CN107304447A - Recombinant nucleic acid fragment RecCR010007 and its detection primer and application - Google Patents
Recombinant nucleic acid fragment RecCR010007 and its detection primer and application Download PDFInfo
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Abstract
The present invention relates to molecular biology, a kind of rice anti-rice blast recombinant nucleic acid fragment RecCR010007 and its detection primer and application are specifically disclosed.The present invention also provides a kind of method for the rice plant for containing blast resistant gene group recombinant nucleic acid fragment based on full-length genome selection and use technology seed selection, target gene group fragment is imported into acceptor material, and realize the reply of background simultaneously.The acceptor material that the present invention is improved is ' deep 95B ', it is that ' the deep matching used maintainers of 95A ', using the above method, can retain ' in the case of the deep original advantages of 95B ' widely used sterile line, rice blast resistance gene pack section is imported, its rice blast resistance is improved.The recombinant nucleic acid fragment that the present invention is provided is closely related with rice blast resistance, and the cultivation of other kinds can be applied to as Resistance resource.
Description
Technical field
The present invention relates to molecular biology, specifically, it is related to a kind of rice anti-rice blast weight
Group nucleic acid fragment.
Background technology
For a long time, the system of selection of traditional breeding method depends on the evaluation of variable rate technology type,
Accepted or rejected according to breeding man personal experience, its maximum shortcoming is that time-consuming, less efficient.
The efficiency of selection is improved, optimal method should directly genotype be selected.
With the development of molecular biotechnology, molecular labeling directly selects offer to realize to genotype
May.In recent years, have started to improve individual target using molecular marker-assisted selection method
Character, can significantly shorten the breeding time limit.
Rice blast is one of disease of paddy rice most serious, the annual paddy rice as caused by rice blast in the whole world
Production loss accounts for 11%~30%, therefore the research of rice blast and its resistance is particularly important.
As what rice blast was studied progressively gos deep into, many rice blast resistant gene DNA fragmentations are successive
It is positioned and clones.Wherein, to be positioned at the chromosome of paddy rice the 11st long for Pi1 and Pik clusters allele
Arm proximal end region (Hua etc., Theoretical and Applied Genetics.2012,125:
1047-1055；Li etc., Molecular Breeding.2007,20:179-188；Alok etc.,
Functional&Integrative Genomics.2012,12:215-228；Yuan etc.,
Theoretical and Applied Genetics.2011,122:1017-1028)。
In order to improve stability, shortening breeding process and the time of breeding, effectively utilize Rice Resistance
Property resource, it is necessary to the rice blast resistance gene recombinant fragment produced to paddy rice in crossbreeding
Studied, realize that paddy rice resistance breeding provides effective means for be capable of efficient stable.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of paddy rice
Blast resisting recombinant nucleic acid fragment RecCR010007 and its detection primer and application.
In order to realize the object of the invention, technical scheme is as follows：
In a first aspect, the present invention provides a kind of recombinant nucleic acid fragment, it is characterised in that its nucleosides
Acid sequence comprising 165-312bp shown in SEQ ID NO.1 sequence or its fragment or its variant,
Or its complementary series.
Preferably, its nucleotide sequence includes the sequence or its piece shown in SEQ ID NO.1
Section or its variant or its complementary series.Sequence shown in SEQ ID NO.1 comes from ' deep
95B ' and ' the restructuring plant that magnificent 3418B ' genome areas exchange is produced.
In general, the variant of specific nucleotide sequence will have extremely with the specific nucleotide sequence
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 identity, or more complementary series.Such variant sequence thereof include one or
Addition, missing or the replacement of multiple nucleic acids, so as to cause corresponding amino acid residue
Addition, remove or replace.Include hybridization skill by alignment programs known in the art
Art determines sequence identity.The nucleotide sequence variants of embodiment and the difference of the sequence of the present invention
It is different to may be as few as 1-15 nucleotides, as little as 1-10 (such as 6-10), it is as little as 5, few
To 4,3,2 or even 1 nucleotides.
Second aspect, the invention provides the primer for expanding the recombinant nucleic acid fragment.
The primer includes those skilled in the art can design obtained institute for the amplification target
There is primer.
