Multiple preferably restructuring individual plant (this generation background recovery value is more than 75%) is carried out again with recurrent parent Backcrossing, obtains second backcross generation, and it is detected using favorable selection mark BphC04ID06, The restructuring individual plant containing brown planthopper resistant gene pack section is selected, is then educated using paddy rice full-length genome Chip is planted, such as RICE6K carries out Foreground selection to it；3) restructuring for selecting background to reply Individual plant (this generation background recovery value is more than 87.5%) is returned again with recurrent parent, is obtained Third backcross generation, using favorable selection mark BphC04ID06 and negative itemsets mark RM6659, MS5 carries out the opposite side homologous recombination fragment screening of brown planthopper resistant gene pack section, and profit to it With paddy rice full-length genome breeding chip, such as RICE60K carries out Foreground selection to it；And 4) select introgressed segment little, and the restructuring individual plant replied of background (background recovery value exceedes 93.75%), by the restructuring individual plant selfing chosen once, selfed seed is obtained, using favorable selection mark Note BphC04ID06 detects to it, and using paddy rice full-length genome breeding chip, for example RICE60K, to it Foreground selection is carried out, final to obtain the restructuring of the group of brown planthopper resistant gene containing homozygosis The rice plant of nucleic acid fragment and background reply (background recovery value is more than 99%).

In another embodiment, adopt when carrying out foreground selection to plant of recombinating using molecular labeling Amplimer, including：For the primer pair that amplifier molecule marks BphC04ID06, its Middle forward primer be 5 '-CCTAGCCGTCAGGTTAATAGATCAT-3 ', reverse primer For 5 '-ACCAGGTCTACTAGCTTTTACGGAG-3 '；For amplifier molecule mark The primer pair of RM6659, wherein forward primer are 5 '-TGTGGAGGCTTAGGAAATTC TGG-3 ', reverse primer is 5 '-TGTGAAACATGCCACGATACTGC-3 '；For Amplifier molecule marks the primer pair of MS5, and wherein forward primer is 5 '-TTGTGGGTCCTC ATCTCCTC-3 ', reverse primer is 5 '-TGACAACTTGTGCAAGATCA-3 '.

Another aspect, this application provides the method for detection recombinant nucleic acid fragment, it includes basis Foregoing recombinant nucleic acid fragment design specific primer, is carried out by template of testing gene group PCR reacts, and the step of analyze pcr amplification product.Specifically, for example, the primer is such as It is front described.Selectively, pcr amplification product is analyzed using Sanger PCR sequencing PCRs.

Specifically, in the method for the detection recombinant nucleic acid fragment that the application is provided, for expanding And detection SEQ ID NO:The primer combination of sequence shown in 1 is as follows：Amplimer, including forward direction Primer：5 '-ACGAGTGTAAAGTGGCAAGC-3 ', reverse primer：5’-AACAGCA CCCACCACTATTA-3’；Sequencing primer, including forward primer：5’-ACGAGTGTAAA GTGGCAAGC-3 ', forward primer：5 '-ACAACGAGGCAACATGGTAA-3 ', Reverse primer：5 '-TTACTCTAAACCGAACAAGC-3 ', reverse primer：5’-ATAC CTCCCAAGAACGTAGA-3 ', and forward primer：5’-TCTACATCACCTCCA CCTTC-3’.Methods described with testing sample genomic DNA as template, using above-mentioned amplification Primer enters performing PCR amplification, then the amplified production for obtaining is surveyed using above-mentioned sequencing primer Sequence, if sequencing result and SEQ ID NO:1 sequence is consistent or complementary, then contain in testing sample SEQ ID NO: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, including forward direction draws Thing：5 '-GTCCCTGCACCGTCTCCTACACT-3 ', reverse primer：5’-ACTCC ACAGACCTCTATCCCATC-3’；Sequencing primer, including forward primer： 5 '-GGTCGTTACGCAAGATTGA-3 ', and reverse primer：5’-CTCACAAGGCC ACAGGTATG-3’.Methods described with testing sample genomic DNA as template, using upper State amplimer and enter performing PCR amplification, then the amplified production using above-mentioned sequencing primer to acquisition It is sequenced, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, then testing sample In contain SEQ ID NO:Homologous recombination fragment shown in 2.

