CN106893769A - Recombinant nucleic acid fragment RecCR012602 and its detection method - Google Patents
Recombinant nucleic acid fragment RecCR012602 and its detection method Download PDFInfo
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Abstract
This application provides recombinant nucleic acid fragment and its detection method.Present invention also provides the selection of the rice plant containing recombinant nucleic acid fragment, foreground selection and Foreground selection are carried out to restructuring plant using molecular labeling, obtain the rice plant containing recombinant nucleic acid fragment.
Description
Technical field
The application is related to full-length genome selection and use technology.Specifically, the application relates to the use of
Full-length genome selection and use technology seed selection contains the rice plant of recombinant nucleic acid fragment, and thus
And the recombinant nucleic acid fragment and its detection method for obtaining.
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 is to realize directly selecting offer to genotype
May.In recent years, have started to improve individual target using molecular marker-assisted selection method
Proterties, can significantly shorten the breeding time limit.
Rice blast is one of disease of paddy rice most serious, the annual paddy rice 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 fragmentation phases
After being positioned and clone.Wherein, the Pi2 intervals of the chromosome of paddy rice the 6th are positioned and clone
Many rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm, Pi50, the interval includes
One gene cluster of rice blast resistance gene (Qu etc., Genetics.2006,172:1901-1914；
Wang etc., Phytopathology.2012,102:779-786；Xiao etc., Mol Breeding.
2012,30:1715-1726；Liu etc., Mol Genet Genomics.2002,267:472-480；
Jiang etc., Rice.2012,5:29-35；Zhu etc., Theor Appl Genet.2012,124:
1295-1304；Deng etc., Theor Appl Genet.2006,113:705-713).
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 sequence 368-1702 shown in 1 nucleotides or its variant or its complementation
Sequence；Ii SEQ ID NO) are included:The sequence of sequence shown in 1 or its fragment or its variant or its
Complementary series；Iii SEQ ID NO) are included:The sequence of sequence 564-968 shown in 2 nucleotides
Or its fragment or its variant or its complementary series；Iv SEQ ID NO) are included:Sequence shown in 2
Sequence or its fragment or its variant or its complementary series；And the combination of above fragment.It is real one
Apply in scheme, the recombinant nucleic acid fragment is genome recombination nucleic acid fragment.
Additionally, this application provides the primer for detecting 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 sequence 1-368 shown in 1 nucleotides and
Specific recognition SEQ ID NO:The sequence of sequence 1702-2839 shown in 1 nucleotides it is reverse
Primer；(II) first group of primer pair and second group of combination of primer pair below, it includes (a) first
Group primer pair：Specific recognition SEQ ID NO:1-368 sequence of nucleotides of sequence shown in 1
The forward primer and specific recognition SEQ ID NO of row:369-1701 core of sequence 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 and specific recognition of 369-1701 sequence of nucleotides of sequence shown in 1
SEQ ID NO:1702-2839 reverse primer of the sequence of nucleotides of sequence shown in 1；(III)
Specific recognition includes SEQ ID NO:Sequence 367-368 or 368-369 shown in 1
The forward primer and specific recognition of the sequence of nucleotides include SEQ ID NO:Sequence shown in 1
1701-1702 or the 1702-1703 reverse primer of the sequence of nucleotides；(IV) it is special
Opposite sex identification includes SEQ ID NO:Sequence shown in 1 367-368 or 368-369 core
The forward primer and specific recognition SEQ ID NO of the sequence of thuja acid:Sequence shown in 1
The 1702-2839 reverse primer of the sequence of nucleotides；(V) specific recognition SEQ ID NO:1
The forward primer and specific recognition of 1-368 sequence of nucleotides of shown sequence include SEQ
ID NO:1701-1702 or 1702-1703 sequence of nucleotides of sequence shown in 1
Reverse primer；And/or optionally, (VI) specific recognition SEQ ID NO:Sequence shown in 2
1-564 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Sequence shown in 2
The 968-1748 reverse primer of the sequence of nucleotides of row；(VII) below the 3rd group of primer pair with
The 4th group of combination of primer pair, it includes (c) the 3rd group of primer pair：Specific recognition SEQ ID
NO:The forward primer and specific recognition of 1-564 sequence of nucleotides of sequence shown in 2
SEQ ID NO:565-967 reverse primer of the sequence of nucleotides of sequence shown in 2；(d)
4th group of primer pair：Specific recognition SEQ ID NO:565-967 nucleosides of sequence shown in 2
The forward primer and specific recognition SEQ ID NO of the sequence of acid:Sequence shown in 2
The 968-1748 reverse primer of the sequence of nucleotides；(VIII) specific recognition includes SEQ ID
NO:The forward primer and specific recognition of 564-565 sequence of nucleotides of sequence shown in 2
Comprising SEQ ID NO:Sequence shown in 2 967-968 or the 968-969 sequence of nucleotides
The reverse primer of row；(IX) specific recognition includes SEQ ID NO:Sequence shown in 2
564-565 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Shown in 2
968-1748 reverse primer of the sequence of nucleotides of sequence；(X) specific recognition SEQ ID
NO:The forward primer and specific recognition bag of 1-564 sequence of nucleotides of sequence shown in 2
The NO of ID containing SEQ:Sequence shown in 2 967-968 or the 968-969 sequence of nucleotides
Reverse primer.
