CN106609270A - Recombinant nucleic acid fragment RecCR020264 and detection method thereof - Google Patents

Abstract

The invention provides a recombinant nucleic acid fragment and a detection method thereof. The invention also provides a breeding method of a paddy rice plant containing the recombinant nucleic acid fragment. Foreground selection and background selection of a recombinant plant are realized through a molecular marker and the paddy rice plant containing the recombinant nucleic acid fragment is obtained.

Description

Recombinant nucleic acid fragment RecCR020264 and its detection method

Technical field

The application is related to full-length genome selection and use technology.Specifically, the application relates to the use of Full-length genome selection and use technology selection-breeding contains the rice plant of recombinant nucleic acid fragment, and thus And the recombinant nucleic acid fragment 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 Oryza sativa L., 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 phloem 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 brown paddy plant hopper Drug resistance 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 are included NO:The sequence or its fragment of the nucleotide of sequence 634-669 position shown in 1 or its variant or its complementary sequence Row；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 of the nucleotide of sequence 687-1488 position shown in 2 or Its fragment or its variant or its complementary series；Iv) SEQ ID NO are included:The sequence of sequence shown in 2 Row or its fragment or its variant or its complementary series；And the combination of above fragment.Implement one In scheme, 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 nucleotide of sequence 1-634 position shown in 1 and Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 669-727 position shown in 1 is reversely drawn Thing；(II) combination of first group of primer pair and second group of primer pair below, it includes first group of (a) Primer pair：Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-634 positions shown in 1 Forward primer and specific recognition SEQ ID NO:The nucleotide of sequence 635-668 positions shown in 1 Sequence reverse primer；(b) second group of primer pair：Specific recognition SEQ ID NO:1 The forward primer and specific recognition SEQ ID of the sequence of shown sequence 635-668 positions nucleotide NO:The reverse primer of the sequence of the nucleotide of sequence 669-727 positions shown in 1；(III) specificity Identification includes SEQ ID NO:The forward direction of the sequence of the nucleotide of sequence 634-635 positions shown in 1 is drawn Thing and specific recognition include SEQ ID NO:The sequence of the nucleotide of sequence 668-669 positions shown in 1 The reverse primer of row；(IV) specific recognition includes SEQ ID NO:Sequence shown in 1 The forward primer and specific recognition SEQ ID NO of the sequence of 634-635 positions nucleotide:Shown in 1 The reverse primer of the sequence of sequence 669-727 positions nucleotide；(V) specific recognition SEQ ID NO:The forward primer and specific recognition bag of the sequence of the nucleotide of sequence 1-634 positions shown in 1 The NO of ID containing SEQ:The reverse primer of the sequence of the nucleotide of sequence 668-669 positions shown in 1；With / or optionally, (VI) specific recognition SEQ ID NO:The nucleoside of sequence 1-687 positions shown in 2 The forward primer and specific recognition SEQ ID NO of the sequence of acid:Sequence shown in 2 The reverse primer of the sequence of 1488-1514 positions nucleotide；(VII) the 3rd group of primer pair and the below The combination of four groups of primer pairs, it includes (c) the 3rd group of primer pair：Specific recognition SEQ ID NO: The forward primer and specific recognition SEQ ID of the sequence of the nucleotide of sequence 1-687 positions shown in 2 NO:The reverse primer of the sequence of the nucleotide of sequence 688-1487 positions shown in 2；(d) the 4th Group primer pair：Specific recognition SEQ ID NO:The nucleotide of sequence 688-1487 positions shown in 2 Sequence forward primer and specific recognition SEQ ID NO:Sequence 1488-1514 shown in 2 The reverse primer of the sequence of position nucleotide；(VIII) specific recognition includes SEQ ID NO:2 institutes The forward primer and specific recognition for showing the sequence of sequence 687-688 positions nucleotide includes SEQ ID NO:The reverse primer of the sequence of the nucleotide of sequence 1487-1488 positions shown in 2；(IX) it is special Opposite sex identification includes SEQ ID NO:The sequence of the nucleotide of sequence 687-688 positions shown in 2 is just To primer and specific recognition SEQ ID NO:The nucleotide of sequence 1488-1514 positions shown in 2 The reverse primer of sequence；(X) specific recognition SEQ ID NO:Sequence 1-687 positions shown in 2 The forward primer and specific recognition of the sequence of nucleotide includes SEQ ID NO:Sequence shown in 2 The reverse primer of the sequence of 1487-1488 positions nucleotide.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-GGGTGTTGCATGAGGTGTTCAG-3 ', 5 '-TGTGCTTAGAT CAAGGCGTAAA-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 1 is, For example, 5 '-GGGTGTTGCATGAGGTGTTCAG-3 '；With 5 '-TGTGCTTAGAT CAAGGCGTAAA-3’。