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

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

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

What the application was provided contains brown planthopper resistant gene based on full-length genome selection and use technology seed selection Group recombinant nucleic acid fragment rice plant method, with it is quick, accurate, stablize three big features. Only pass through the transformation of five generations, you can only by target gene group fragment importing acceptor material, and while Realize the reply of background.The application improvement acceptor material for ' flourishing age B ', be quality early rice not It is ' the maintainer of flourishing age A ', with the good outstanding feature of rice matter to educate.Using said method, ' in the case of the original advantages of flourishing age B ', resistance gene of brown planthopper pack can be imported retaining Section, improves its Brown Planthopper Resistance.Further, it is by least first cross, generation backcrossing Stable ' the flourishing age A ', then realize hybridizing by combo containing Brown Planthopper Resistance fragment can be obtained Plant increasing substantially for Brown Planthopper Resistance.The recombinant nucleic acid fragment that the application is provided resists with brown paddy plant hopper Property is closely related, and the cultivation of other kinds can be applied to as Resistance resource.

Description of the drawings

Fig. 1 is CR020142 paddy rice RICE60K full-length genome breedings in the embodiment of the present application 1 Chip detection result；Wherein, the indicated square frame of abscissa numeral represents successively 12 dyeing of paddy rice Body, ordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome, Grey lines represent receptor parent, and ' flourishing age B ' genotype, black lines represent donor parents ' China 2048B ' genotype, it is consistent i.e. without polymorphism section that white line represents two parent genotypes. Lines display block is the brown planthopper resistant gene for importing at rice chromosome black round dot in figure Group recombinant fragment RecCR020142.

Fig. 2 is RecCR020142 upstreams 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 CR020142 is in figure The new lines of acquisition, T003 is that ' flourishing age B ', R006 are donor parents ' China to receptor parent 2048B’。

Fig. 3 is RecCR020142 downstreams homologous recombination sequencing fragment ratio in the embodiment of the present application 2 To result.

Fig. 4 is the structure of RecCR020142 both sides homologous recombination fragment in the embodiment of the present application 2 Figure；Wherein, (A) be upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment Structure chart, top base is donor ' the SNP or InDel mark of magnificent 2048B ', lower section alkali Base is acceptor ' the SNP or InDel mark of flourishing age B '.Grey section is from ' the flourishing age B ' genomic segments, black section is from ' magnificent 2048B ' genomic segments, white area Section is homologous recombination section, and abscissa is fragment length, with base pairs (bp) as unit.

Fig. 5 is qualification result in CR020142 Brown Planthopper Resistances room in the embodiment of the present application 3； Blade is followed successively by shown in figure：(A) high sense brown paddy plant hopper kind ' coming No. 1 in platform '；(B) original Plant ' flourishing age B '；(C) new lines CR020142 are improved；(D) donor parents ' magnificent 2048B '.

Specific embodiment

Defined below and method is provided preferably to define the application and 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 Nucleotides or ribonucleotide polymer, and unless otherwise restriction, nucleotide sequence with 5 ' extremely 3 ' directions are write from left to right, including the known analog (example with natural nucleotide fundamental property Such as, peptide nucleic acid), the analog with naturally occurring nucleotides similar mode and single-stranded core Acid hybridization.

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

Specifically, the application is related to SEQ ID NO:1 or SEQ ID NO:2 is further excellent Nucleotide sequence obtained by change.The more details of the method are described in Murray etc. (1989) Nucleic Acids Res.17:477-498.Optimization nucleotide sequence can be used to improve brown planthopper resistant base Because of a group expression of the recombinant nucleic acid fragment in paddy rice.