When it is sequence shown in SEQ ID NO.1 to expand target, the primer may be selected from：
(I) sequence 1-165bp regions nucleotide sequence shown in specific recognition SEQ ID NO.1
In sequence 312-393bp regions shown in the forward primer and specific recognition SEQ ID NO.1 of row
The reverse primer of nucleotide sequence；
(II) combination of first group of primer pair and second group of primer pair below, it is included：
(a) first group of primer pair：Sequence shown in specific recognition SEQ ID NO.1
The forward primer of 1-165bp regions nucleotide sequence and specific recognition SEQ ID NO.1 institutes
Show the reverse primer of sequence 166-311bp regions nucleotide sequence；
(b) second group of primer pair：Sequence shown in specific recognition SEQ ID NO.1
The forward primer and specific recognition SEQ ID NO.1 of 166-311bp regions nucleotide sequence
The reverse primer of shown sequence 312-393bp regions nucleotide sequence；
(III) specific recognition includes the 165th nucleotides of sequence shown in SEQ ID NO.1
The forward primer and specific recognition of sequence include the 312nd core of sequence shown in SEQ ID NO.1
The reverse primer of the sequence of thuja acid；
(IV) specific recognition includes the 165th nucleotides of sequence shown in SEQ ID NO.1
Sequence 312-393bp areas shown in the forward primer and specific recognition SEQ ID NO.1 of sequence
The reverse primer of domain nucleotide sequence；
(V) nucleosides in sequence 1-165bp regions shown in specific recognition SEQ ID NO.1
The forward primer and specific recognition of acid sequence include sequence the 312nd shown in SEQ ID NO.1
The reverse primer of the sequence of nucleotides.
Specifically, the present invention provides amplification and detection SEQ ID NO:The primer of sequence shown in 1
Combination is as follows：
Amplimer includes：
Forward primer：5 '-CACAAGACCCTCGCCCTTGACG-3 ',
Reverse primer：5 '-AAGCTCACGCTTACCGTGCTCC-3 ',
Sequencing primer includes：
Forward primer：5 '-GGTCCTCCGTGCCGATACGT-3 ',
Reverse primer 1：5 '-AACAGGTGGGAGGCTGCC-3 ',
Reverse primer 2：5’-AAGCTCACGCTTACCGTGCTCC-3’.
Further, the present invention also provides the kit containing foregoing primer.
The third aspect, the invention provides the recombinant nucleic acid fragment in anti-rice blast rice breeding
In application.
For example, the fragment is imported in other rice plants, to obtain the water with blast resisting
Rice plants.
Fourth aspect, the invention provides rice plant of the screening containing the recombinant nucleic acid fragment
Method.
Specific primer is designed according to foregoing recombinant nucleic acid fragment, with testing gene group
The step of entering performing PCR for template to react, and analyze pcr amplification product.Specifically, it is described
Primer is as previously described.Selectively, pcr amplification product is analyzed using Sanger PCR sequencing PCRs.
Methods described is carried out using testing sample genomic DNA as template using above-mentioned amplimer
PCR is expanded, and then the amplified production of acquisition is sequenced using above-mentioned sequencing primer, if surveying
Sequence result is consistent or complementary with SEQ ID NO.1 sequences, then contains SEQ ID in testing sample
Homologous recombination fragment shown in NO.1.
The recombinant nuclear containing sequence shown in SEQ ID NO.1 in testing sample is determined by detection
Acid fragment, you can determine to include the recombinant nucleic acid fragment containing resistant gene in testing sample.
Preferably, using foregoing primer or aforementioned agents box, detecting testing sample genome
In whether contain the recombinant nucleic acid fragment described in claim 1.
It is understood that screened by methods described obtain containing disclosed by the invention heavy
The rice plant or its seed of group nucleic acid fragment fall within protection scope of the present invention.
5th aspect, the invention provides the choosing of the rice plant containing the recombinant nucleic acid fragment
Method is educated, is specially：So that ' for recurrent parent, ' magnificent 3418B ' is carried out deep 95B ' for donor parents
Hybridization, by resulting cenospecies, ' deep 95B ' is returned, then will be resulting with recurrent parent
Backcrossing kind carry out selfing, obtain paddy rice containing recombinant nucleic acid fragment described in claim 1 and plant
Strain；
Wherein, the cenospecies, backcrossing kind and selfed seed need to be utilized respectively molecular labeling and paddy rice
Full-length genome breeding chip carries out foreground selection and Foreground selection.
The molecular labeling is one kind in PiC11ID115a, PiC11S122 and PiC11S166
Or it is a variety of.
