CN109929837A - Rice genome recombinant nucleic acid segment RecCR012613 and its detection method - Google Patents

Abstract

This application provides rice genome recombinant nucleic acid segment RecCR012613 and its detection methods.Present invention also provides the selections of the rice plant containing recombinant nucleic acid segment, carry out foreground selection and Foreground selection to recombination plant using molecular labeling, obtain the rice plant containing recombinant nucleic acid segment.

Description

Rice genome recombinant nucleic acid segment RecCR012613 and its detection method

Technical field

This application involves full-length genome selection and use technologies.Specifically, this application involves educated using full-length genome selection Kind technology breeding has the rice plant of the recombinant nucleic acid segment of rice blast resistance function, and the recombinant nucleic acid obtained therefrom Segment and its detection method.

Background technique

For a long time, the selection method of traditional breeding method depends on the evaluation of variable rate technology type, according to breeder individual Experience is accepted or rejected, and maximum disadvantage is that time-consuming, and efficiency is lower.The efficiency of selection is improved, optimal method is answered It is directly to select genotype.With the development of molecular biotechnology, molecular labeling is to realize to the straight of genotype Selecting, which is selected, provides possibility.In recent years, started to improve individual target character using molecular marker-assisted selection method, it can It is significant to shorten the breeding time limit.

Rice blast is one of rice most serious disease, and the Rice Yield Loss Caused as caused by rice blast accounts for 11% every year in the whole world ~30%, therefore the research of rice blast and its resistance is particularly important.As that studies rice blast gradually gos deep into, many water Rice blast resistant gene DNA fragmentation is positioned and is cloned in succession.Wherein, the section Pi2 of the 6th chromosome of rice is positioned and is cloned Many rice blast resistance genes, such as Pi2, Piz-t, Pi9, Pigm, Pi50, which includes a rice blast resistance gene Gene cluster (Qu etc., Genetics.2006,172:1901-1914；Wang etc., Phytopathology.2012,102:779- 786；Xiao etc., Mol Breeding.2012,30:1715-1726；Liu et al., 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).

Summary of the invention

On the one hand, this application provides rice genome recombinant nucleic acid segments, and it includes following recombinant nucleic acid segments, or It is made of following recombinant nucleic acid segment:

- the first recombinant nucleic acid segment, is selected from: i) comprising sequence shown in SEQ ID NO:1 the 504th to 525 and the 697th To the sequence of 721 nucleotide or its segment or its variant or its complementary series；Ii) comprising sequence shown in SEQ ID NO:1 the The sequence or its segment of 514 to 706 nucleotide or its variant or its complementary series；Iii) comprising sequence shown in SEQ ID NO:1 Arrange the sequence or its segment or its variant or its complementary series and SEQ of the 504th to 525 and the 697th to 721 nucleotide At least one of the 5th to 26, the 338th to 360 and the 730th to 754 nucleotide of sequence shown in ID NO:1 sequence or its Segment or its variant or its complementary series；Or iv) comprising sequence or its segment or its variant shown in SEQ ID NO:1 or its mutually Complementary series；And/or

- the second recombinant nucleic acid segment, is selected from: v) comprising sequence shown in SEQ ID NO:2 the 181st to 203 and the 406th To the sequence of 430 nucleotide or its segment or its variant or its complementary series；Vi) comprising sequence shown in SEQ ID NO:2 the The sequence or its segment of 192 to 416 nucleotide or its variant or its complementary series；Vii) comprising sequence shown in SEQ ID NO:2 Arrange the sequence or its segment or its variant or its complementary series and SEQ of the 181st to 203 and the 406th to 430 nucleotide In the 3rd to 21, the 143rd to 162, the 442nd to 465 and the 692nd to 713 nucleotide of sequence shown in ID NO:2 extremely A kind of few sequence or its segment or its variant or its complementary series；Or viii) include sequence or its piece shown in SEQ ID NO:2 Section or its variant or its complementary series.

In one embodiment, rice genome recombinant nucleic acid segment provided by the present application include selected from i), ii), iii), Iv the first recombinant nucleic acid segment of any sequence in), or by selected from i), ii), iii), iv) in any sequence first recombination Nucleic acid fragment composition.

In another embodiment, rice genome recombinant nucleic acid segment provided by the present application include selected from v), vi), Vii), viii) in any sequence the second recombinant nucleic acid segment, or by selected from v), vi), vii), viii) in any sequence The second recombinant nucleic acid segment composition.

In yet another embodiment, rice genome recombinant nucleic acid segment provided by the present application include selected from i), ii), Iii), iv) in any sequence the first recombinant nucleic acid segment with selected from v), vi), vii), viii) in any sequence the second weight Group nucleic acid fragment combination, or by selected from i), ii), iii), iv) in any sequence the first recombinant nucleic acid segment be selected from V), vi), vii), viii) in the group of the second recombinant nucleic acid segment of any sequence be combined into.