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

The application is further related to comprising SEQ ID NO:1 or SEQ ID NO:It is special in sequence shown in 2 The sequence of anchor point or its fragment or its variant or its complementary series, for example, comprising SEQ ID NO: The sequence or its fragment of the 534-936 positions nucleotides of sequence shown in 1 or its variant or its complementary sequence Row, or comprising SEQ ID NO:The sequence of the 163-288 positions nucleotides of sequence shown in 2 or its Fragment or its variant or its complementary series.According to the fragment comprising above-mentioned specific site, Neng Goute Corresponding SEQ ID NO are identified different in naturely:1 or SEQ ID NO:Sequence shown in 2.Enter one Step ground, by identifying containing SEQ ID NO:1 or SEQ ID NO:The base of sequence shown in 2 Because of a group recombinant fragment, you can determine and include in testing sample the recombinant nucleic acid piece containing resistant gene Section.

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

Go for any rice varieties for needing seed selection in the present processes.That is, (i.e. Comprehensive Traits are preferable, it is contemplated that have and send out for the improved seeds that certain beneficial traits can be lacked by any The kind of exhibition future) it is used as recurrent parent.With another with beneficial traits lacking in this receptor Kind is used as donor parents.In the embodiment of the application, using paddy rice, ' flourishing age B ' makees For recurrent parent, using the paddy rice with good Brown Planthopper Resistance, ' magnificent 2048B ' is used as confession Body.

In the selection of restructuring plant provided herein, using molecular labeling to restructuring Plant carries out foreground selection.The reliability of foreground selection depends primarily on mark and target gene group The intersegmental chain tightness degree of piece, is the accuracy rate for improving selection, general simultaneously adjacent with both sides Two marks selection is tracked to target gene group fragment.

In the embodiment of the application, the foreground selection mark of employing includes that favorable selection is marked Mark with negative itemsets, wherein, favorable selection mark is (brown containing resisting away from target gene group fragment Plant hopper gene) screen in the range of upstream and downstream 50kb (in paddy rice its genetic distance be 0.2cM) Polymorphism molecular labeling.Negative itemsets mark is away from target gene group fragment upstream and downstream The polymorphism molecular labeling screened in the range of 500kb (its genetic distance is 2cM in paddy rice). In specific embodiment, the positive foreground selection mark of optimized Select to use is and target gene The mark BphC04ID06 of pack section close linkage, negative itemsets mark is in target fragment The molecular labeling RM6659 of trip about 220kb, and dividing away from target fragment downstream about 450kb Son mark MS5.

In the embodiment of the application, using above-mentioned foreground selection mark homologous recombination is carried out During detection, the criterion of side or unilateral homologous recombination is BphC04ID06 detections and ' China The identical banding patterns of 2048B ', and RM6659 or MS5 detections and ' the identical banding patterns of flourishing age B '； The criterion of both sides or bilateral homologous recombination is BphC04ID06 detections and ' magnificent 2048B ' Identical banding pattern, and RM6659 and MS5 detections and ' the identical banding patterns of flourishing age B '.In the application In, it is possible to use any available chip is carried out in breeding method provided herein Foreground selection.In preferred embodiments, can be using the applicant in Chinese patent Shen Paddy rice full-length genome breeding chip RICE6K, Huo Zhe that please be disclosed in CN102747138A Paddy rice full-length genome breeding chip disclosed in PCT international application WO/2014/121419 RICE60K.Full content in this two parts of application documents is incorporated herein by reference.

Following examples are merely to illustrate and the purpose of unrestricted the application scope.If not referring in particular to Bright, embodiment is according to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J&Russell DW,Molecular cloning:a laboratory manual, 2001), or according to the condition of 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 the fine genome of paddy rice Japan MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).

The open SSR molecular marker that the application is previously mentioned can be found in website http://www.gramene.org/。

Embodiment 1Seed selection imports the restructuring plant of brown planthopper resistant gene pack section

Material used in the present embodiment is paddy rice ' flourishing age B ' and paddy rice ' magnificent 2048B '.

' magnificent 2048B ' has good Brown Planthopper Resistance to paddy rice, thus it is speculated that possibly No. 4 dye Colour solid Bph3 (Liu etc., Nature Biotechnology.2015,33:301-305)、QBph4 (Hu etc., Molecular Breeding.2015,35:3) and Bph15 (Yang etc., Theoretical and Applied Genetics.2004,110:182-191) the gene cluster area being located Key effect is played in domain to the Brown Planthopper Resistance of the material.