In one embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 1 is,
For example, 5 '-TATGCTTGGCGTCATATTGCTCTT-3 ', and 5 '-TATTTTGCT
ATCGGATCTAAACCCT-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 1
Primer is, for example, 5 '-ATGGAACGGTCCCATACTTG-3 ', 5 '-GAATATC
GGTTTCGGTTGAT-3 ', 5 '-GGACAATAACATTCCACCAG-3 ', 5 '-TG
AAGAGGACGGATTGTGAG-3 ', 5 '-GGGATCGTACTCGCACAGA
G-3 ' and 5 '-CGGACAGATGATTACCCACA-3 '.
In another embodiment, for expanding SEQ ID NO:The primer pair of sequence shown in 2
For, for example, 5 '-GAAGTCGCATAATAGTAACCACG-3 ', and 5 '-GAAACC
GCCTACGATACATACCC-3’.For detecting SEQ ID NO:The sequencing of sequence shown in 2
Primer is, for example, 5 '-TGGGAGACAGGATTCATACC-3 ', 5 '-CGAATTTA
CTACCCGTGAG-3 ', 5 '-CCCCAGTTGGGTCGGAAAG-3 ', 5 '-GGGT
CAGAGCCAAAGTGCG-3 ' and 5 '-GAAACCGCCTACGATACATAC
CC-3’。
On the other hand, the side of the rice plant of recombinant nucleic acid fragment is contained this application provides seed selection
Method, it is included using the paddy rice recipient plant parent without genes of interest pack section as recurrent parent,
It is hybridized with the paddy rice donor plant containing genes of interest pack section, then will be resulting
Cenospecies be returned with recurrent parent, then the step of resulting backcrossing kind is carried out into selfing,
Foreground selection and Foreground selection wherein are carried out to restructuring plant using molecular labeling.For example, described
Recombinant nucleic acid fragment is as previously described.
In the above-mentioned methods, for the foreground selection molecular labeling be selected from Pi31,
One or more in W068C06B and Pi2S122；And/or utilize paddy rice full-length genome breeding
Chip carries out the Foreground selection.
In one embodiment, the seed selection that the application is provided contains blast resistant gene group recombinant nuclear
The method of the rice plant of acid fragment, it is comprised the following steps：1) recurrent parent and donor are planted
Thing is hybridized, then resulting cenospecies and recurrent parent are returned, and obtains backcrossing one
In generation, Pi31 and negative itemsets mark W068C06B, Pi2S122 couple are marked using favorable selection
Its unilateral homologous recombination fragment screening for carrying out blast resistant gene pack section, and it is complete using paddy rice
Genomic breeding chip, such as RICE6K, Foreground selection is carried out to it；2) selection background is replied
Preferably restructuring individual plant (this generation background recovery value is more than 75%) is returned again with recurrent parent
Hand over, obtain second backcross generation, detect that selection contains to it using favorable selection mark Pi31
The restructuring individual plant of blast resistant gene pack section, then using paddy rice full-length genome breeding chip,
Such as RICE6K, Foreground selection is carried out to it；3) restructuring individual plant (this that selection background is replied
Generation background recovery value obtains backcrossing three more than 87.5%) being returned again with recurrent parent
In generation, Pi31 and negative indicia W068C06B, Pi2S122 is marked to enter it using favorable selection
The opposite side homologous recombination fragment screening of row blast resistant gene pack section, and utilize the full base of paddy rice
Because of group a breeding chip, such as RICE60K, Foreground selection is carried out to it；And 4) selection is imported
Fragment is small, and the restructuring individual plant (background recovery value is more than 93.75%) that background is replied, and will choose
Restructuring individual plant selfing once, obtain selfed seed, using favorable selection mark Pi31 it is carried out
Detection, and using paddy rice full-length genome breeding chip, such as RICE60K carries out background to it
Selection, it is final to obtain the recombinant nucleic acid fragment homozygosis of group containing blast resistant gene and background reply (background
Recovery value is more than rice plant 99%).
In another embodiment, adopted when carrying out foreground selection to restructuring plant using molecular labeling
Amplimer, including：The primer pair of Pi31, wherein forward direction is marked to draw for amplifier molecule
Thing is 5 '-ATCCAAACCCGTTGTTGCAC-3 ', and reverse primer is 5 '-CGGCAATT
GCCACGATGATA-3’；The primer pair of W068C06B is marked for amplifier molecule, wherein
Forward primer is 5 '-CCTATCGCTGACAAAGAG-3 ', and reverse primer is
5’-CACCCAGCCAGTTCATCTA-3’；And mark Pi2S122 for amplifier molecule
Primer pair, wherein forward primer be 5 '-GACTTGAAAACCAGTGCGTG-3 ', instead
It is 5 '-CCTACCTAATGGAAAGGATTGC-3 ' to primer.
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 such as
It is preceding described.Selectively, pcr amplification product is analyzed using Sanger PCR sequencing PCRs.