Specifically, the selection comprises the following steps：1) by recurrent parent and donor
Plant is hybridized, and resulting cenospecies and recurrent parent are returned, and obtains backcrossing one
Generation, using favorable selection mark PiC11ID115a and negative itemsets mark PiC11S122,
PiC11S166 carries out the unilateral homologous recombination fragment screening of blast resistant gene pack section to it,
And paddy rice full-length genome breeding chip is utilized, 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%) and samsara
Parent is returned again, obtains second backcross generation, and PiC11ID115a is marked using favorable selection
It is detected, the restructuring individual plant containing blast resistant gene pack section is selected, then utilizes
Paddy rice full-length genome breeding chip, such as RICE6K carries out Foreground selection to it；3) select
Restructuring individual plant (this generation background recovery value is more than 87.5%) and recurrent parent that background has been replied
Again be returned, obtain third backcross generation, using favorable selection mark PiC11ID115a with
Negative indicia PiC11S122, PiC11S166 carries out the another of blast resistant gene pack section to it
Side homologous recombination fragment screening, and utilization paddy rice full-length genome breeding chip, such as RICE60K,
Foreground selection is carried out to it；And 4) selection introgressed segment is small, and the restructuring that background has been replied
Individual plant (background recovery value is more than 93.75%), by the restructuring individual plant selfing chosen once, is obtained
Selfed seed, is detected, and utilize paddy rice using favorable selection mark PiC11ID115a to it
Full-length genome breeding chip, such as RICE60K carries out Foreground selection to it, finally contained
Blast resistant gene group recombinant nucleic acid fragment homozygosis and background reply that (background recovery value exceedes
99%) rice plant.
Wherein, the amplimer used when carrying out foreground selection using molecular labeling is as follows：
Amplifier molecule marks PiC11ID115a primer pair, and it includes：
Forward primer：5 '-CTCAGTGCCCTTTTCTTCCTCA-3 ',
Reverse primer：5’-TCACAGCAAAGACCATACAACCAT-3’；
Amplifier molecule marks PiC11S122 primer pair, and it includes：
Forward primer：5 '-TACGACCGTGACATGTCCTT-3 ',
Reverse primer：5’-ATTAACCACCATGCTCACCA-3’；
Amplifier molecule marks PiC11S166 primer pair, and it includes：
Forward primer：5 '-TTAGCCCCTCTCTCTCTCCA-3 ',
Reverse primer：5’-GCCAGATCTAGCAGAGGTGA-3’.
The beneficial effects of the present invention are：
The present invention is obtained by full-length genome selection and use technology seed selection and provides a kind of rice
The recombinant nucleic acid fragment of seasonal febrile diseases resistant gene, recombinant nucleic acid fragment and rice blast that the present invention is provided
Sick resistance is closely related, and the cultivation of other kinds can be applied to as Resistance resource.
The present invention provides one kind and contains blast resisting based on full-length genome selection and use technology seed selection
The method of the rice plant of genome recombination nucleic acid fragment, this method has quick, accurate, steady
Fixed advantage, only passes through the transformation of five generations, you can target gene group fragment only is imported into acceptor material
Material, and the reply of background is realized simultaneously.
On the basis of the above, the present invention by with ' deep 95B ' be recurrent parent, ' magnificent 3418B '
Hybridized for donor parents, be returned the restructuring plant that there must be rice blast resistance with selfing, and
A kind of recombinant nucleic acid fragment with rice blast resistance is obtained.
The acceptor material that the present invention is improved is ' deep 95B ' is that widely used sterile line is ' deep
The matching used maintainers of 95A '.Using the above method, ' deep 95B ' originals can retained
In the case of having advantage, rice blast resistance gene pack section is imported, its rice blast resistance is improved.
Further, it can be obtained by least first cross, generation backcrossing and stable contain rice blast
Resistance fragments ' deep 95A ', then cenospecies rice blast resistance is realized significantly by combo
Improve.The genome recombination nucleic acid fragment that the present invention is provided is closely related with rice blast resistance, can
The cultivation of other kinds is applied to as Resistance resource.
Brief description of the drawings
Fig. 1 is CR010007 paddy rice RICE60K full-length genome breeding cores in the embodiment of the present invention 1
Piece testing result；Wherein, the indicated square frame of abscissa numeral represents 12 chromosomes of paddy rice successively,
Ordinate numeral is the physical location [with megabasse (Mb) for unit] on rice genome, ash
Colo(u)r streak bar represents receptor parent, and ' deep 95B ' genotype, black lines represent donor parents ' China
3418B ' genotype, it is consistent i.e. without polymorphism section that white line represents two parent genotypes.
O.11 chromosome black lines display block is the blast resistant gene group weight of importing in figure
Group nucleic acid fragment RecCR010007.
Fig. 2 is RecCR010007 downstreams homologous recombination sequencing fragment ratio in the embodiment of the present invention 2
To result；Asterisk shown in figure represents in comparison result that CR010007 is in identical base, figure
The new lines of acquisition, T001 is that ' deep 95B ', R002 are donor parents ' China to receptor parent
3418B’。
Fig. 3 is the structure of RecCR010007 downstreams homologous recombination fragment in the embodiment of the present invention 2
Figure；Wherein, top base is donor ' magnificent 3418B ' SNP or InDel mark, lower section alkali
Base is acceptor ' deep 95B ' SNP or InDel mark.Grey section is from ' deep 95B '
Genomic segment, black section is from ' magnificent 3418B ' genomic segments, white section
For homologous recombination section, abscissa is fragment length, with base pairs (bp) for unit.
