CN106480054A - Recombinant nucleic acid fragment RecCR020322 and its detection method - Google Patents

Abstract

This application provides recombinant nucleic acid fragment and its detection method.The selection of the rice plant containing recombinant nucleic acid fragment that the application provides, carries out foreground selection and Foreground selection using molecular marker to restructuring plant, obtains the rice plant containing recombinant nucleic acid fragment.

Description

Recombinant nucleic acid fragment RecCR020322 and its detection method

Technical field

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

Background technology

Brown paddy plant hopper, scientific name Nilaparvata lugens (), belong to Homoptera, Delphacidae.Brown Plant hopper is monophagy insect, only takes food Oryza sativa L., has happiness temperature, cold tolerance is weak, growth cycle 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 producing 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.

Since the eighties in 20th century, from wild rice and cultivated rice identified more than 30 Individual brown planthopper resistant site.Due to the complexity of brown paddy plant hopper phenotypic evaluation, lead to just 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, producing with brown planthopper resistant kind In continuous utilization, brown paddy plant hopper gradually strengthens to the adaptability of kind.Some are wide on producing The resistant variety of general utilization is just gradually 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 Plus production cost, pollute environment, and promote brown paddy plant hopper Drug resistance to strengthen.

As can be seen here, the cultivation demand of brown planthopper resistant new varieties is very urgent.

Content of the invention

On the one hand, this application provides recombinant nucleic acid fragment, it is selected from：I) comprise SEQ ID NO: The sequence of the nucleotide of sequence 281-1019 position shown in 1 or its fragment or its variant or its complementary sequence Row；Ii) comprise SEQ ID NO:The sequence of sequence shown in 1 or its fragment or its variant or it is mutual Complementary series.In one embodiment, described recombinant nucleic acid fragment is genome recombination nucleic acid fragment.

Additionally, this application provides detecting the primer of described recombinant nucleic acid fragment, it is selected from：(I) special Opposite sex identification SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 1-281 position shown in 1 and Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1019-1488 position shown in 1 reverse Primer；(II) the combining of first group of primer pair and second group of primer pair below, it comprises (a) first Group primer pair：Specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-281 position shown in 1 The forward primer of row and specific recognition SEQ ID NO:The core of sequence 282-1018 position shown in 1 The reverse primer of the sequence of thuja acid；(b) second group of primer pair：Specific recognition SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 282-1018 position shown in 1 and specific recognition SEQ ID NO:The reverse primer of the sequence of the nucleotide of sequence 1019-1488 position shown in 1；(III) Specific recognition comprises SEQ ID NO:The sequence of the nucleotide of sequence 281-282 position shown in 1 Forward primer and specific recognition comprise SEQ ID NO:The core of sequence 1018-1019 position shown in 1 The reverse primer of the sequence of thuja acid；(IV) specific recognition comprises SEQ ID NO:Sequence shown in 1 Arrange the forward primer of sequence and the specific recognition SEQ ID NO of 281-282 position nucleotide:1 The reverse primer of the sequence of shown sequence 1019-1488 position nucleotide；(V) specific recognition SEQ ID NO:The forward primer of sequence of the nucleotide of sequence 1-281 position shown in 1 and specificity Identification comprises SEQ ID NO:The sequence of the nucleotide of sequence 1018-1019 position shown in 1 reverse Primer.

In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is, For example, 5 '-CACGCTCTTCGTCTAGCCACCTCC-3 ', and 5 '-TTCGTACAAG CCCGTTGCCATCTC-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 1 is drawn Thing is, for example, 5 '-CACGCTCTTCGTCTAGCCACCTCC-3 '； 5’-TTGATTGGCATGTCTACTGG-3’；With 5 '-TTCGTACAAGCCCGTTG CCATCTC-3’.

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

In the above-mentioned methods, for described foreground selection molecular marker be selected from BphC03ID03, One or more of RM16175 and RM16211；And/or utilize Oryza sativa L. full-length genome breeding Chip carries out described Foreground selection.