Specifically, in the method for the detection recombinant nucleic acid fragment that the application is provided, for expanding
And detection SEQ ID NO:The primer combination of sequence shown in 1 is as follows：Amplimer, including forward direction
Primer：5 '-TATGCTTGGCGTCATATTGCTCTT-3 ', reverse primer：5’-TATTTT
GCTATCGGATCTAAACCCT-3’；Sequencing primer, including forward primer：5’-ATGG
AACGGTCCCATACTTG-3 ', reverse primer：5’-GAATATCGGTTTCGGTT
GAT-3 ', forward primer：5 '-GGACAATAACATTCCACCAG-3 ', forward primer：
5 '-TGAAGAGGACGGATTGTGAG-3 ', forward primer：5’-GGGATCGTA
CTCGCACAGAG-3 ' and forward primer：
5’-CGGACAGATGATTACCCACA-3’.Methods described is with testing sample genome
DNA is template, and entering performing PCR using above-mentioned amplimer expands, then using above-mentioned sequencing
The amplified production that primer pair is obtained is sequenced, if sequencing result and SEQ ID NO:1 sequence one
Cause or complementary, then contain SEQ ID NO in testing sample:Homologous recombination fragment shown in 1.
In addition, the application provide detection recombinant nucleic acid fragment method in, for expand and
Detection SEQ ID NO:The primer combination of sequence shown in 2 is as follows：Amplimer, including forward direction is drawn
Thing：5 '-GAAGTCGCATAATAGTAACCACG-3 ', reverse primer：5’-GAAA
CCGCCTACGATACATACCC-3’；Sequencing primer, including forward primer：5’-TGGGA
GACAGGATTCATACC-3 ', forward primer：5’-CGAATTTACTACCCGTGA
G-3 ', forward primer：5 '-CCCCAGTTGGGTCGGAAAG-3 ', forward primer：
5’-GG
GTCAGAGCCAAAGTGCG-3 ' and reverse primer：5’-GAAACCGCCTACGA
TACATACCC-3’.Methods described with testing sample genomic DNA as template, using upper
State amplimer and enter performing PCR amplification, then amplified production of the above-mentioned sequencing primer of utilization to acquisition
It is sequenced, if sequencing result and SEQ ID NO:2 sequences are consistent or complementary, then testing sample
In contain SEQ ID NO:Homologous recombination fragment shown in 2.
Contain SEQ ID NO during testing sample is determined by detection:1 and/or SEQ ID NO:2
The recombinant nucleic acid fragment of shown sequence, you can determine in testing sample comprising containing resistant gene
Recombinant nucleic acid fragment.
Additionally, present invention also provides the kit of detection recombinant nucleic acid fragment, it is included as preceding
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 described in the genome for detect rice plant to be measured
Recombinant nucleic acid fragment the step of.In one embodiment, examined using foregoing primer
Survey in the genome of rice plant to be measured and whether contain 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 implementation
In scheme, detected using foregoing kit in the genome of rice plant to be measured whether
Contain foregoing recombinant nucleic acid fragment.
It yet still another aspect, containing the application this application provides what is obtained by methods described screening
The rice plant or its seed of disclosed recombinant nucleic acid fragment.
The seed selection based on full-length genome selection and use technology that the application is provided contains blast resisting base
Because of a group method for the rice plant of recombinant nucleic acid fragment, the advantage with quick, accurate stabilization.
Only pass through the transformation of five generations, you can target gene group fragment is only imported into acceptor material, and simultaneously
Realize the reply of background.The acceptor material of the application improvement is ' HD9802S ', is paddy rice morning Xian
Type temp-sensing sterile line.Using the above method, ' HD9802S ' primary characteristic can retained
In the case of increase substantially its rice blast resistance.Meanwhile, the genome recombination core that the application is provided
Acid fragment is closely related with rice blast resistance, and the training of other kinds can be applied to as Resistance resource
Educate.
Brief description of the drawings
Fig. 1 is CR012602 paddy rice RICE60K full-length genome breedings in the embodiment of the present application 1
Chip detection result；Wherein, the indicated square frame of abscissa numeral represents 12 dyeing of paddy rice successively
Body, ordinate numeral is the physical location [with megabasse (Mb) as unit] on rice genome,
Grey lines represent receptor parent ' HD9802S ' genotype, and black lines represent donor parents
' R6 ' genotype, it is consistent i.e. without polymorphism section that white line represents two parent genotypes.Figure
In at No. 6 chromosome black round dot lines display block be the blast resistant gene group of importing
Recombinant nucleic acid fragment RecCR012602.
Fig. 2A and Fig. 2 B are RecCR012602 upstreams homologous recombination in the embodiment of the present application 2
Sequencing fragment comparison result；Asterisk shown in figure represents identical base in comparison result, in figure
CR012602 is the new lines for obtaining, and HD9802S is receptor parent ' HD9802S ', R6
It is donor parents ' R6 '.
Fig. 3 A and Fig. 3 B are RecCR012602 downstreams homologous recombination in the embodiment of the present application 2
Sequencing fragment comparison result；Asterisk shown in figure represents identical base in comparison result, in figure
CR012602 is the new lines for obtaining, and HD9802S is receptor parent ' HD9802S ', R6
It is donor parents ' R6 '.
Fig. 4 is the structure of RecCR012602 both sides homologous recombination fragment in the embodiment of the present application 2
Figure；Wherein, (A) is upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment
Structure chart, top base for donor ' R6 ' SNP or InDel mark, lower section base be by
SNP or the InDel mark of body ' HD9802S '.Grey section is from ' HD9802S '
Genomic segment, black section is that, from ' R6 ' genomic segment, white section is homologous
Restructuring section, abscissa is fragment length, with base pairs (bp) as unit.