Fig. 4 is qualification result in CR010007 rice blast resistances room in the embodiment of the present invention 3；Figure
Shown in blade be followed successively by：(A) rice blast susceptible variety Lijiang xintuanheigu；(B) original
Plant ' deep 95B '；(C) improvement new lines CR010007；(D) rice blast disease-resistant variety paddy plum
No. 4.
Embodiment
Defined below and method is provided preferably to define the present invention and in present invention practice
In instruct those of ordinary skill in the art.Unless otherwise mentioned, term is common according to association area
The common usage of technical staff understands.
As used herein, " nucleotide sequence " includes the deoxidation core for being related to single-stranded or double-stranded form
Ribotide or ribonucleotide polymer, and unless otherwise limitation, nucleotide sequence
Write from left to right with 5 ' to 3 ' directions, including with natural nucleotide fundamental property
Know analog (for example, peptide nucleic acid), the analog with naturally occurring nucleotides
Similar mode hybridizes with single-chain nucleic acid.
In some embodiments, the nucleotide sequence of the present invention can be changed,
To carry out conserved amino acid replacement.In certain embodiments, can be close according to unifacial leaf
Numeral Preference is not changed replacing for amino acid sequence to the nucleotide sequence of the present invention
Change, for example, can replace coding with monoamino-acid sequence with the codon of monocotyledon preference
The codon of row, without changing the amino acid sequence coded by the nucleotide sequence.
In particular it relates to further optimize the nucleosides of gained to SEQ ID NO.1
Acid sequence.The more details of this method are described in Murray etc. (1989) Nucleic Acids
Res.17:477-498.Optimize nucleotide sequence to can be used for improving the restructuring of blast resistant gene group
Expression of the nucleic acid fragment in paddy rice.
In some embodiments, the invention further relates to sequence shown in SEQ ID NO.1
Variant.In general, the variant of specific nucleotide sequence will be with the specific nucleotide sequence
With at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%
Or 99.9% or higher sequence identity, or more complementary series.Such variant sequence
Row include addition, missing or the replacement of one or more nucleic acids, so as to cause
Addition, removal or the replacement of corresponding amino acid residue.Pass through sequence known in the art
Row alignment programs determine sequence identity including hybridization technique.The nucleotides sequence of embodiment
Row variant and the difference of the sequence of the present invention may be as few as 1-15 nucleotides, as little as 1-10
Individual (such as 6-10), as little as 5, as little as 4,3,2 or even 1 nucleotides.
The invention further relates to include the sequence of specific site in sequence shown in SEQ ID NO.1
Or its fragment or its variant or its complementary series, for example, comprising shown in SEQ ID NO.1
The sequence or its fragment or its variant or its complementary series of the 165-312 positions nucleotides of sequence.
According to the fragment for including above-mentioned specific site, corresponding SEQ can be specifically identified
Sequence shown in ID NO.1.Further, by identifying containing shown in SEQ ID NO.1
The recombinant nucleic acid fragment of sequence, you can determine to include containing resistant gene in testing sample
Recombinant nucleic acid fragment.
As used herein, " paddy rice " be any rice plant and including can be with rice breeding institute
There is plant variety.As used herein, " plant " or " plant ", including whole plant, plant are thin
Plant cell tissue's training that born of the same parents, plant organ, plant protoplast, plant can therefrom regenerate
Plant cell complete in thing, plant callus, vegetation bed and plant or plant part is supported,
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 the rice varieties of any required seed selection in the method for the present invention.Also
To say, the improved seeds that certain beneficial traits can be lacked by any (i.e. Comprehensive Traits compared with
It is good, it is contemplated that promising kind) it is used as recurrent parent.There is this receptor with another
Lacking in beneficial traits kind as donor parents, and beneficial traits provided
Preferably dominant Dominant gene.In embodiments of the invention, it is ' deep using paddy rice
95B ' is as recurrent parent, using the paddy rice for having been shown to have good rice blast resistance
' magnificent 3418B ' is used as donor.
In the selection of restructuring plant provided by the present invention, molecular labeling pair is utilized
Recombinate plant and carry out foreground selection.The reliability of foreground selection depends primarily on mark and mesh
Chain tightness degree between mark gene is general to use two simultaneously to improve the accuracy rate of selection
Two adjacent marks of side are tracked selection to target gene.
In embodiments of the invention, the foreground selection mark of use includes favorable selection
Mark and negative itemsets mark.In a particular embodiment, optimized Select to use is being just
It is the mark with target gene group fragment close linkage to prospect selected marker
PiC11ID115a, negative itemsets mark is the mark positioned at target fragment upstream
PiC11S122, and the mark PiC11S166 positioned at target fragment downstream.