In one embodiment, the selection-breeding that the application provides contains brown planthopper resistant gene group recombinant nuclear The method of the rice plant of acid fragment, it comprises the following steps：1) recurrent parent is planted with donor Thing is hybridized, and obtained cenospecies and recurrent parent are returned, and obtains first backcross generation, Using favorable selection labelling BphC03ID03 and negative itemsets labelling RM16175, RM16211 It is carried out with the unilateral homologous recombination fragment screening of brown planthopper resistant gene group fragment, and utilizes Oryza sativa L. Full-length genome breeding chip, such as RICE6K, Foreground selection is carried out 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, is obtained second backcross generation, using favorable selection labelling BphC03ID03, it is detected, Select the restructuring individual plant containing brown planthopper resistant gene group fragment, then educated using Oryza sativa L. full-length genome Plant chip, such as RICE6K, Foreground selection is carried out to it；3) select the restructuring that background is replied Individual plant (this generation background recovery value is more than 87.5%) is returned again with recurrent parent, obtains Third backcross generation, is detected to it using favorable selection labelling BphC03ID03, select containing The restructuring individual plant of brown planthopper resistant gene group fragment, then utilizes Oryza sativa L. full-length genome breeding chip, Such as RICE60K, carries out Foreground selection to it；And 4) select introgressed segment little, and background The restructuring individual plant (background recovery value is more than 93.75%) replied, by the restructuring chosen individual plant selfing Once, obtain selfed seed, using favorable selection labelling BphC03ID03, it detected, And utilize Oryza sativa L. full-length genome breeding chip, such as RICE60K, Foreground selection is carried out to it, The final acquisition group recombinant nucleic acid fragment of brown planthopper resistant gene containing homozygosis and background reply (background reply Value is more than 99%) rice plant.

In another embodiment, using molecular marker, restructuring plant is carried out adopting during foreground selection Amplimer, including：For the primer pair of amplifier molecule labelling BphC03ID03, its Middle forward primer is 5 '-GCAAGAATCCGACGCCATAA-3 ', and reverse primer is 5’-CTCTGCTCCTTGCTCTAATCCTCT-3’；For amplifier molecule labelling The primer pair of RM16175, wherein forward primer are 5 '-AGCTTTGGTTTCTTGGCTTT GG-3 ', reverse primer is 5 '-ATTAGCGTTGAACCCAAGTGTGG-3 '；For expanding Increase the primer pair of molecular marker RM16211, wherein forward primer is 5 '-AATGCTAATGG CGACTGACTTCG-3 ', reverse primer is 5 '-ATGGGCTTGTTTGATTGCAT CC-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 with testing gene group for template PCR reacts, and the step analyzing pcr amplification product.Specifically, for example, described primer is such as Front described.Selectively, analyze pcr amplification product using Sanger sequencing.

Specifically, in the method for detection recombinant nucleic acid fragment that the application provides, for expanding And detection SEQ ID NO:The primer combination of sequence shown in 1 is as follows：Amplimer, including positive Primer：5 '-CACGCTCTTCGTCTAGCCACCTCC-3 ', and reverse primer： 5’-TTCGTACAAGCCCGTTGCCATCTC-3’；Sequencing primer, including forward primer： 5 '-CACGCTCTTCGTCTAGCCACCTCC-3 ', reverse primer：5’-TTGATTGG CATGTCTACTGG-3 ', and reverse primer：5’-TTCGTACAAGCCCGTTGCC ATCTC-3’.Methods described with testing sample genomic DNA as template, using above-mentioned amplification Primer enters performing PCR amplification, then using above-mentioned sequencing primer, the amplified production obtaining is surveyed 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.

Determined by detection and in testing sample, contain SEQ ID NO:The restructuring of sequence shown in 1 Nucleic acid fragment, you can determine and comprise resistant gene group recombinant nucleic acid fragment in testing sample.

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

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

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

What the application provided contains brown planthopper resistant gene based on full-length genome selection and use technology selection-breeding Group recombinant nucleic acid fragment rice plant method, have quick, accurate, stablize three big features. Only pass through the transformation of five generations, you can only target gene group fragment is imported acceptor material, and simultaneously Realize the reply of background.The acceptor material of the application improvement is restorer ' high mountain for widely used three Extensive 9113 '.Using said method, ' the premise of extensive 9113 ' the original features in high mountain can retained Under, import resistance gene of brown planthopper group fragment.Further, cenospecies are realized by combo brown The increasing substantially of plant hopper resistance.The recombinant nucleic acid fragment that the application provides is tight with Brown Planthopper Resistance Close correlation, can be applied to the cultivation of other kinds as Resistance resource.