Fig. 5 is qualification result in CR012602 rice blast resistances room in the embodiment of the present application 3；
Blade is followed successively by shown in figure：(A) rice blast susceptible variety Lijiang xintuanheigu；(B) original kind
‘HD9802S’；(C) improvement new lines CR012602；(D) rice blast disease-resistant variety paddy plum 4
Number.
Specific embodiment
Defined below and method is provided preferably to define the application and put into practice middle finger in the application
Lead those of ordinary skill in the art.Unless otherwise mentioned, term is according to association area ordinary skill people
The common usage of member understands.
As used herein, " nucleotide sequence " includes being related to the deoxyribose of single-stranded or double-stranded form
Nucleotides or ribonucleotide polymer, and unless otherwise limitation, nucleotide sequence with 5 ' extremely
3 ' directions are write from left to right, including the known analog (example with natural nucleotide fundamental property
Such as, peptide nucleic acid), the analog with naturally occurring nucleotides similar mode and single-stranded core
Acid hybridization.
In some embodiments, the nucleotide sequence of the application can be changed, to enter
Row conserved amino acid replacement.In certain embodiments, can be according to unifacial leaf codon preference
Property is not changed the replacement of amino acid sequence to the nucleotide sequence of the application, for example, can use
The codon of monocotyledon preference replaces codon of the coding with amino acid sequence, without changing
Become the amino acid sequence coded by the nucleotide sequence.
Specifically, the application is related to SEQ ID NO:1 or SEQ ID NO:2 is further excellent
Nucleotide sequence obtained by change.The more details of the method are described in Murray etc. (1989)
Nucleic Acids Res.17:477-498.Optimization nucleotide sequence can be used to improve blast resisting base
Because of a group expression of the recombinant nucleic acid fragment in paddy rice.
In some embodiments, the application further relates to SEQ ID NO:1 or SEQ ID NO:2
The variant of shown sequence.In general, the variant of specific nucleotide sequence will be with the specific nucleosides
Acid sequence have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%th, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%
Or 99.9% or higher sequence identity, or more complementary series.Such variant sequence thereof
Addition including one or more nucleic acids, missing are replaced, corresponding such that it is able to cause
Addition, removal or the replacement of amino acid residue.By alignment programs known in the art
Determine sequence identity including hybridization technique.The nucleotide sequence variants and the application of embodiment
The difference of sequence may be as few as 1-15 nucleotides, as little as 1-10 (such as 6-10),
As little as 5, as little as 4,3,2 or even 1 nucleotides.
The application is further related to comprising SEQ ID NO:1 or SEQ ID NO:It is specific in sequence shown in 2
The sequence in site or its fragment or its variant or its complementary series, for example, comprising SEQ ID NO:1
The sequence or its fragment of 368-1702 nucleotides of shown sequence or its variant or its complementary sequence
Row, or comprising SEQ ID NO:The sequence of 564-968 nucleotides of sequence shown in 2 or its
Fragment or its variant or its complementary series.According to the fragment comprising above-mentioned specific site, Neng Goute
Corresponding SEQ ID NO are identified different in naturely:1 or SEQ ID NO:Sequence shown in 2.Further
Ground, SEQ ID NO are contained by identifying:1 or SEQ ID NO:The recombinant nuclear of sequence shown in 2
Acid fragment, you can determine to include the recombinant nucleic acid fragment containing resistant gene in testing sample.
As used herein, " paddy rice " is any rice plant and including can be all with rice breeding
Plant variety.As used herein, " plant " or " plant ", including whole plant, plant cell,
Plant cell tissue cultures that plant organ, plant protoplast, plant can therefrom regenerate,
Complete plant cell in plant callus, vegetation bed and plant or plant part, the plant
Part for example embryo, pollen, ovule, seed, leaf, flower, branch, fruit, cane, root, the tip of a root,
Flower pesticide etc..
Go for the rice varieties of any required seed selection in the present processes.That is,
Can (i.e. Comprehensive Traits be preferable, it is contemplated that have hair by any improved seeds for lacking certain beneficial traits
Open up the kind of future) it is used as recurrent parent.With another with beneficial traits lacking in this receptor
Kind is used as donor parents, and the beneficial traits for being provided are preferably dominant Dominant gene.
In the embodiment of the application, using paddy rice ' HD9802S ' as recurrent parent, use
The paddy rice ' R6 ' of good rice blast resistance is had been shown to have as donor.
In the selection of restructuring plant provided herein, using molecular labeling to restructuring
Plant carries out foreground selection.The reliability of foreground selection is depended primarily between mark and target gene
Chain tightness degree is general simultaneously with adjacent two in both sides to improve the accuracy rate of selection
Mark is tracked selection to target gene.
In the embodiment of the application, the foreground selection mark of use includes that favorable selection is marked
Marked with negative itemsets.In a particular embodiment, the positive prospect choosing of optimized Select to use
It is the mark Pi31 with target gene group fragment close linkage to select mark, and negative itemsets mark is position
In the mark W068C06B of target fragment upstream, and positioned at the mark in target fragment downstream
Pi2S122。
In the embodiment of the application, homologous recombination is carried out using above-mentioned foreground selection mark
During detection, the criterion of side or unilateral homologous recombination is that Pi31 detects band identical with ' R6 '
Type, and W068C06B or Pi2S122 detects banding pattern identical with ' HD9802S '；Both sides or
The criterion of bilateral homologous recombination is that Pi31 detects banding pattern identical with ' R6 ', and W068C06B
Banding pattern identical with ' HD9802S ' is detected with Pi2S122.