In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2 For, for example, 5 '-CTGTGGATACCAGCAAAGAC-3 ', 5 '-TGTACGCACT GTACCAAGAC-3’.For detecting SEQ ID NO:The sequencing primer of sequence shown in 2 is, For example, 5 '-GCAGATGGACCGAACAAACA-3 '；5’-ATGTGCCTGGTCA GTCTGCT-3’；With 5 '-TGTACGCACTGTACCAAGAC-3 '.

On the other hand, this application provides selection-breeding contains the side of the rice plant of recombinant nucleic acid fragment Method, it is included using the Oryza sativa L. recipient plant parent without genes of interest pack section as recurrent parent, It is hybridized with the Oryza sativa L. 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 marker.For example, The recombinant nucleic acid fragment is as previously mentioned.

In the above-mentioned methods, for the foreground selection molecular marker selected from BphC03ID03, One or more in RM16165 and RM16211；And/or using Oryza sativa L. full-length genome breeding Chip carries out the Foreground selection.

In one embodiment, the selection-breeding 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 Generation, using favorable selection labelling BphC03ID03 and negative itemsets labelling RM16165, RM16211 carries out the unilateral homologous recombination fragment screening of brown planthopper resistant gene pack section to it, and Using Oryza sativa L. full-length genome breeding chip, such as RICE6K carries out Foreground selection to it；2) Background is selected to reply preferably restructuring individual plant (this generation background recovery value is more than 75%) with samsara parent This is returned again, obtains second backcross generation, using favorable selection labelling BphC03ID03 pair It is detected, the restructuring individual plant containing brown planthopper resistant gene pack section is selected, then using water Rice full-length genome breeding chip, such as RICE6K carries out Foreground selection to it；3) background is selected The restructuring individual plant (this generation background recovery value is more than 87.5%) replied is with recurrent parent again It is returned, is obtained third backcross generation, is selected using favorable selection labelling BphC03ID03 and negative sense Select labelling RM16165, RM16211 it is carried out brown planthopper resistant gene pack section opposite side it is same Source recombinant fragment screening, and using Oryza sativa L. full-length genome breeding chip, such as RICE60K is right It carries out Foreground selection；And 4) select introgressed segment little, and the restructuring individual plant that background is replied (background recovery value is more than 93.75%), by the restructuring individual plant selfing chosen once, obtains selfed seed, It is detected using favorable selection labelling BphC03ID03, and using Oryza sativa L. full-length genome Breeding chip, such as RICE60K, to it Foreground selection is carried out, and final acquisition resists brown containing homozygosis The Oryza sativa L. of plant hopper genome recombination nucleic acid fragment and background reply (background recovery value is more than 99%) is planted Strain.

In another embodiment, adopt when carrying out foreground selection to plant of recombinating using molecular marker Amplimer, including：For the primer pair of amplifier molecule labelling BphC03ID03, its Middle forward primer is 5 '-GCAAGAATCCGACGCCATAA-3 ', and reverse primer is 5’-CTCTGCTCCTTGCTCTAATCCTCT-3’；For amplifier molecule labelling The primer pair of RM16165, wherein forward primer are 5 '-GGTTAACCAAGAGGAAA GGAACC-3 ', reverse primer is 5 '-GCTTTGCAACTCACTGTTGTTACG-3 '； For the primer pair of amplifier molecule labelling RM16211, wherein forward primer is 5 '-AATGCTAATGGCGACTGACTTCG-3 ', reverse primer is 5 '-ATGGGCTT GTTTGATTGCATCC-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 sequencing.