Brief description

Fig. 1 is CR020322 Oryza sativa L. RICE60K full-length genome breeding in the embodiment of the present application 1 Chip detection result；Wherein, square frame indicated by abscissa numeral represents 12 dyeing of Oryza sativa L. successively Body, vertical coordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome, Lycoperdon polymorphum Vitt lines represent receptor parent, and ' the extensive 9113 ' genotype in high mountain, black lines represent donor parents ' magnificent 3418B ' genotype, white line represents the consistent i.e. no polymorphic region of two parent genotypes Section.At No. 3 chromosome black round dot of in figure, lines display block is the brown planthopper resistant importing Genome recombination nucleic acid fragment RecCR020322.

Fig. 2 is RecCR020322 upstream homologous recombination sequencing fragment ratio in the embodiment of the present application 2 To result；Asterisk shown in figure represents identical base in comparison result, and in figure CR020322 is The new lines obtaining, T005 is that ' high mountain extensive 9113 ', R002 is donor parents ' China to receptor parent 3418B’.

Fig. 3 is the structure of RecCR020322 upstream homologous recombination fragment in the embodiment of the present application 2 Figure；Top base is that ' SNP the or InDel labelling of magnificent 3418B ', lower section base is donor Receptor ' SNP the or InDel labelling on high mountain extensive 9113 '.Lycoperdon polymorphum Vitt section is from ' high mountain extensive 9113 ' Genomic segment, black section is from ' magnificent 3418B ' genomic segment, white section For homologous recombination section, abscissa is fragment length, with base pairs (bp) as unit.

Fig. 4 is CR020322 Brown Planthopper Resistance interior qualification result in the embodiment of the present application 3； Shown in figure, blade is followed successively by：(A) high sense brown paddy plant hopper kind ' coming No. 1 in platform '；(B) original Plant ' high mountain extensive 9113 '；(C) new lines CR020322 are improved；(D) donor parents ' magnificent 3418B '.

Specific embodiment

There is provided defined below and method in order to preferably to define the application and to put into practice middle finger in the application Lead those of ordinary skill in the art.Unless otherwise mentioned, term is according to association area ordinary skill people The common usage of member understands.

As used herein, " nucleotide sequence " includes being related to the deoxyribose of single-stranded or double-stranded form Nucleotide or ribonucleotide polymer, and unless otherwise restriction, nucleotide sequence is with 5 ' extremely 3 ' directions are write from left to right, including the known analog (example with natural nucleotide fundamental property As peptide nucleic acid(PNA)), described analog with naturally occurring nucleotide similar mode and single-stranded core Acid hybridization.

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

Specifically, the application is related to SEQ ID NO:1 nucleotides sequence optimizing gained further Row.The more details of the method are described in Murray etc. (1989) Nucleic Acids Res. 17:477-498.Optimization nucleotide sequence can be used for raising brown planthopper resistant gene group recombinant fragment and exists Expression in Oryza sativa L..

In some embodiments, the application further relates to SEQ ID NO:The change of sequence shown in 1 Body.In general, the variant of specific nucleotide sequence will with this specific nucleotide sequence have to Few about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%th, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or Higher sequence iden, or more complementary seriess.Such variant sequence thereof include one or The interpolation of multiple nucleic acid, disappearance or replacement, such that it is able to lead to corresponding amino acid residue Interpolation, remove or replace.Include hybridizing skill by alignment programs known in the art Art determines sequence iden.The nucleotide sequence variants of embodiment and the difference of the sequence of the application Different may be as few as 1-15 nucleotide, as little as 1-10 (such as 6-10), as little as 5, As little as 4,3,2 or even 1 nucleotide.