In embodiments of the invention, carried out using above-mentioned foreground selection mark homologous heavy
During the detection of group, the criterion of side or unilateral homologous recombination is PiC11ID115a detections
With ' the identical banding pattern of magnificent 3418B ', and PiC11S122 or PiC11S166 detections and ' deep 95B '
Identical banding pattern；The criterion of both sides or bilateral homologous recombination be PiC11ID115a detection with
' the identical banding pattern of magnificent 3418B ', and PiC11S122 and PiC11S166 detections and ' deep 95B '
Identical banding pattern.
In the present invention, any available chip can be used to carry out institute of the present invention
Foreground selection in the breeding method of offer.In preferred embodiments, it can use
Paddy rice full-length genome of the present inventor disclosed in Chinese patent application CN102747138A
Breeding chip RICE6K, or disclosed in PCT international applications WO/2014/121419
Paddy rice full-length genome breeding chip RICE60K.It is all interior in this two parts of application documents
Appearance is incorporated herein by reference.
Following examples are merely to illustrate the purpose for the scope that is not intended to limit the present invention.If not special
Do not indicate, embodiment is according to conventional laboratory conditions, and such as Sambrook equimoleculars clone is real
Test handbook (Sambrook J＆Russell DW, Molecular cloning:a laboratory
Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Rice plant material information used in the present invention can be found in rice in China kind
And its pedigree database (http://www.ricedata.cn/variety/index.htm).
The rice genome physical location being previously mentioned in the present invention is with reference to paddy rice Nipponbare base
Because a group MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).
The seed selection of embodiment 1 imports the restructuring plant of blast resistant gene pack section
The material used in the present embodiment is paddy rice ' deep 95B ' and paddy rice ' magnificent 3418B '.
' magnificent 3418B ' has good rice blast resistance to paddy rice, and it is probably the 11st to speculate
Rice blast resistance of the Pi1 and Pik cluster allele regions of number chromosome to the material
Serve key effect.
In the Breeding Process of restructuring plant, before being carried out using molecular labeling to restructuring plant
Scape is selected, and the foreground selection molecular labeling used is screened.With reference to paddy rice day
This fine genome MSU/TIGR annotates the 6.1st edition, downloads the 11st chromosome 27,155,000
To 28,495,000DNA sequences.Using SSRLocator softwares in above-mentioned sequence
SSR sites are scanned.Using the softwares of Primer Premier 3.0 to the SSR that searches out
Primer is designed in site, and 385 pairs of primer is designed altogether.By PCR method, screen above-mentioned
Primer pair is in ' magnificent 3418B ' and ' polymorphism in deep 95B ' is finally picked out in two parts of materials
There is the high foreground selection molecular labeling of polymorphism, amplification efficiency in material, be positive respectively
Selected marker PiC11ID115a and negative itemsets mark PiC11S122, PiC11S166.
1 is shown in Table for the PCR specific primer information for expanding above-mentioned molecular labeling.
The foreground selection molecular labeling primer information of table 1
By paddy rice, ' genomic fragment in magnificent 3418B ' where forementioned gene imported into paddy rice
' in deep 95B ', detailed process is as follows：
So that ' for recurrent parent, ' magnificent 3418B ' is that donor parents are hybridized to deep 95B ', will
' deep 95B ' is returned resulting cenospecies, obtains BC with recurrent parent1F1Seed,
After nursery PiC11ID115a and negative itemsets mark are marked using favorable selection
PiC11S122, PiC11S166 carry out restructuring Single-plant selection, filter out 13 in target base
Because of a group individual plant for DNA fragmentation side homologous recombination, i.e. PiC11ID115a detections and ' China
The identical banding patterns of 3418B ', and PiC11S122 or PiC11S166 detection with ' deep 95B ' is identical
Banding pattern, and utilize paddy rice full-length genome breeding chip RICE6K (CN102747138A)
Foreground selection (Yu etc., Plant Biotechnology Journal.2014,12 are carried out to it:
28-37)。
Comparable chip result, the selection back of the body in the 13 unilateral homologous recombination individual plants filtered out
Scape replys best restructuring individual plant (this generation background recovery value is more than 75%), makes itself and wheel
Returning parent, ' deep 95B ' is returned again, obtains BC2F1Seed, utilizes forward direction after nursery
Selected marker PiC11ID115a detects that selection contains target gene group fragment to it
Recombinate individual plant, i.e. PiC11ID115a detections with ' the identical banding pattern of magnificent 3418B ', utilizes paddy rice
Full-length genome breeding chip RICE6K carries out Foreground selection to it.