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 used
Paddy rice full-length genome breeding chip disclosed in Chinese patent application CN102747138A
RICE6K, or the paddy rice full genome disclosed in PCT international applications 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) condition, or according to manufacturer's specification advised.
Rice plant material information used in this application can be found in rice in China kind and its
Pedigree database (http://www.ricedata.cn/variety/index.htm).
The rice genome physical location being previously mentioned in the application is with reference to paddy rice Nipponbare genome
MSU/TIGR annotates the 6.1st edition (http://rice.plantbiology.msu.edu/).
Embodiment 1Seed selection imports the restructuring plant of blast resistant gene pack section
The material used in the present embodiment is paddy rice ' HD9802S ' and paddy rice ' R6 '.
Paddy rice ' R6 ' has good rice blast resistance, and it is probably No. 6 chromosome to speculate
Pi2, Pi9 and Pigm where gene cluster region the rice blast resistance of the material is served
Key effect.
In the Breeding Process of restructuring plant, prospect choosing is carried out to restructuring plant using molecular labeling
Select, the foreground selection molecular labeling to being used is screened.With reference to paddy rice Nipponbare gene
Group MSU/TIGR annotates the 6.1st edition, downloads the 6th chromosome 9,559,000 to 10,990,000
DNA sequence dna.The SSR sites in above-mentioned sequence are scanned using SSRLocator softwares.
Primer is designed to the SSR sites for searching out using the softwares of Primer Premier 3.0, design altogether is drawn
Thing 162 pairs.By the method for PCR, above-mentioned primer pair is screened at ' R6 ' and ' HD9802S '
In polymorphism, finally pick out before having polymorphism, amplification efficiency high in two parts of materials
Scape selects molecular labeling, is respectively favorable selection mark Pi31 and negative itemsets mark
W068C06B、Pi2S122.The specific primer information for expanding above-mentioned molecular labeling for PCR is shown in
Table 1.
The foreground selection molecular labeling primer information of table 1
Genomic fragment where forementioned gene cluster in paddy rice ' R6 ' is imported into paddy rice
In ' HD9802S ', detailed process is as follows：
With ' HD9802S ' for recurrent parent, ' R6 ' is hybridized for donor parents, by institute
The cenospecies for obtaining is returned with recurrent parent ' HD9802S ', obtains BC1F1Seed,
After nursery Pi31 and negative itemsets mark W068C06B, Pi2S122 are marked using favorable selection
Carry out restructuring Single-plant selection, filter out 24 it is homologous heavy in target gene group DNA fragmentation side
The individual plant of group, i.e. Pi31 detects banding pattern identical with ' R6 ', and W068C06B or Pi2S122
Detection banding pattern identical with ' HD9802S ', and utilize paddy rice full-length genome breeding chip
RICE6K (CN102747138A) carries out Foreground selection (Yu etc., Plant Biotechnology to it
Journal.2014,12:28-37)。
The comparable chip result in the 24 unilateral homologous recombination individual plants for filtering out, selection background is returned
Multiple best restructuring individual plant (this generation background recovery value is more than 75%), makes itself and recurrent parent
' HD9802S ' is returned again, obtains BC2F1Seed, utilizes favorable selection after nursery
Mark Pi31 to detect it, select the restructuring individual plant containing target gene group DNA fragmentation,
That is Pi31 detects banding pattern identical with ' R6 ', using paddy rice full-length genome breeding chip RICE6K
Foreground selection is carried out to it.
Selection background replys preferable individual plant (this generation background recovery value is more than 87.5%), make its with
Recurrent parent ' HD9802S ' is returned again, obtains BC3F1Seed, profit after nursery
Pi31 and negative indicia W068C06B, Pi2S122 are marked to the seed that harvests with favorable selection
Carry out the screening of target gene group DNA fragmentation opposite side homologous recombination fragment, obtain 24
The individual plant of target fragment both sides restructuring, i.e. Pi31 detects banding pattern identical with ' R6 ', and
W068C06B and Pi2S122 detects banding pattern identical with ' HD9802S '.
Using paddy rice full-length genome breeding chip RICE60K (WO/2014/121419) to above-mentioned
24 bilaterals exchange individual plant carries out background and target fragment selection (Chen etc., Molecular Plant.
2014,7:541-553), importing target fragment is screened smaller, and the target list that background is replied
One (this generation background recovery value is more than 93.75%) of strain.
The individual plant selfing that will be chosen once, obtains BC3F2, marked using favorable selection after nursery
Pi31 detects to it, selects the individual plant containing target gene group DNA fragmentation, i.e. Pi31
Detection banding pattern identical with ' R6 ', using paddy rice full-length genome breeding chip RICE60K to it
Carry out Foreground selection.
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 CR012602.Chip detection result is shown in Fig. 1.