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 '-GGGTGTTGCATGAGGTGTTCAG-3 ', reverse primer：5’-TGTGC TTAGATCAAGGCGTAAA-3’；Sequencing primer, including forward primer：5’-GGGTGTT GCATGAGGTGTTCAG-3 ', and forward primer：5’-TGTGCTTAGATCAA GGCGTAAA-3’.Methods described with testing sample genomic DNA as template, using above-mentioned Amplimer enters performing PCR amplification, then the amplified production for obtaining is entered using above-mentioned sequencing primer Row sequencing, if sequencing result and SEQ ID NO:1 sequence is consistent or complementary, then in testing sample Containing 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 '-CTGTGGATACCAGCAAAGAC-3 ', reverse primer：5’-TGTACG CACTGTACCAAGAC-3’；Sequencing primer, including forward primer：5’-GCAGATGGA CCGAACAAACA-3 ', forward primer：5’-ATGTGCCTGGTCAGTCTGC T-3 ', and reverse primer：5’-TGTACGCACTGTACCAAGAC-3’.Methods described with Testing sample genomic DNA is template, enters performing PCR amplification using above-mentioned amplimer, so Afterwards the amplified production for obtaining is sequenced using above-mentioned sequencing primer, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, then contain SEQ ID NO in testing sample:It is homologous shown in 2 Recombinant fragment.

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

Additionally, present invention also provides the test 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 test 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 selection-breeding 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 acceptor material of the application improvement is for ' Lu perfume 618B ', is with Odor type sterile line ' 618A ' the supporting maintainer of Lu perfume of low amylose content.Using above-mentioned Method, ' in the case of the perfume original advantages of 618B ' of Lu, can improve its brown paddy plant hopper retaining Resistance.Further, stable containing can be obtained by least first cross, generation backcrossing Brown Planthopper Resistance fragment ' Lu perfume 618A ', then cenospecies Brown Planthopper Resistance is realized by combo Increase substantially.Meanwhile, the genome recombination 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 CR020264 Oryza sativa L. 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 Oryza sativa L. Body, vertical coordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome, Lycoperdon polymorphum Vitt lines represent receptor parent, and ' Lu perfume 618B ' genotype, black lines represent donor parents ' magnificent 3418B ' genotype, it is consistent i.e. without polymorphic region that white line represents two parent genotypes Section.Lines display block is the brown planthopper resistant for importing at No. 3 chromosome black round dot in figure Genome recombination nucleic acid fragment RecCR020264.

Fig. 2 is RecCR020264 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 CR020264 is in figure The new lines of acquisition, T004 is that ' Lu perfume 618B ', R002 are donor parents ' China to receptor parent 3418B’。

Fig. 3 is RecCR020264 downstreams homologous recombination sequencing fragment ratio in the embodiment of the present application 2 To result.

Fig. 4 is the structure of RecCR020264 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 ' SNP the or InDel labellings of magnificent 3418B ', lower section alkali Base is receptor ' SNP the or InDel labellings of Lu perfume 618B '.Lycoperdon polymorphum Vitt section is from ' Lu Fragrant 618B ' genomic segments, black section be from ' magnificent 3418B ' genomic segments, White section is homologous recombination section, and abscissa is fragment length, is with base pairs (bp) Unit.

Fig. 5 is qualification result in CR020264 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 ' Lu perfume 618B '；(C) new lines CR020264 are improved；(D) donor parents ' magnificent 3418B '.

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 Nucleotide 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(PNA)), the analog with naturally occurring nucleotide similar mode and single-stranded core Acid hybridization.