The application further relates to comprise SEQ ID NO:In sequence shown in 1 sequence of specific site or Its fragment or its variant or its complementary series, for example, comprise SEQ ID NO:Sequence shown in 1 The sequence of 281-1019 position nucleotide or its fragment or its variant or its complementary series.According to comprising The fragment of above-mentioned specific site, can specifically identify corresponding SEQ ID NO:Shown in 1 Sequence.Further, by identifying containing SEQ ID NO:The recombinant nucleic acid of sequence shown in 1 Fragment, you can determine and comprise resistance recombinant nucleic acid fragment in testing sample.

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

Go for any rice varieties needing selection-breeding in the present processes.That is, Can (i.e. Comprehensive Traits be preferably sent out it is contemplated that having by any improved seeds lacking certain beneficial traits The kind of exhibition future) it is used as recurrent parent.With another, there are beneficial traits lacking in this receptor Kind is as donor parents.In the embodiment of the application, using Oryza sativa L. ' high mountain extensive 9113 ' As recurrent parent, using the Oryza sativa L. ' magnificent 3418B ' conduct 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, for improving the accuracy rate of selection, typically uses both sides adjacent simultaneously Two labellings target gene group fragment is tracked select.

In the embodiment of the application, the foreground selection labelling of employing includes favorable selection labelling With negative itemsets labelling, wherein, favorable selection labelling is (brown containing resisting away from target gene group fragment Plant hopper gene) screening in the range of upstream and downstream 50kb (in Oryza sativa L. its genetic distance be 0.2cM) Pleiomorphism molecular marker.Negative itemsets labelling is away from target gene group fragment upstream and downstream The pleiomorphism molecular marker of screening in the range of 500kb (its genetic distance is 2cM in Oryza sativa L.).? In specific embodiments, the positive foreground selection labelling of optimized Select to use is and target gene The labelling BphC03ID03 of group fragment close linkage, negative itemsets labelling is in target fragment The molecular marker RM16175 of trip about 260kb, and dividing away from target fragment downstream about 370kb Sub- labelling RM16211.

In the embodiment of the application, carry out homologous recombination using above-mentioned foreground selection labelling During detection, the criterion of side or unilateral homologous recombination is BphC03ID03 detection and ' China The identical banding pattern of 3418B ', and RM16175 or RM16211 detection and the ' extensive 9113 ' phases in high mountain Same banding pattern；The criterion of both sides or bilateral homologous recombination is BphC03ID03 detection and ' China The identical banding pattern of 3418B ', and RM16175 and RM16211 detection and the ' extensive 9113 ' phases in high mountain 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 all according to conventional laboratory conditions, such as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J&Russell DW,Molecular cloning:a laboratory manual, 2001) condition, or according to manufacturer's description suggestion.

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

The rice genome physical location being previously mentioned in the application is all with reference to the fine genome of Oryza sativa L. Japan MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).

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 group fragment

Used in the present embodiment, material is Oryza sativa L. ' high mountain extensive 9113 ' and Oryza sativa L. ' magnificent 3418B '.

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

In the Breeding Process of restructuring plant, using molecular marker, prospect choosing is carried out to restructuring plant Select, the foreground selection molecular marker being adopted is screened.The molecular marker portion being used Divide and derive from website http://www.gramene.org/, partially self designs.Method for designing is, Annotate the 6.1st edition with reference to the fine genome MSU/TIGR of Oryza sativa L. Japan, download aforementioned zones segment DNA Sequence.Using SSRLocator software, the SSR site in above-mentioned sequence is scanned.Profit With Primer Premier 3.0 software to the SSR site design primer searching out, design primer altogether 128 pairs.By the method for PCR, screen above-mentioned primer pair in ' magnificent 3418B ' and ' high mountain is extensive Polymorphism in 9113 ', finally picks out and has polymorphism, amplification efficiency in two parts of materials High foreground selection molecular marker, is favorable selection labelling BphC03ID03 and negative sense choosing respectively Select labelling RM16175, RM16211.Expand specifically drawing of above-mentioned molecular marker for PCR Thing information is shown in Table 1.