Because ' HD9802S ' is two-line sterile line, BC in above process1F1、BC2F1、
BC3F1And BC3F2Selected individual plant is sterile individual plant from generation to generation, takes following method and realizes not
The sterile changeover of individual plant is educated to be returned or selfing：When sterile individual plant enters 4 phase of reproduction period,
(21-23 DEG C) of low-temperature treatment area is processed 10 days or so, using plant growth cabinet, is planted
Thing growth room, cold water leaching fill pond etc.；The pollen for putting forth ears is carried out using IKI decoration method
Fertility identification, judges that the fertile fringe of more than 70% pollen is fertility successful conversion fringe, carries out immediately
Backcrossing or selfing.
Embodiment 2The determination of homologous recombination fragment after importing rice blast resistance gene pack section
For the rice blast resistance gene group clip size for determining to import, ' HD9802S ' is led
Entering the homozygosis individual plant of fragment has carried out the sequencing of target gene group fragment both sides homologous recombination fragment.
Blast resistant gene group recombinant nucleic acid fragment contained by CR012602 is named as
RecCR012602。
Primarily determined that by paddy rice full-length genome breeding chip RICE60K testing results,
RecCR012602 is located at two SNP markers R0610084130TG and R0610454932GA
Between.
Meanwhile, using Miseq sequencing technologies to ' HD9802S ', ' R6 ' and CR012602
Three samples 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, and uses MiSeq V2 Reagent Kit
(illumina) kit carries out sequencing reaction.Entered using the desk-top sequenators of Miseq (illumina)
Row detection.Specific steps and method are referring to each kit and sequenator operation instructions.
It is according to foregoing SNP chip and Miseq sequencing results, RecCR012602 upstreams is same
10090076bp to the 10093784bp that source recombinant fragment is positioned at the 6th chromosome is interval, downstream
It is interval that homologous recombination fragment is positioned at 10437335bp to 10439846bp.
On this basis, the 6.1st edition is annotated with reference to paddy rice Nipponbare genome MSU/TIGR,
Download respective segments DNA sequence dna.Expanded using the Software for Design of Primer Premier 5.0 and surveyed
Sequence primer, design requirement is primer 22nt long or so, G/C content 40-60% and no mispairing.
It is control with receptor parent ' HD9802S ' and donor parents ' R6 ', to RecCR012602
Upstream and downstream homologous recombination fragment separately designs amplimer, uses high-fidelity enzyme KOD FX
Neo (TOYOBO) is expanded, and finds optimal amplification condition using two-step method or three-step approach, really
Protect amplified production and be shown as single bright band in agarose gel electrophoresis detection.Wherein determine
Upstream homologous recombination fragment amplification primer reaction condition be：94℃2min；98 DEG C of 10sec,
68 DEG C of 180sec, 37 circulations；20℃1min.Downstream homologous recombination fragment amplification primer is anti-
The condition is answered to be：94℃2min；98 DEG C of 10sec, 68 DEG C of 180sec, 37 circulations；20℃
1min.Thus, pair for amplification primer is finally respectively filtered out for upstream and downstream homologous recombination piece
The amplification of section.
In addition, with amplified production as template, being sequenced using Sanger PCR sequencing PCRs, according to reality
Border is sequenced effect, finally respectively filters out 6 and 5 sequencing primers are respectively used to upstream and downstream
The sequencing of homologous recombination fragment.Specific amplimer and sequencing primer sequence are shown in Table 2, sequencing
Result is shown in Fig. 2A, Fig. 2 B and Fig. 3 A, Fig. 3 B.
RecCR012602 upstreams homologous recombination sequencing fragment length is 2839bp (SEQ ID
NO:1).1-368bp is the genomic segment of acceptor ' HD9802S ', is compared with donor ' R6 '
Compared with there are 2 SNP, 1 Indel.This 1333bp section of 369-1701bp is homologous
Restructuring section.1702-2839bp is donor ' R6 ' genomic fragment, with ' HD9802S '
Compare, there are 4 SNP.
RecCR012602 downstreams homologous recombination sequencing fragment length is 1748bp (SEQ ID
NO:2).1-564bp is the genomic segment of donor ' R6 ', is compared with ' HD9802S ',
In the presence of 2 SNP, 1 Indel.This 403bp section of 565-967bp is homologous recombination area
Section.968-1748bp is the genomic segment of acceptor ' HD9802S ', is compared with donor ' R6 '
Compared with there are 3 SNP, 2 Indel.
Fig. 4 is the structure chart of RecCR012602 both sides homologous recombination fragment.Wherein, (A)
It is upstream homologous recombination fragment structure figure；(B) it is downstream homologous recombination fragment structure figure.Top alkali
Base is marked for the SNP or InDel of donor ' R6 ', and lower section base is acceptor ' HD9802S '
SNP or InDel mark.Grey section be from ' HD9802S ' genomic segment,
Black section is that, from ' R6 ' genomic segment, white section is homologous recombination section.It is horizontal
Coordinate 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 3The Resistance Identification that ' HD9802S ' is imported after blast resistant gene pack section
In order to identify resistance effect, to new lines CR012602, the recurrent parent of the application seed selection
' HD9802S ', rice blast disease-resistant variety paddy plum No. 4 (as positive control), and rice blast
Susceptible variety Lijiang xintuanheigu (as negative control) carries out indoor plantation, is cultivated to 3-4
Adopted after the leaf phase and identified with the following method：
Choose from Yichang sick nursery rice blast disease sample separate within 2015 M15Bb-1-1,
M15Bb-1-2, M15Bb-2-1, M15Bb-3-1, M15Bb-4-1, M15Bb-5-1 and
M15Bb-6-1, totally 7 plants of rice blast bacterial strains are used as inoculating strain.Bacterial strain uses -20 DEG C of sorghum grain method
Preserve, the sorghum grain of preservation is taken out to potato dextrose medium (PDA) flat board using preceding
Activation (PDA：Peeled potatoes 200g, glucose 20g, agar powder 15g, distilled water constant volume
To 1L), 28 DEG C of illumination cultivations take diameter 5mm fresh mycelia block after 5 days is forwarded to sorghum grain
(sorghum grain 500g adds 1.5L distilled water, boils and liquid is filtered off to boiling, by height in culture medium
Fine strain of millet grain pulls loading 250ml triangular flasks, 100ml/ bottles, moist heat sterilization 20 minutes out), 10 pieces/
Bottle, connects bacterium and sorghum grain is shaken into scattered daily after 2 days, and 28 DEG C of dark culturing to mycelia cover with sorghum grain.