Background is selected to reply preferable individual plant (this generation background recovery value is more than 87.5%),
Making it, ' deep 95B ' is returned again, obtains BC with recurrent parent3F1Seed, nursery
Afterwards using favorable selection mark PiC11ID115a and negative indicia PiC11S122,
PiC11S166 carries out target gene group fragment opposite side homologous recombination piece to the seed of harvest
The screening of section, obtains 7 individual plants recombinated in target fragment both sides, i.e. PiC11ID115a
Detection and ' the identical banding pattern of magnificent 3418B ', and PiC11S122 and PiC11S166 detections and ' depth
The identical banding patterns of 95B '.
It is right using paddy rice full-length genome breeding chip RICE60K (WO/2014/121419)
Above-mentioned 7 bilaterals exchange individual plant carry out background and target fragment selection (Chen etc.,
Molecular Plant.2014,7:541-553), importing target fragment is screened smaller, and the back of the body
The target individual plant one that scape has been replied (this generation background recovery value is more than 93.75%).
By the individual plant selfing chosen once, BC is obtained3F2, favorable selection mark is utilized after nursery
Note PiC11ID115a is detected to it, selects the individual plant containing target gene group fragment,
That is PiC11ID115a is detected and ' the identical banding pattern of magnificent 3418B ', is educated using paddy rice full-length genome
Plant chip RICE60K and Foreground selection is carried out to it.
It is final to obtain target fragment homozygosis, and background replys (background recovery value is more than 99%)
Strain one, be named as CR010007.Chip testing result is shown in Fig. 1.
Embodiment 2 imports the determination of homologous recombination fragment after rice blast resistance gene pack section
In order to determine the rice blast resistance gene group clip size imported, to ' deep 95B '
The homozygosis individual plant of introgressed segment has carried out target gene group fragment both sides homologous recombination fragment
Sequencing.Blast resistant gene group recombinant nucleic acid fragment contained by CR010007 is named as
RecCR010007。
Primarily determined that by paddy rice full-length genome breeding chip RICE60K testing results,
RecCR010007 is located at SNP marker F1127982620TC upstreams.
Meanwhile, using Miseq sequencing technologies to ' deep 95B ', ' magnificent 3418B ' and
Tri- samples of CR010007 carry out genome sequencing.Use TruSeq Nano DNA LT
Kit (illumina) kit carries out library foundation, uses Library Quantification
Kit-Universal (KAPA Biosystems) kit is quantified, used
MiSeq V2Reagent Kit (illumina) kit carries out sequencing reaction.Use Miseq
Desk-top sequenator (illumina) is detected.Specific steps and method are referring to each kit
And sequenator operation instructions.
According to foregoing SNP chip and Miseq sequencing results, by under RecCR010007
Trip homologous recombination fragment is positioned at 27961872bp to the 27963142bp of the 11st chromosome
It is interval.
On this basis, with reference to paddy rice Nipponbare genome MSU/TIGR annotations the 6.1st
Version, downloads respective segments DNA sequence dna.Expanded using the Software for Design of Primer Premier 5.0
Increase and sequencing primer, design requirement be long 22nt of primer or so, G/C content 40-60% and
There is no mispairing.
With receptor parent, ' ' magnificent 3418B ' is control, right for deep 95B ' and donor parents
RecCR010007 downstreams homologous recombination fragment design amplimer, uses high-fidelity enzyme
KOD FX Neo (TOYOBO) are expanded, and are found most using two-step method or three-step approach
Good amplification condition, it is ensured that amplified production is shown as single in agarose gel electrophoresis detection
Bright band.The downstream homologous recombination fragment amplification primer reaction condition wherein determined is：
94℃2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 60sec, 37 circulations；
20℃1min.Thus, finally filtering out pair for amplification primer is used for downstream homologous recombination piece
The amplification of section.
In addition, using amplified production as template, being sequenced using Sanger PCR sequencing PCRs, root
Factually border sequencing effect, finally filters out 3 sequencing primers for downstream homologous recombination piece
The sequencing of section.Specific amplimer and sequencing primer sequence are shown in Table 2, and sequencing result is shown in figure
2。
RecCR010007 downstreams homologous recombination sequencing fragment length is 393bp (SEQ ID
NO:1).1-165bp is donor ' magnificent 3418B ' genomic segment, with acceptor ' depth
95B ' compares, and there are 6 SNP.This 146bp section of 166-311bp is homologous heavy
Group section.312-393bp is acceptor ' deep 95B ' genomic fragments, with donor ' magnificent 3418B '
Compare, there are 4 SNP.