In the Breeding Process of restructuring plant, prospect choosing is carried out to plant of recombinating using molecular labeling Select, the foreground selection molecular labeling to being adopted is screened.The molecular labeling portion for being used Divide and derive from website http://www.gramene.org/, partially self design.Method for designing is, The 6.1st edition is annotated with reference to the fine genome MSU/TIGR of paddy rice Japan, aforementioned zones segment DNA is downloaded Sequence.The SSR sites in above-mentioned sequence are scanned using SSRLocator softwares.Profit With the softwares of Primer Premier 3.0 to the SSR sites design primer for searching out, primer is designed altogether 106 pairs.By the method for PCR, above-mentioned primer pair is screened in ' magnificent 2048B ' and ' flourishing age Polymorphism in B ', finally picks out and has polymorphism, amplification efficiency high in two parts of materials Foreground selection molecular labeling, be respectively favorable selection mark BphC04ID06 and negative itemsets Mark RM6659, MS5.The concrete primer information for expanding above-mentioned molecular labeling for PCR is shown in Table 1.

The foreground selection molecular labeling primer information of table 1

By ' genomic fragment that forementioned gene cluster is located in magnificent 2048B ' imports to ' flourishing age B ' In, detailed process is as follows：

So that ' as recurrent parent, ' magnificent 2048B ' is hybridized flourishing age B ' for donor parents, will Resulting cenospecies is returned with recurrent parent, obtains BC₁F₁Seed, utilizes after nursery Favorable selection marks BphC04ID06 and negative itemsets mark RM6659, MS5 to be recombinated Single-plant selection, filters out 9 individual plants in target gene group fragment side homologous recombination, i.e., BphC04ID06 is detected and ' the identical banding pattern of magnificent 2048B ', and RM6659 or MS5 detects With ' the identical banding patterns of flourishing age B ', and using paddy rice full-length genome breeding chip RICE6K (CN102747138A) carries out Foreground selection (Yu etc., Plant Biotechnology to it Journal.2014,12:28-37)。

The comparable chip result in the 9 unilateral homologous recombination individual plants for filtering out, selects background to return Multiple best restructuring individual plant (this generation background recovery value is more than 75%) so as to ' contain with recurrent parent Generation B ' it is returned again, obtain BC₂F₁Seed, is marked after nursery using favorable selection BphC04ID06 detects to it, selects the restructuring individual plant containing target gene group fragment, That is BphC04ID06 is detected with ' the identical banding pattern of magnificent 2048B ', is educated using paddy rice full-length genome Plant chip RICE6K carries out Foreground selection to it.

Select background to 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 marks the seed of BphC04ID06 and negative indicia RM6659, MS5 to results The screening of target gene group fragment opposite side homologous recombination fragment is carried out, 9 is obtained in target patch The individual plant of section both sides restructuring, i.e. BphC04ID06 detections with ' the identical banding pattern of magnificent 2048B ', And RM6659 and MS5 detections and ' the identical banding patterns of flourishing age B '.

Using paddy rice 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), importing target fragment is screened 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, BC is obtained₃F₂, marked using favorable selection after nursery BphC04ID06 detects to it, selects the individual plant containing target gene group fragment, i.e., BphC04ID06 is detected and ' the identical banding pattern of magnificent 2048B ', using paddy rice full-length genome breeding core Piece RICE60K carries out Foreground selection to it.

It is final to obtain target fragment homozygosis, and background replys the strain of (background recovery value is more than 99%) It is one, is named as CR020142.Chip detection result is shown in Fig. 1.

Embodiment 2Import the determination of homologous recombination fragment after resistance gene of brown planthopper pack section

In order to determine the brown planthopper resistant gene group clip size of importing, to ' flourishing age B ' importing tablet The homozygosis individual plant of section has carried out the sequencing of target gene group fragment both sides homologous recombination fragment.Will Brown planthopper resistant gene group recombinant nucleic acid fragment contained by CR020142 is named as RecCR020142.

Primarily determined that by paddy rice full-length genome breeding chip RICE60K testing results, RecCR020142 is located at two SNP markers R0404288326CA and F0407038360TG Between.