Table 1 foreground selection molecular labeling primer information

By ' genomic fragment that in magnificent 3418B ', forementioned gene cluster is located imports to ' high mountain extensive 9113 ' In, detailed process is as follows：

With ' high mountain extensive 9113 ' as recurrent parent, ', magnificent 3418B ' is hybridized for donor parents, By obtained cenospecies, ' high mountain extensive 9113 ' carries out first backcross generation, obtains BC with recurrent parent₁F₁ Seed, utilizes favorable selection labelling BphC03ID03 and negative itemsets labelling after nursery RM16175, RM16211 carry out Single-plant selection of recombinating, and filter out 12 in target gene group The individual plant of fragment side homologous recombination, that is, BphC03ID03 detection is with ' magnificent 3418B ' is identical Banding pattern, and RM16175 or RM16211 detection and ' extensive 9113 ' the identical banding patterns in high mountain, and profit With Oryza sativa L. full-length genome breeding chip RICE6K (CN102747138A), Foreground selection is carried out to it (Yu etc., Plant Biotechnology Journal.2014,12:28-37).

Comparable chip result in the unilateral homologous recombination individual plant of 12 filtering out, selects background to return Multiple best restructuring individual plant (this generation background recovery value is more than 75%) so as to receptor parent ' high mountain Extensive 9113 ' are returned again, obtain BC₂F₁Seed.Because target gene group fragment is located at the 3 ends of chromosome, occur the probability of restructuring low, so not screening opposite side homology after nursery Recombinant fragment, detects to it merely with favorable selection labelling BphC03ID03, selects to contain There is the individual plant of target gene group fragment, that is, BphC03ID03 detects with ' magnificent 3418B ' is identical Banding pattern, carries out Foreground selection using Oryza sativa L. full-length genome breeding chip RICE6K to it.

Select background to reply best individual plant (this generation background recovery value is more than 87.5%) so as to ' high mountain extensive 9113 ' is returned again recurrent parent, obtains BC₃F₁Seed, profit after nursery With favorable selection labelling BphC03ID03, it is detected, select to contain target gene pack The restructuring individual plant 5 of section, i.e. BphC03ID03 detection and ' the identical banding pattern of magnificent 3418B '.

Using Oryza sativa L. full-length genome breeding chip RICE60K (WO/2014/121419) to above-mentioned 5 Individual individual plant carry out background and target fragment select (Chen etc., Molecular Plant.2014,7: 541-553), screen importing target fragment less, and one (this of target individual plant that background is replied Generation, background recovery value was more than 93.75%).

By the individual plant selfing chosen once, obtain BC₃F₂, after nursery, utilize favorable selection labelling BphC03ID03 detects to it, selects the individual plant containing target gene group fragment, that is, BphC03ID03 detection is with ' the identical banding pattern of magnificent 3418B ', using Oryza sativa L. full-length genome breeding core Piece RICE60K carries out Foreground selection to it.

Final acquisition target fragment homozygosis, and background replys the strain of (background recovery value is more than 99%) It is one, be named as CR020322.Chip detection result is shown in Fig. 1.

Embodiment 2The determination of homologous recombination fragment after importing resistance gene of brown planthopper group fragment

In order to determine the brown planthopper resistant gene group clip size of importing, to ' high mountain extensive 9113 ' is led The homozygosis individual plant entering brown planthopper resistant gene group fragment has carried out target gene group fragment both sides homology weight The sequencing of group fragment.By the brown planthopper resistant gene group recombinant nucleic acid fragment life contained by CR020322 Entitled RecCR020322.

Primarily determined that by Oryza sativa L. full-length genome breeding chip RICE60K testing result, RecCR020322 is located at SNP marker F0335556318CA to end of chromosome region.

Meanwhile, using Miseq sequencing technologies to ' high mountain extensive 9113 ', ' magnificent 3418B ' and Tri- samples of CR020322 carry out genome sequencing.Using TruSeq Nano DNA LT Kit (illumina) test kit carries out library foundation, using Library Quantification Kit Universal (KAPA Biosystems) test kit carries out quantitation, using MiSeq V2 Reagent Kit (illumina) test kit carries out sequencing reaction.Using the desk-top sequenator 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 result, by CR020322 brown planthopper resistant base Because the 35565584bp that group recombinant fragment upstream homologous recombination fragment is positioned at the 3rd chromosome arrives 35567071bp is interval.