Then sorghum grain is spread out on sterile gauze, covers aseptic damp gauze, in 25 DEG C, RH
Cultivated under >=95%, 12h illumination condition 4-5 days and produced to a large amount of spores, (0.02% is contained with sterilized water
Polysorbas20) lower spore is washed, wait spore amount combined inoculation bacterial strain, adjustment concentration to 5 × 105Individual/ml.
With mixing conidial suspension spray inoculation CR012602, ' HD9802S ', Gu Mei 4
Number and Lijiang xintuanheigu, be inoculated with three repetitions.Transparent cover, 28 DEG C of dark on inoculation back cover
24h is cultivated, then 16h illumination cultivations are investigated after 5 days.
Investigation standard is 0 grade (height is anti-, HR)：There is no symptom；1 grade (anti-, R)：Very little it is brown
Color scab；2 grades (in resist, MR)：Diameter is about the brown scab of 1mm；3 grades (MS, in
Sense)：Directly it is about the band circle scab of 2-3mm, central canescence, edge brown；4 grades (sense,
S)：It is about the oval scab of 1-3cm, central canescence, edge brown；5 grades (height sense,
HS)：Big oval scab long and wide, scab fusion is in blocks, withered to blade.Wherein 0-2
Level is disease-resistant, and 3-5 grades is susceptible.Inoculation the results are shown in Table 3 and Fig. 5.
Resistant expression after the inoculation rice blast fungus of table 3
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 piece of sequence 368-1702 shown in 1 nucleotides
Section 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 sequence 564-968 shown in 2 nucleotides
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 sequence 1-368 shown in 1 nucleotides
Forward primer and specific recognition SEQ ID NO:Sequence 1702-2839 shown in 1 nucleotides
The reverse primer of sequence；
(II) first group of primer pair and second group of combination of primer pair below, it is included
(a) first group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1
1-368 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Sequence shown in 1
Arrange the 369-1701 reverse primer of the sequence of nucleotides；With
(b) second group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 1
369-1701 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:1 institute
Show 1702-2839 reverse primer of the sequence of nucleotides of sequence；
(III) specific recognition includes SEQ ID NO:Sequence shown in 1 367-368 or
The forward primer and specific recognition of 368-369 sequence of nucleotides include SEQ ID NO:1
Shown sequence 1701-1702 or the 1702-1703 reverse primer of the sequence of nucleotides；
(IV) specific recognition includes SEQ ID NO:Sequence shown in 1 367-368 or
368-369 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Shown in 1
1702-2839 reverse primer of the sequence of nucleotides of sequence；
(V) specific recognition SEQ ID NO:1-368 sequence of nucleotides of sequence shown in 1
Forward primer and specific recognition include SEQ ID NO:Sequence 1701-1702 shown in 1
Or the 1702-1703 reverse primer of the sequence of nucleotides；And/or optionally,
(VI) specific recognition SEQ ID NO:The sequence of sequence 1-564 shown in 2 nucleotides
Forward primer and specific recognition SEQ ID NO:Sequence 968-1748 shown in 2 nucleotides
The reverse primer of sequence；
(VII) the 3rd group of primer pair and the 4th group of combination of primer pair below, it is included
(c) the 3rd group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2
1-564 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Sequence shown in 2
Arrange the 565-967 reverse primer of the sequence of nucleotides；With
(d) the 4th group of primer pair：Specific recognition SEQ ID NO:Sequence shown in 2
565-967 the forward primer and specific recognition SEQ ID NO of the sequence of nucleotides:Shown in 2
968-1748 reverse primer of the sequence of nucleotides of sequence；
(VIII) specific recognition includes SEQ ID NO:564-565 nucleosides of sequence shown in 2
The forward primer and specific recognition of the sequence of acid include SEQ ID NO:Sequence shown in 2
967-968 or the 968-969 reverse primer of the sequence of nucleotides；
(IX) specific recognition includes SEQ ID NO:564-565 nucleotides of sequence shown in 2
Sequence forward primer and specific recognition SEQ ID NO:Sequence 968-1748 shown in 2
The reverse primer of the sequence of position nucleotides；
(X) specific recognition SEQ ID NO:1-564 sequence of nucleotides of sequence shown in 2
Forward primer and specific recognition include SEQ ID NO:Sequence shown in 2 967-968 or
The 968-969 reverse primer of the sequence of nucleotides.