Meanwhile, using Miseq sequencing technologies to ' flourishing age B ', ' magnificent 2048B ' and CR020142 Three samples carry out genome sequencing.Using TruSeq Nano DNA LT Kit (illumina) Kit carries out library foundation, using Library Quantification Kit-Universal (KAPA Biosystems) kit is carried out quantitatively, using MiSeq V2Reagent Kit (illumina) kit carries out sequencing reaction.Entered using the desk-top sequenators of Miseq (illumina) Row detection.Concrete steps and method are referring to each kit and sequenator operation instructions.

According to aforementioned SNP chip and Miseq sequencing results, by CR020142 brown planthopper resistant bases Because the 4294015bp that group recombinant fragment upstream homologous recombination fragment is positioned at the 4th chromosome is arrived 4295597bp is interval, and downstream homologous recombination fragment is positioned at 7015808bp to 7016712bp areas Between.

On this basis, the 6.1st edition is annotated with reference to the fine genome MSU/TIGR of paddy rice Japan, Download respective segments DNA sequence dna.Expanded using the Software for Design of Primer Premier 5.0 and surveyed Sequence primer, design requirement is long 22nt of primer or so, G/C content 40-60% and no mispairing.

With receptor parent, ' ' magnificent 2048B ' is control, to CR020142 for flourishing age B ' and donor parents Upstream and downstream homologous recombination fragment separately design amplimer, using high-fidelity enzyme KOD FX Neo (TOYOBO) are expanded, and using two-step method or three-step approach optimal amplification condition is found, Guarantee that amplified production is shown as single bright band in agarose gel electrophoresis detection.It is wherein true Fixed upstream homologous recombination fragment amplification primer reaction condition is：94℃2min；98℃ 10sec, 68 DEG C of 270sec, 37 circulations；20℃1min.Downstream homologous recombination fragment expands Increasing primer reaction condition is：94℃2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 150sec, 37 circulations；20℃1min.Thus, finally filter out two pairs of amplimers to be respectively used to Trip, the amplification of downstream homologous recombination fragment.

In addition, with amplified production as template, being sequenced using Sanger PCR sequencing PCRs, according to reality Border is sequenced effect, finally filter out 5 and 2 sequencing primers to be respectively used to upstream and downstream same 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.

RecCR020142 upstreams homologous recombination sequencing fragment length is 1609bp (SEQ ID NO: 1).1-534bp is the acceptor ' genomic segment of flourishing age B ', with donor ' magnificent 2048B ' ratios Compared with there are 4 SNP.This 401bp section of 535-935bp is homologous recombination section. 936-1609bp is donor ' magnificent 2048B ' genomic fragments, with ' flourishing age, B ' compared, and deposited In 3 SNP, 1 InDel.

RecCR020142 downstreams homologous recombination sequencing fragment length is 907bp (SEQ ID NO: 2).1-163bp is that the flourishing age, B ' compared donor ' genomic segment of magnificent 2048B ', with ', There are 9 SNP.This 124bp section of 164-287bp is homologous recombination section.288-907bp For acceptor, ' genomic segment of flourishing age B ', ' magnificent 2048B ' compares, and has 9 with donor Individual SNP, 2 Indel.

Fig. 4 is the structure chart of RecCR020142 both sides homologous recombination fragment.Wherein, (A) For upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment structure figure.Top alkali Base is that ' the SNP or InDel mark of magnificent 2048B ', lower section base is the acceptor ' flourishing age to donor The SNP or InDel mark of B '.Grey section be from ' flourishing age B ' genomic segment, Black section is from ' magnificent 2048B ' genomic segments, white section is homologous recombination area Section.Abscissa is fragment length, with base pairs (bp) as unit.

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

Embodiment 3' flourishing age B ' imports the Resistance Identification after brown planthopper resistant gene pack section

New lines CR020142, acceptor parent in order to identify resistance effect, to the application seed selection This ' flourishing age B ', donor parents ' magnificent 2048B ' (as positive control), and high sense is brown flies Lice kind ' coming No. 1 in platform ' (as negative control) carries out Resistance Identification in brown paddy plant hopper room, Authentication method is as follows.