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

With receptor parent, ' ' magnificent 3418B ' is for comparison, right for high mountain extensive 9113 ' and donor parents The upstream homologous recombination fragment of CR020322 separately designs amplimer, using high-fidelity enzyme KOD FX Neo (TOYOBO) is expanded, and finds optimal amplification using two-step method or three-step approach Condition is it is ensured that amplified production is shown as single bright band in agarose gel electrophoresiies detection. The upstream homologous recombination fragment amplification primer reaction condition of wherein determination is：94℃2min； 98 DEG C of 10sec, 60 DEG C of 30sec, 68 DEG C of 90sec, 37 circulations；20℃1min.Finally Filter out pair for amplification primer for the amplification of upstream homologous recombination fragment.

In addition, with amplified production as template, being sequenced using Sanger sequencing, according to reality Border sequencing effect, finally filters out 3 sequencing primers and is respectively used to upstream homologous recombination fragment Sequencing.

Specific amplimer and sequencing primer sequence are shown in Table 2, and sequencing result is shown in Fig. 2.

RecCR020322 upstream homologous recombination sequencing fragment length is 1488bp (SEQ ID NO: 1).1-281bp is that ' genomic segment on high mountain extensive 9113 ', with donor ' magnificent 3418B ' for receptor Relatively, there are 2 SNP, 2 InDel.This 737bp section of 282-1018bp is homology Restructuring section.1019-1488bp is that ' magnificent 3418B ' genomic fragment, with ' high mountain is extensive for donor 9113 ' compare, and there are 3 SNP.

Fig. 3 is the structure chart of RecCR020322 upstream homologous recombination fragment.Top base be ' SNP the or InDel labelling of magnificent 3418B ', lower section base is receptor ' high mountain extensive 9113 ' to donor SNP or InDel labelling.Lycoperdon polymorphum Vitt section be from ' the extensive 9113 ' genomic segment in high mountain, Black section is from ' magnificent 3418B ' genomic segment, white section is homologous recombination area Section.Abscissa is fragment length, with base pairs (bp) as unit.

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

Embodiment 3' high mountain extensive 9113 ' imports the Resistance Identification after brown planthopper resistant gene group fragment

In order to identify the Brown Planthopper Resistance of the plant of fragment containing recombinant nucleic acid selecting, to the application ' high mountain is extensive 9113 ', donor parents ' magnificent 3418B ' for new lines CR020322 of selection-breeding, receptor parent (as positive control), and high sense brown paddy plant hopper kind ' coming No. 1 in platform ' is (right as feminine gender According to) carrying out brown paddy plant hopper interior Resistance Identification, 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, every part of material divides 3 row to sow 45 plants altogether, when two one heart stages of leaf, often capable reservation 10 Totally 30 plants of consistent plant of healthy growing way are used for connecing worm for strain.Worm sources come since field collect brown Plant hopper, and captive breeding indoors.Take 2-3 age nymph to plant to be identified, every plant has 5-10 head nymph.When ' coming No. 1 in platform ' dead seedling 95%, start recording respectively identifies Seedling Resistance class, take the meansigma methodss of the resistance class of 30 young plants, as the resistance class of this 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, the jaundice of second leaf, shrinkage or withered, the blue or green volume of lobus cardiacus；6 grades, lobus cardiacus Curling, lobus cardiacus tip burn on leaf；7 grades, lobus cardiacus curling is withered, and plant is not dead；8 grades, lobus cardiacus Withered, somewhat lodge；9 grades, whole strain lodging.According to above-mentioned resistance class, 0-0.9 level judges Resist for high, 1.0-2.9 level is anti-, 3.0-5.9 level is anti-in being, 6.0-6.9 level is middle sense, 7.0-7.9 Level is sense, and 8.0-9.0 level is high sense.

Brown paddy plant hopper interior Resistance Identification result is shown in Fig. 4, wherein ' is coming No. 1 in platform ' and is feeling for high, ' high mountain extensive 9113 ' is sense to original kind, and improvement new lines CR020322 are anti-, donor parents ' China 3418B ' is high anti-.

Although, above with a general description of the specific embodiments the application has been made in detail Most description, but on the basis of the application, it can be made some modifications or improvements, this is to this It is obvious for skilled person.Therefore, on the basis without departing from the application spirit Upper these modifications or improvements, belong to this application claims scope.