Fig. 3 is the structure chart of RecCR010007 downstreams homologous recombination fragment.Top base
For donor, ' magnificent 3418B ' SNP or InDel mark, lower section base is that acceptor is ' deep
95B ' SNP or InDel mark.Grey section is from ' deep 95B ' genomes
Section, black section is from ' magnificent 3418B ' genomic segments, white section is same
Source recombinates section.Abscissa is fragment length, with base pairs (bp) for unit.
The blast resistant gene group recombinant nucleic acid fragment amplification of table 2 and sequencing primer information
' the Resistance Identification that deep 95B ' is imported after blast resistant gene pack section of embodiment 3
In order to identify resistance effect, new lines CR010007, samsara to seed selection of the present invention
Parent ' deep 95B ', rice blast disease-resistant variety paddy plum No. 4 (being used as positive control), and
Rice blast susceptible variety Lijiang xintuanheigu (being used as negative control) carries out indoor plantation,
Cultivated to the 3-4 leaf phases to adopt and identified with the following method：
Choose the 21K02-1 that is separated on the sick sample of sick nursery rice blast of bestowing favour for 2013 and
21K14-1, the 6102-1 of Sichuan sick nursery, 8111-1 and 2011 year of Fujian sick nursery
The LJ7-1-2 separated from the sick sample of sick nursery rice blast of bestowing favour, totally 5 plants of rice blast bacterial strain conducts
Inoculating strain.Bacterial strain uses the preceding sorghum grain by preservation using -20 DEG C of preservations of sorghum grain method
Take out to potato dextrose medium (PDA) flat board and activate (PDA：Peeled potatoes
200g, glucose 20g, agar powder 15g, distilled water is settled to 1L), 28 DEG C of illumination trainings
Diameter 5mm fresh mycelia block is taken to be forwarded to (sorghum in sorghum grain culture medium after supporting 5 days
Grain 500g adds 1.5L distilled water, boils and liquid is filtered off to boiling, pull sorghum grain out dress
Enter 250ml triangular flasks, 100ml/ bottles, moist heat sterilization 20 minutes), 10 pieces/bottle connect bacterium
Sorghum grain is shaken scattered daily after 2 days, 28 DEG C of dark culturing to mycelia cover with sorghum grain.Then
Sorghum grain is spread out on sterile gauze, sterile damp gauze is covered, at 25 DEG C,
Cultivate 4-5 days and produced to a large amount of spores under RH >=95%, 12h illumination conditions, use sterilized water
(containing 0.02% polysorbas20) washes lower spore, waits spore amount combined inoculation bacterial strain, adjusts dense
Spend to 5 × 105Individual/ml.
With mixing conidial suspension spray inoculation CR010007, ' deep 95B ', Gu Mei 4
Number and Lijiang xintuanheigu, be inoculated with three repetitions.It is inoculated with transparent cover, 28 DEG C on back cover
Dark culturing 24h, then 16h illumination cultivations investigated after 5 days.
Investigation standard is 0 grade (height is anti-, HR)：There is no symptom；1 grade (anti-, R)：
The brown scab of very little；2 grades (moderate resistance, MR)：The brown scab that diameter is about 1mm；
3 grades (MS, middle sense)：Directly about 2-3mm band circle scab, central canescence,
Edge brown；4 grades (sense, S)：It is about 1-3cm oval scab, central canescence,
Edge brown；5 grades (height sense, HS)：Long and wide big oval scab, scab fusion
In flakes, it is withered to blade.Wherein 0-2 grades is disease-resistant, and 3-5 grades are susceptible.It is inoculated with result
It is shown in Table 3 and Fig. 4.
Resistant expression after the inoculation rice blast fungus of table 3
Although above having made in detail to the present invention with a general description of the specific embodiments
Most description, but on the basis of the present invention, 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 spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. a kind of recombinant nucleic acid fragment, it is characterised in that its nucleotide sequence includes SEQ
165-312bp sequence shown in ID NO.1 or its variant or its complementary series.
2. recombinant nucleic acid fragment according to claim 1, it is characterised in that its nucleosides
Acid sequence includes the sequence or its fragment or its variant shown in SEQ ID NO.1 or its complementation
Sequence.
3. the primer for recombinant nucleic acid fragment described in amplification or test right requirement 1 or 2.