Each part material is soaked seed indoors, vernalization, subsequently sowing pulling the plastic cover of grid line In basin, 3 rows of every part of material point sow altogether 45 plants, and when two one heart stages of leaf, often row retains 10 Totally 30 plants of consistent plant of healthy growing way are used to connect worm for strain.Worm sources are brown come what is collected since field Plant hopper, and captive breeding indoors.2-3 ages nymph is taken to plant to be identified, per plant has 5-10 head nymphs.Start recording respectively identifies seedling when ' coming No. 1 in platform ' dead seedling 95% Resistance class, take the mean value of the resistance class of 30 young plants, as the resistance class of the material, Its resistance level is evaluated according to resistance class.Wherein, resistance class is divided into 10 grades：0 or 1 grade, Blade is not aggrieved or first piece leaf blade tip turns to be yellow；2 grades, first piece leaf 1/2 turns to be yellow or blade tip wrinkle Contracting；3 grades, first piece leaf turns to be yellow or withered；4 grades, second leaf portion distributes yellow or lobus cardiacus leaf Point jaundice；5 grades, second leaf jaundice, shrinkage or withered, lobus cardiacus green grass or young crops volume；6 grades, lobus cardiacus Curling, lobus cardiacus tip burn on leaf；7 grades, lobus cardiacus curling is withered, and plant is not dead；8 grades, lobus cardiacus It is withered, somewhat lodge；9 grades, whole strain lodging.According to above-mentioned resistance class, 0-0.9 levels judge For high anti-, 1.0-2.9 levels be anti-, and 3.0-5.9 levels are anti-in being, 6.0-6.9 levels are middle sense, 7.0-7.9 Level is sense, and 8.0-9.0 levels are high sense.

Resistance Identification result is shown in Fig. 5 in brown paddy plant hopper room, wherein ' coming No. 1 in platform ' is high sense, ' flourishing age B ' is that sense, improvement new lines CR020142 resist in being to original kind, donor parents ' China 2048B ' is high anti-.

Although above having made in detail to the application with a general description of the specific embodiments 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 art personnel.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) SEQ ID NO are included:The sequence or its fragment of the nucleotides of sequence 534-936 position shown in 1 Or its variant or its complementary series；

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

Iii) SEQ ID NO are included:The sequence or its piece of the nucleotides of sequence 163-288 position shown in 2 Section or its variant or its complementary series；

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

The combination of above fragment.

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

(I) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-534 position shown in 1 Forward primer and specific recognition SEQ ID NO:The nucleotides of sequence 936-1609 position shown in 1 The reverse primer of sequence；

(II) combination of first group of primer pair and second group of primer pair below, it is included

(a) first group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer and specific recognition SEQ ID NO of the sequence of 1-534 positions nucleotides:Sequence shown in 1 Arrange the reverse primer of the sequence of 535-935 positions nucleotides；With

(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer and specific recognition SEQ ID NO of the sequence of 535-935 positions nucleotides:Shown in 1 The reverse primer of the sequence of sequence 936-1609 positions nucleotides；

(III) specific recognition includes SEQ ID NO:The nucleotides of sequence 534-535 positions shown in 1 Sequence forward primer and specific recognition include SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 935-936 positions nucleotides；

(IV) specific recognition includes SEQ ID NO:The nucleotides of sequence 534-535 positions shown in 1 Sequence forward primer and specific recognition SEQ ID NO:Sequence 936-1609 shown in 1 The reverse primer of the sequence of position nucleotides；

(V) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-534 positions shown in 1 Forward primer and specific recognition include SEQ ID NO:The cores of sequence 935-936 positions 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 nucleotides of sequence 1-163 positions shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleotides of sequence 288-907 positions shown in 2 Sequence reverse primer；

(VII) combination of the 3rd group of primer pair and the 4th group of primer pair below, it is included

(c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer and specific recognition SEQ ID NO of the sequence of 1-163 positions nucleotides:Sequence shown in 2 Arrange the reverse primer of the sequence of 164-287 positions nucleotides；With