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

In some embodiments, the application further relates to SEQ ID NO:1 or SEQ ID NO:2 The variant of shown sequence.In general, the variant of specific nucleotide sequence will be with the specific nucleoside 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 seriess.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 nucleotide, as little as 1-10 (such as 6-10), As little as 5, as little as 4,3,2 or or even 1 nucleotide.

The application is further related to comprising SEQ ID NO:1 or SEQ ID NO:It is specific in sequence shown in 2 The fragment in site or its variant or its complementary series, for example, comprising SEQ ID NO:Sequence shown in 1 The sequence or its fragment of the 634-669 positions nucleotide of row or its variant or its complementary series, or Comprising SEQ ID NO:The sequence or its fragment of the 687-1488 positions nucleotide of sequence shown in 2 or its Variant or its complementary series.According to the fragment comprising above-mentioned specific site, can specifically reflect Make corresponding SEQ ID NO:1 or SEQ ID NO:Sequence shown in 2.Further, pass through Identify containing SEQ ID NO:1 or SEQ ID NO:The recombinant nucleic acid fragment of sequence shown in 2, Can determine that and include in testing sample the recombinant nucleic acid fragment containing resistant gene.

As used herein, " Oryza sativa L. " 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 selection-breeding 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 Oryza sativa L. ' Lu perfume 618B ' As recurrent parent, using the Oryza sativa L. ' magnificent 3418B ' conducts with good Brown Planthopper Resistance Donor.

In the selection of restructuring plant provided herein, using molecular marker to restructuring Plant carries out foreground selection.The reliability of foreground selection depends primarily on labelling 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 labellings selection is tracked to target gene group fragment.

In the embodiment of the application, the foreground selection labelling of employing includes favorable selection labelling With negative itemsets labelling, wherein, favorable selection labelling be away from target gene group fragment (containing anti- Brown paddy plant hopper gene) screen in the range of upstream and downstream 50kb (in Oryza sativa L. its genetic distance be 0.2cM) Pleiomorphism molecular marker.Negative itemsets labelling is away from target gene group fragment upstream and downstream The pleiomorphism molecular marker screened in the range of 500kb (its genetic distance is 2cM in Oryza sativa L.). In specific embodiment, the positive foreground selection labelling of optimized Select to use is and target gene The labelling BphC03ID03 of pack section close linkage, negative itemsets labelling is in target fragment The molecular marker RM16165 of trip about 430kb, and dividing away from target fragment downstream about 370kb Sub- labelling RM16211.

In the embodiment of the application, using above-mentioned foreground selection labelling homologous recombination is carried out During detection, the criterion of side or unilateral homologous recombination is BphC03ID03 detections and ' China The identical banding patterns of 3418B ', and RM16165 or RM16211 detections and ' Lu perfume 618B ' phases Same banding pattern；The criterion of both sides or bilateral homologous recombination is BphC03ID03 detections and ' China The identical banding patterns of 3418B ', and RM16165 and RM16211 detections and ' Lu perfume 618B ' phases Same banding pattern.

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

Following examples are merely to illustrate and the purpose of unrestricted the application scope.If not referring in particular to Bright, embodiment 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 description suggestion.

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

The rice genome physical location being previously mentioned in the application is with reference to the fine genome of Oryza sativa L. 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 1Selection-breeding imports the restructuring plant of brown planthopper resistant gene pack section

Material used in the present embodiment is Oryza sativa L. ' Lu perfume 618B ' and Oryza sativa L. ' magnificent 3418B '.

' magnificent 3418B ' has good Brown Planthopper Resistance to Oryza sativa L., thus it is speculated that possibly No. 3 dye Colour solid QBph3 (Hu etc., Molecular Breeding.2015,35:3) with Bph14 (Du Deng, PNAS.2009,106 (52):22163-22168) gene cluster region being located is to the material Brown Planthopper Resistance has played pivotal role.