4. primer according to claim 3, it is characterised in that the primer is selected from：
(I) sequence 1-165bp regions nucleotide sequence shown in specific recognition SEQ ID NO.1
In sequence 312-393bp regions shown in the forward primer and specific recognition SEQ ID NO.1 of row
The reverse primer of nucleotide sequence；
(II) combination of first group of primer pair and second group of primer pair below, it is included：
(a) first group of primer pair：Sequence shown in specific recognition SEQ ID NO.1
The forward primer of 1-165bp regions nucleotide sequence and specific recognition SEQ ID NO.1 institutes
Show the reverse primer of sequence 166-311bp regions nucleotide sequence；
(b) second group of primer pair：Sequence shown in specific recognition SEQ ID NO.1
The forward primer and specific recognition SEQ ID NO.1 of 166-311bp regions nucleotide sequence
The reverse primer of shown sequence 312-393bp regions nucleotide sequence；
(III) specific recognition includes the 165th nucleotides of sequence shown in SEQ ID NO.1
The forward primer and specific recognition of sequence include the 312nd core of sequence shown in SEQ ID NO.1
The reverse primer of the sequence of thuja acid；
(IV) specific recognition includes the 165th nucleotides of sequence shown in SEQ ID NO.1
Sequence 312-393bp areas shown in the forward primer and specific recognition SEQ ID NO.1 of sequence
The reverse primer of domain nucleotide sequence；
(V) nucleosides in sequence 1-165bp regions shown in specific recognition SEQ ID NO.1
The forward primer and specific recognition of acid sequence include sequence the 312nd shown in SEQ ID NO.1
The reverse primer of the sequence of nucleotides.
5. primer according to claim 3, it is characterised in that the primer is selected from：
(I) amplification SEQ ID NO:The primer pair of sequence shown in 1
5 '-CACAAGACCCTCGCCCTTGACG-3 ',
5’-AAGCTCACGCTTACCGTGCTCC-3’；And
(II) sequencing SEQ ID NO:The primer of sequence shown in 1
5’-GGTCCTCCGTGCCGATACGT-3’；
5’-AACAGGTGGGAGGCTGCC-3’；
5’-AAGCTCACGCTTACCGTGCTCC-3’。
6. the kit containing any one of claim 3~5 primer.
7. the recombinant nucleic acid fragment described in claim 1 or 2 is in anti-rice blast rice breeding
Using.
8. screen the side containing the rice plant of recombinant nucleic acid fragment described in claim 1 or 2
Method, it is characterised in that using described in any one of claim 3~5 primer or claim 6
Kit, detection testing sample genome in whether contain the restructuring described in claim 1 or 2
Nucleic acid fragment.
9. the seed selection side containing the rice plant of recombinant nucleic acid fragment described in claim 1 or 2
Method, it is characterised in that comprise the following steps：
1) it is that donor parents are hybridized with ' deep 95B ' be recurrent parent, ' magnificent 3418B ',
Recurrent parent is hybridized with donor plant, resulting cenospecies and recurrent parent are carried out
Backcrossing, obtains first backcross generation, and PiC11ID115a and negative itemsets mark are marked using favorable selection
Note PiC11S122, PiC11S166 carry out the unilateral homologous heavy of blast resistant gene pack section to it
Pack section screening, and Foreground selection is carried out to it using paddy rice full-length genome breeding chip；
2) restructuring individual plant of the selection background recovery value more than 75% is returned again with recurrent parent
Hand over, obtain second backcross generation, it is detected using favorable selection mark PiC11ID115a,
The restructuring individual plant containing blast resistant gene pack section is selected, is then educated using paddy rice full-length genome
Plant chip and Foreground selection is carried out to it；
3) restructuring individual plant of the selection background recovery value more than 87.5% is carried out again with recurrent parent
Backcrossing, obtains third backcross generation, and PiC11ID115a and negative indicia are marked using favorable selection
The opposite side that PiC11S122, PiC11S166 carry out blast resistant gene pack section to it is homologous heavy
Pack section screening, and Foreground selection is carried out to it using paddy rice full-length genome breeding chip；
4) selection introgressed segment is small, and restructuring individual plant of the background recovery value more than 93.75%, will
The restructuring individual plant selfing chosen once, is obtained selfed seed, marked using favorable selection
PiC11ID115a is detected to it, and it is carried out using paddy rice full-length genome breeding chip
Foreground selection, it is final to obtain the recombinant nucleic acid fragment homozygosis of group containing blast resistant gene and background reply
It is worth the rice plant more than 99%.
10. selection according to claim 9, it is characterised in that utilize molecule mark
Remember the amplimer used during row foreground selection into as follows：
Amplifier molecule marks PiC11ID115a primer pair, and it includes：
Forward primer：5 '-CTCAGTGCCCTTTTCTTCCTCA-3 ',
Reverse primer：5’-TCACAGCAAAGACCATACAACCAT-3’；
Amplifier molecule marks PiC11S122 primer pair, and it includes：
Forward primer：5 '-TACGACCGTGACATGTCCTT-3 ',
Reverse primer：5’-ATTAACCACCATGCTCACCA-3’；And
Amplifier molecule marks PiC11S166 primer pair, and it includes：
Forward primer：5 '-TTAGCCCCTCTCTCTCTCCA-3 ',
Reverse primer：5’-GCCAGATCTAGCAGAGGTGA-3’.
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