(d) the 4th group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2 The forward primer and specific recognition SEQ ID NO of the sequence of 164-287 positions nucleotides:Shown in 2 The reverse primer of the sequence of sequence 288-907 positions nucleotides；

(VIII) specific recognition includes SEQ ID NO:The nucleosides of sequence 163-164 positions shown in 2 The forward primer and specific recognition of the sequence of acid includes SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 287-288 positions nucleotides；

(IX) specific recognition includes SEQ ID NO:The nucleotides of sequence 163-164 positions shown in 2 Sequence forward primer and specific recognition SEQ ID NO:Sequence 288-907 positions shown in 2 The reverse primer of the sequence of nucleotides；

(X) specific recognition SEQ ID NO:The sequence of the nucleotides of sequence 1-163 positions shown in 2 Forward primer and specific recognition include SEQ ID NO:The cores of sequence 287-288 positions shown in 2 The reverse primer of the sequence of thuja acid.

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

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

5 '-ACGAGTGTAAAGTGGCAAGC-3 ',

5’-AACAGCACCCACCACTATTA-3’；And

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

5’-ACGAGTGTAAAGTGGCAAGC-3’；

5’-ACAACGAGGCAACATGGTAA-3’；

5’-TTACTCTAAACCGAACAAGC-3’；

5’-ATACCTCCCAAGAACGTAGA-3’；

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

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

5 '-GTCCCTGCACCGTCTCCTACACT-3 ',

5’-ACTCCACAGACCTCTATCCCATC-3’；And

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

5’-GGTCGTTACGCAAGATTGA-3’；

5’-CTCACAAGGCCACAGGTATG-3’。

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

5. method as claimed in claim 4, wherein for the molecular labeling of the foreground selection One or more in BphC04ID06, RM6659 and MS5；And/or

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

6. the method as described in claim 4 or 5, wherein the recombinant nucleic acid fragment contain it is anti- Brown paddy plant hopper gene, and the method comprising the steps of：

1) recurrent parent and donor plant are hybridized, then by resulting cenospecies and samsara Parent is returned, and obtains first backcross generation, is marked BphC04ID06 using favorable selection and is born The unilateral homologous of brown planthopper resistant gene pack section is carried out to it to selected marker RM6659, MS5 Recombinant fragment is screened, and carries out Foreground selection to it using paddy rice full-length genome breeding chip；

2) select background to reply preferably restructuring individual plant to be returned again with recurrent parent, obtain Second backcross generation, using favorable selection mark BphC04ID06 it is detected, select containing The restructuring individual plant of brown planthopper resistant gene pack section, then using paddy rice full-length genome breeding chip pair It carries out Foreground selection；

3) select the restructuring individual plant that background is replied to be returned again with recurrent parent, obtain Third backcross generation, using favorable selection mark BphC04ID06 and negative itemsets mark RM6659, MS5 carries out the opposite side homologous recombination fragment screening of brown planthopper resistant gene pack section, and profit to it Foreground selection is carried out to it with paddy rice full-length genome breeding chip；And

4) select introgressed segment little, and the restructuring individual plant that background is replied, by the restructuring list chosen Strain selfing once, obtains selfed seed, and it is carried out using favorable selection mark BphC04ID06 Detection, and Foreground selection is carried out to it using paddy rice full-length genome breeding chip, finally contained The rice plant that homozygosis brown planthopper resistant gene group recombinant nucleic acid fragment and background are replied.

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

Amplifier molecule marks the primer pair of BphC04ID06, and it includes：

Forward primer：5 '-CCTAGCCGTCAGGTTAATAGATCAT-3 ',

Reverse primer：5’-ACCAGGTCTACTAGCTTTTACGGAG-3’；

Amplifier molecule marks the primer pair of RM6659, and it includes：

Forward primer：5 '-TGTGGAGGCTTAGGAAATTCTGG-3 ',

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

Amplifier molecule marks the primer pair of MS5, and it includes：

Forward primer：5 '-TTGTGGGTCCTCATCTCCTC-3 ',

Reverse primer：5’-TGACAACTTGTGCAAGATCA-3’.

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

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

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

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