In the Breeding Process of restructuring plant, prospect choosing is carried out to plant of recombinating using molecular marker Select, the foreground selection molecular marker to being adopted is screened.The molecular marker 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 Oryza sativa L. 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 128 pairs.By the method for PCR, above-mentioned primer pair is screened in ' magnificent 3418B ' and ' Lu perfume Polymorphism in 618B ', finally picks out and has polymorphism, amplification efficiency in two parts of materials High foreground selection molecular marker, is respectively favorable selection labelling BphC03ID03 and negative sense choosing Select labelling RM16165, RM16211.Specifically drawing for above-mentioned molecular marker is expanded for PCR Thing information 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 3418B ' imports to ' Lu perfume 618B ' In, detailed process is as follows：

With ' Lu perfume 618B ' as recurrent parent, ', magnificent 3418B ' is hybridized for donor parents, By resulting cenospecies, ' Lu perfume 618B ' is returned, and obtains BC with recurrent parent₁F₁ Seed, using favorable selection labelling BphC03ID03 and negative itemsets labelling after nursery RM16165, RM16211 carry out restructuring Single-plant selection, filter out 8 in target gene pack The individual plant of section side homologous recombination, i.e. BphC03ID03 detections and ' the identical band of magnificent 3418B ' Type, and RM16165 or RM16211 detections and ' identical banding patterns of Lu perfume 618B ', and utilizing Oryza sativa L. full-length genome breeding chip RICE6K (CN102747138A) carries out Foreground selection to it (Yu etc., Plant Biotechnology Journal.2014,12:28-37).

The comparable chip result in the 8 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 recurrent parent ' Lu Fragrant 618B ' is returned again, obtains BC₂F₁Seed, utilizes favorable selection labelling after nursery BphC03ID03 detects to it, selects the restructuring individual plant containing target gene group fragment, That is BphC03ID03 is detected with ' the identical banding pattern of magnificent 3418B ', is educated using Oryza sativa L. 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 ' Lu perfume 618B ' is returned again recurrent parent, obtains BC₃F₁Seed, profit after nursery With favorable selection labelling BphC03ID03 and negative indicia RM16165, RM16211 to harvesting Seed carry out the screening of target gene group fragment opposite side homologous recombination fragment, obtain 3 The individual plant of target fragment both sides restructuring, i.e. BphC03ID03 is detected with ' magnificent 3418B ' is identical Banding pattern, and RM16165 and RM16211 detections and ' the identical banding patterns of Lu perfume 618B '；.

Using Oryza sativa L. full-length genome breeding chip RICE60K (WO/2014/121419) to above-mentioned 3 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, favorable selection labelling is utilized after nursery BphC03ID03 detects to it, selects the individual plant containing target gene group fragment, i.e., BphC03ID03 is detected and ' the identical banding pattern of magnificent 3418B ', using Oryza sativa L. 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 CR020264.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 ' Lu perfume 618B ' leads Entering the homozygosis individual plant of fragment has carried out the sequencing of target gene group fragment both sides homologous recombination fragment. Brown planthopper resistant gene group recombinant nucleic acid fragment contained by CR020264 is named as RecCR020264。

Primarily determined that by Oryza sativa L. full-length genome breeding chip RICE60K testing results, RecCR020264 is located at two SNP markers R0335257356CA and R0335762765GA Between.

Meanwhile, using Miseq sequencing technologies to ' Lu perfume 618B ', ' magnificent 3418B ' and Tri- samples of CR020264 carry out genome sequencing.Using TruSeq Nano DNA LT Kit (illumina) test kit carries out library foundation, using Library Quantification Kit- Universal (KAPA Biosystems) test kit is carried out quantitatively, using MiSeq V2Reagent Kit (illumina) test kit carries out sequencing reaction.Using the desk-top sequenators of Miseq (illumina) Detected.Concrete steps and method are referring to each test kit and sequenator operation instructions.

According to aforementioned SNP chip and Miseq sequencing results, by CR020264 brown planthopper resistant bases Because the 35258029bp that group recombinant fragment upstream homologous recombination fragment is positioned at the 3rd chromosome is arrived 35258755bp is interval, and downstream homologous recombination fragment is positioned at 35758065bp to 35759549bp It is interval.