3. test right requires the primer of fragment described in 1, wherein the primer is selected from：
(I) amplification SEQ ID NO:The primer pair of sequence shown in 1
5 '-TATGCTTGGCGTCATATTGCTCTT-3 ',
5’-TATTTTGCTATCGGATCTAAACCCT-3’；And
(II) sequencing SEQ ID NO:The primer of sequence shown in 1
5 '-ATGGAACGGTCCCATACTTG-3 ',
5 '-GAATATCGGTTTCGGTTGAT-3 ',
5 '-GGACAATAACATTCCACCAG-3 ',
5 '-TGAAGAGGACGGATTGTGAG-3 ',
5 '-GGGATCGTACTCGCACAGAG-3 ',
5’-CGGACAGATGATTACCCACA-3’；And/or optionally,
(III) amplification SEQ ID NO:The primer pair of sequence shown in 2
5 '-GAAGTCGCATAATAGTAACCACG-3 ',
5’-GAAACCGCCTACGATACATACCC-3’；And
(IV) sequencing SEQ ID NO:The primer of sequence shown in 2
5 '-TGGGAGACAGGATTCATACC-3 ',
5 '-CGAATTTACTACCCGTGAG-3 ',
5 '-CCCCAGTTGGGTCGGAAAG-3 ',
5 '-GGGTCAGAGCCAAAGTGCG-3 ',
5’-GAAACCGCCTACGATACATACCC-3’。
4. the method that seed selection contains the rice plant of the recombinant nucleic acid fragment described in claim 1,
It includes, using the paddy rice recipient plant parent without genes of interest pack section as recurrent parent, inciting somebody to action
It is hybridized with the paddy rice donor plant containing genes of interest pack section, then will be resulting
Cenospecies is returned with recurrent parent, then the step of resulting backcrossing kind is carried out into selfing,
Foreground selection and Foreground selection wherein are carried out to restructuring plant using molecular labeling.
5. method as claimed in claim 4, wherein for the molecular labeling of the foreground selection
One or more in selected from Pi31, W068C06B and Pi2S122；And/or
Optionally, the Foreground selection is carried out using paddy rice full-length genome breeding chip.
6. the method as described in claim 4 or 5, wherein the recombinant nucleic acid fragment contain it is anti-
Rice blast ospc gene, and the described method comprises the following steps：
1) recurrent parent and donor plant are hybridized, by resulting cenospecies and samsara parent
Originally it is returned, is obtained first backcross generation, marks Pi31 and negative itemsets to mark using favorable selection
W068C06B, Pi2S122 carry out the unilateral homologous recombination piece of blast resistant gene pack section to it
Section screening, and Foreground selection is carried out to it using paddy rice full-length genome breeding chip；
2) selection background is replied preferably restructuring individual plant and is returned again with recurrent parent, is obtained
Second backcross generation, detects that selection contains blast resisting to it using favorable selection mark Pi31
The restructuring individual plant of genomic fragment, is then carried on the back using paddy rice full-length genome breeding chip to it
Scape is selected；
3) the restructuring individual plant that selection background is replied is returned again with recurrent parent, is obtained
Third backcross generation, Pi31 and negative indicia W068C06B, Pi2S122 are marked using favorable selection
The opposite side homologous recombination fragment screening of blast resistant gene pack section is carried out to it, and utilizes water
Rice full-length genome breeding chip carries out Foreground selection to it；And
4) selection introgressed segment is small, and the restructuring individual plant that background is replied, the restructuring list that will be chosen
Strain selfing once, obtains selfed seed, and it is detected using favorable selection mark Pi31, and
Foreground selection is carried out to it using paddy rice full-length genome breeding chip, it is final to obtain the anti-rice containing homozygosis
The rice plant that seasonal febrile diseases genome recombination nucleic acid fragment and background are replied.
7. the method as any one of claim 4 to 6, wherein using molecular labeling pair
The amplimer that restructuring plant used during foreground selection is as follows：
Amplifier molecule marks the primer pair of Pi31, and it includes：
Forward primer：5 '-ATCCAAACCCGTTGTTGCAC-3 ',
Reverse primer：5’-CGGCAATTGCCACGATGATA-3’；
Amplifier molecule marks the primer pair of W068C06B, and it includes：
Forward primer：5 '-CCTATCGCTGACAAAGAG-3 ',
Reverse primer：5’-CACCCAGCCAGTTCATCTA-3’；And
Amplifier molecule marks the primer pair of Pi2S122, and it includes：
Forward primer：5 '-GACTTGAAAACCAGTGCGTG-3 ',
Reverse primer：5’-CCTACCTAATGGAAAGGATTGC-3’.
8. the method that test right requires the recombinant nucleic acid fragment described in 1, it includes using right
It is required that the primer described in 2 or 3, enters performing PCR reaction, and analyze by template of testing gene group
The step of pcr amplification product.
9. test right requires the kit of the recombinant nucleic acid fragment described in 1, and it includes that right will
Seek the primer described in 2 or 3.
10. rice plant or seed containing the recombinant nucleic acid fragment described in claim 1 are screened
Method, whether it is included in the genome for detect rice plant to be measured or seed containing have 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 claim 8 is used
Described method, or detected using the kit described in claim 9.
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