Claims (10)

1. recombinant nucleic acid fragment, it is selected from：

I) comprise SEQ ID NO:The sequence of the nucleotide of sequence 281-1019 position shown in 1 or its piece Section or its variant or its complementary series；

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

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

(I) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-281 position shown in 1 Forward primer and specific recognition SEQ ID NO:The nucleotide of sequence 1019-1488 position shown in 1 The reverse primer of sequence；

(II) the combining of first group of primer pair and second group of primer pair below, it comprises

(a) first group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 1-281 position nucleotide and specific recognition SEQ ID NO:Sequence shown in 1 Arrange the reverse primer of the sequence of 282-1018 position nucleotide；With

(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1 The forward primer of sequence of 282-1018 position nucleotide and specific recognition SEQ ID NO:1 institute Show the reverse primer of the sequence of sequence 1019-1488 position nucleotide；

(III) specific recognition comprises SEQ ID NO:The nucleotide of sequence 281-282 position shown in 1 The forward primer of sequence and specific recognition comprise SEQ ID NO:Sequence shown in 1 The reverse primer of the sequence of 1018-1019 position nucleotide；

(IV) specific recognition comprises SEQ ID NO:The nucleotide of sequence 281-282 position shown in 1 The forward primer of sequence and specific recognition SEQ ID NO:Sequence 1019-1488 shown in 1 The reverse primer of the sequence of position nucleotide；

(V) specific recognition SEQ ID NO:The sequence of the nucleotide of sequence 1-281 position shown in 1 Forward primer and specific recognition comprise SEQ ID NO:Sequence 1018-1019 position shown in 1 The reverse primer of the sequence of nucleotide.

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

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

5 '-CACGCTCTTCGTCTAGCCACCTCC-3 ',

5’-TTCGTACAAGCCCGTTGCCATCTC-3’；And

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

5’-CACGCTCTTCGTCTAGCCACCTCC-3’；

5’-TTGATTGGCATGTCTACTGG-3’；

5’-TTCGTACAAGCCCGTTGCCATCTC-3’.

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

5. method as claimed in claim 4, is wherein used for the molecular marker of described foreground selection Selected from one or more of BphC03ID03, RM16175 and RM16211；And/or

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

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

1) recurrent parent is hybridized with donor plant, by obtained cenospecies and samsara parent Originally it is returned, obtained first backcross generation, using favorable selection labelling BphC03ID03 and negative sense The one side that selected marker RM16175, RM16211 carries out brown planthopper resistant gene group fragment to it is same Source recombinant fragment screening, and using Oryza sativa L. full-length genome breeding chip, Foreground selection is carried out to it；

2) select background to reply preferably restructuring individual plant to be returned again with recurrent parent, obtain Second backcross generation, is detected to it using favorable selection labelling BphC03ID03, select containing The restructuring individual plant of brown planthopper resistant gene group fragment, then utilizes 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, is detected to it using favorable selection labelling BphC03ID03, select containing The restructuring individual plant of brown planthopper resistant gene group fragment, then utilizes Oryza sativa L. full-length genome breeding chip pair It carries out Foreground selection；And

4) select introgressed segment little, and the restructuring individual plant that background is replied, by the restructuring chosen list Strain selfing once, is obtained selfed seed, using favorable selection labelling BphC03ID03, it is carried out Detection, and using Oryza sativa L. full-length genome breeding chip, Foreground selection is carried out to it, final acquisition contains Homozygosis brown planthopper resistant gene group recombinant nucleic acid fragment and the rice plant of background reply.

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

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

Forward primer：5 '-GCAAGAATCCGACGCCATAA-3 ',

Reverse primer：5’-CTCTGCTCCTTGCTCTAATCCTCT-3’；

The primer pair of amplifier molecule labelling RM16175, it includes：

Forward primer：5 '-AGCTTTGGTTTCTTGGCTTTGG-3 ',

Reverse primer：5’-ATTAGCGTTGAACCCAAGTGTGG-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 Require the primer described in 2 or 3, performing PCR reaction is entered for template with testing gene group, and analyzes The step of PCR primer.

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

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

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