On this basis, the 6.1st edition is annotated with reference to the fine genome MSU/TIGR of Oryza sativa L. Japan, Download respective segments DNA sequence.Expanded using the software designs 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 3418B ' is control, right for Lu perfume 618B ' and donor parents The upstream and downstream homologous recombination fragment of CR020264 separately designs amplimer, using high-fidelity Enzyme KOD FX Neo (TOYOBO) is expanded, and finds optimal using two-step method or three-step approach Amplification condition, it is ensured that amplified production is shown as single bright bar in agarose gel electrophoresiies detection Band.The upstream homologous recombination fragment amplification primer reaction condition for wherein determining is：94℃2min； 98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 60sec, 37 circulations；20℃1min.Downstream Homologous recombination fragment amplification primer reaction condition is：94℃2min；98 DEG C of 10sec, 60 DEG C 30sec, 68 DEG C of 90sec, 37 circulations；20℃1min.Thus, two pairs are finally filtered out Amplimer is respectively used to the amplification of upstream and downstream homologous recombination fragment.

In addition, with amplified production as template, being sequenced using Sanger sequencing, according to reality Border is sequenced effect, finally filter out 2 and 3 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.

RecCR020264 upstreams homologous recombination sequencing fragment length is 727bp (SEQ ID NO:1).1-634bp is the receptor ' genomic segment of Lu perfume 618B ', with donor ' magnificent 3418B ' Relatively, there are 13 SNP, 2 Indel.This 34bp section of 635-668bp is homologous Restructuring section.669-727bp is donor ' magnificent 3418B ' genomic fragments, with ' Lu perfume 618B ' Relatively, there are 3 SNP, 1 Indel.

RecCR020264 downstreams homologous recombination sequencing fragment length is 1514bp (SEQ ID NO:2).1-687bp is the donor ' genomic segment of magnificent 3418B ', with ' Lu perfume 618B ' Relatively, there are 3 SNP, 2 Indel.This 800bp section of 688-1487bp is homologous Restructuring section.1488-1514bp is the receptor ' genomic segment of Lu perfume 618B ', with donor ' magnificent 3418B ' compares, and there are 7 SNP.

Fig. 4 is the structure chart of RecCR020264 both sides homologous recombination fragment.Wherein, (A) For upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment structure figure.Top alkali Base is that ' SNP the or InDel labellings of magnificent 3418B ', lower section base is that ' Lu is fragrant for receptor to donor SNP the or InDel labellings of 618B '.Lycoperdon polymorphum Vitt section is from ' Lu perfume 618B ' genes Group section, black section is from ' magnificent 3418B ' genomic segments, white section is same Source restructuring 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' Lu perfume 618B ' imports the Resistance Identification after brown planthopper resistant gene pack section

New lines CR020264, receptor parent in order to identify resistance effect, to the application selection-breeding This ' Lu perfume 618B ', donor parents ' magnificent 3418B ' (as positive control), and high sense Brown paddy plant hopper kind ' coming No. 1 in platform ' (as negative control) carries out resistance mirror in brown paddy plant hopper room Fixed, authentication method is as follows.

Each part material is soaked seed indoors, accelerating germination, 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 meansigma methodss 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, ' Lu perfume 618B ' is sense to original kind, and improvement new lines CR020264 resist in being, donor parents ' magnificent 3418B ' 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 nucleotide of sequence 634-669 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 nucleotide of sequence 687-1488 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 nucleotide of sequence 1-634 position shown in 1 Forward primer and specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 669-727 position shown in 1 The reverse primer of row；

(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 1-634 positions nucleotide:Sequence shown in 1 Arrange the reverse primer of the sequence of 635-668 positions nucleotide；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 635-668 positions nucleotide:Shown in 1 The reverse primer of the sequence of sequence 669-727 positions nucleotide；

(III) specific recognition includes SEQ ID NO:The nucleotide of sequence 634-635 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 668-669 positions nucleotide；

(IV) specific recognition includes SEQ ID NO:The nucleotide of sequence 634-635 positions shown in 1 Sequence forward primer and specific recognition SEQ ID NO:Sequence 669-727 positions shown in 1 The reverse primer of the sequence of nucleotide；

(V) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-634 positions shown in 1 Forward primer and specific recognition include SEQ ID NO:The cores of sequence 668-669 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 nucleotide of sequence 1-687 positions shown in 2 Forward primer and specific recognition SEQ ID NO:The nucleoside of sequence 1488-1514 positions shown in 2 The reverse primer of the sequence of acid；

(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 1-687 positions nucleotide:Sequence shown in 2 Arrange the reverse primer of the sequence of 688-1487 positions nucleotide；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 688-1487 positions nucleotide:2 institutes Show the reverse primer of the sequence of sequence 1488-1514 positions nucleotide；

(VIII) specific recognition includes SEQ ID NO:The nucleoside of sequence 687-688 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 1487-1488 positions nucleotide；

(IX) specific recognition includes SEQ ID NO:The nucleotide of sequence 687-688 positions shown in 2 Sequence forward primer and specific recognition SEQ ID NO:Sequence 1488-1514 shown in 2 The reverse primer of the sequence of position nucleotide；

(X) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-687 positions shown in 2 Forward primer and specific recognition include SEQ ID NO:Sequence 1487-1488 positions shown in 2 The reverse primer of the sequence of nucleotide.

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 '-GGGTGTTGCATGAGGTGTTCAG-3 ',

5’-TGTGCTTAGATCAAGGCGTAAA-3’；And

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

5’-GGGTGTTGCATGAGGTGTTCAG-3’；

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

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

5 '-CTGTGGATACCAGCAAAGAC-3 ',

5’-TGTACGCACTGTACCAAGAC-3’；And

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

5’-GCAGATGGACCGAACAAACA-3’；

5’-ATGTGCCTGGTCAGTCTGCT-3’；

5’-TGTACGCACTGTACCAAGAC-3’。

4. the method that selection-breeding contains the rice plant of the recombinant nucleic acid fragment described in claim 1, It includes using the Oryza sativa L. recipient plant parent without genes of interest pack section as recurrent parent, incites somebody to action It is hybridized with the Oryza sativa L. donor plant containing genes of interest pack section, then will be resulting Cenospecies are 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 plant of recombinating using molecular marker.

5. method as claimed in claim 4, wherein for the molecular marker of the foreground selection One or more in BphC03ID03, RM16165 and RM16211；And/or

Optionally, the Foreground selection is carried out using Oryza sativa L. 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, using favorable selection labelling BphC03ID03 and negative The one side of brown planthopper resistant gene pack section is carried out to it to selected marker RM16165, RM16211 Homologous recombination fragment is screened, and carries out Foreground selection to it using Oryza sativa L. 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, is detected using favorable selection labelling BphC03ID03 to it, select containing The restructuring individual plant of brown planthopper resistant gene pack section, then using Oryza sativa L. 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 labelling BphC03ID03 and negative itemsets labelling RM16165, RM16211 carries out the opposite side homologous recombination fragment screening of brown planthopper resistant gene pack section to it, And Foreground selection is carried out to it using Oryza sativa L. 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 labelling BphC03ID03 Detection, and Foreground selection is carried out to it using Oryza sativa L. 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 marker pair The amplimer that restructuring plant carries out being adopted during foreground selection is as follows：

The primer pair of amplifier molecule labelling BphC03ID03, it includes：

Forward primer：5 '-GCAAGAATCCGACGCCATAA-3 ',

Reverse primer：5’-CTCTGCTCCTTGCTCTAATCCTCT-3’；

The primer pair of amplifier molecule labelling RM16165, it includes：

Forward primer：5 '-GGTTAACCAAGAGGAAAGGAACC-3 ',

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

The primer pair of amplifier molecule labelling RM16211, it includes：

Forward primer：5 '-AATGCTAATGGCGACTGACTTCG-3 ',

Reverse primer：5’-ATGGGCTTGTTTGATTGCATCC-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 test 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 test kit described in claim 9.