In addition, this application provides the primers for detecting the recombinant nucleic acid segment, wherein the primer includes:

The primer for detecting the first recombinant nucleic acid segment, is selected from: sequence shown in (I) specific recognition SEQ ID NO:1 the The 697th to 721 nucleosides of sequence shown in the primer and specific recognition SEQ ID NO:1 of the sequence of 504 to 525 nucleotide The primer of the sequence of acid；(II) sequence of the 514th to 706 nucleotide of sequence shown in specific recognition SEQ ID NO:1 is drawn Object；(III) primer of the sequence of the 504th to 525 nucleotide of sequence shown in specific recognition SEQ ID NO:1, and specificity Identify the primer and specific recognition SEQ ID of the sequence of the 697th to 721 nucleotide of sequence shown in SEQ ID NO:1 The primer of at least one of sequence the 5th to 26 shown in NO:1, the 338th to 360 and the 730th to 754 nucleotide sequence；Or (IV) primer of sequence shown in specific recognition SEQ ID NO:1；And/or

The primer for detecting the second recombinant nucleic acid segment, is selected from: sequence shown in (V) specific recognition SEQ ID NO:2 the The 406th to 430 nucleosides of sequence shown in the primer and specific recognition SEQ ID NO:2 of the sequence of 181 to 203 nucleotide The primer of the sequence of acid；(VI) sequence of the 192nd to 416 nucleotide of sequence shown in specific recognition SEQ ID NO:2 is drawn Object；(VII) primer of the sequence of the 181st to 203 nucleotide of sequence shown in specific recognition SEQ ID NO:2, and specificity Identify the primer and specific recognition SEQ ID of the sequence of the 406th to 430 nucleotide of sequence shown in SEQ ID NO:2 At least one of sequence the 3rd to 21 shown in NO:2, the 143rd to 162, the 442nd to 465 and the 692nd to 713 nucleotide The primer of sequence；Or the primer of sequence shown in (VIII) specific recognition SEQ ID NO:2.

In one embodiment, for expanding the primer pair of the first recombinant nucleic acid segment, such as expanding SEQ ID The primer pair of sequence shown in NO:1 is, for example, 5 '-CTCTTGCCAAACATGCCAGGGTA-3 ' and 5 '- CTCACGTTCACATTCGATTCTCC-3'；And the primer for the first recombinant nucleic acid segment to be sequenced, such as SEQ to be sequenced The primer of the sequence of the 5th to 26, the 338th to 360 and the 504th to 525 nucleotide of sequence shown in ID NO:1 is, for example, 5'-CTCTTGCCAAACATGCCAGGGTA-3'；For sequence shown in SEQ ID NO:1 the 697th to 721 and the 730th to be sequenced Primer to the sequence of 754 nucleotide is, for example, 5 '-GCCATCAGCATCGTTAGTC-3 '.

In another embodiment, for expanding the primer pair of the second recombinant nucleic acid segment, for expanding SEQ ID NO:2 The primer pair of shown sequence is, such as 5 '-CTAGACCACAAAGACCACGACAG-3 ' and 5 '- TGTGCTCACTGGGTAGGATGTCT-3'.For the primer of the second recombinant nucleic acid segment to be sequenced, for example, for SEQ ID to be sequenced Sequence the 3rd to 21 shown in NO:2, the sequence of the 143rd to 162 and the 181st to 203 nucleotide primer be, for example, 5 '- CTAGACCACAAAGACCACGACAG-3'；And for sequence the 406th to 430, the 442nd shown in SEQ ID NO:2 to be sequenced Primer to the sequence of 465 and the 692nd to 713 nucleotide is, for example, 5 '-ACGTCGTCCCTGAGCTAGA-3 '.

On the other hand, the method for the rice plant of the recombinant nucleic acid segment is contained this application provides breeding, wherein institute Stating recombinant nucleic acid segment has the function of rice blast resistance, and the described method comprises the following steps: 1) by recurrent parent rice ' Y58S ' and donor rice ' Gu Mei No. 4 numbers ' is hybridized, and obtained cenospecies is returned with recurrent parent, is returned A generation marks Pi31 and negative itemsets label Pi2S67, Pi2S122 to carry out blast resistant gene pack to it using favorable selection The unilateral homologous recombination segment screening of section, and Foreground selection is carried out to it using rice full-length genome breeding chip；2) selection back Scape is replied preferable recombination single plant and is returned again with recurrent parent, and second backcross generation is obtained, and marks Pi31 using favorable selection It is detected, the recombination single plant containing blast resistant gene group segment is selected, then utilizes rice full-length genome breeding core Piece carries out Foreground selection to it；3) the recombination single plant that selection background has been replied is returned again with recurrent parent, is returned Three generations is handed over, marks Pi31 and negative itemsets label Pi2S67, Pi2S122 to carry out blast resistant gene group to it using favorable selection The other side homologous recombination segment of segment is screened, and carries out Foreground selection to it using rice full-length genome breeding chip；And 4) selection introgressed segment is small, and the recombination single plant that background has been replied, and the recombination individual plant selfing chosen is primary, obtains selfed seed, It is detected using favorable selection label Pi31, and Foreground selection is carried out to it using rice full-length genome breeding chip, The final rice plant for obtaining the recombinant nucleic acid segment of group containing homozygous gene and background and replying.

In one embodiment, the amplimer used when carrying out foreground selection to recombination plant using molecular labeling, packet Include the primer pair of amplifier molecule label Pi31 comprising forward primer: 5 '-ATCCAAACCCGTTGTTGCAC-3 ' reversely draw Object: 5 '-CGGCAATTGCCACGATGATA-3 '；The primer pair of amplifier molecule label Pi2S67 comprising: forward primer: 5 '- CCGATGCAAGAACAAGCTAA-3 ', reverse primer: 5 '-CCACCACATCACCAGTGTTT-3 '；And amplifier molecule label The primer pair of Pi2S122 comprising: forward primer: 5 '-GACTTGAAAACCAGTGCGTG-3 ', reverse primer: 5 '- CCTACCTAATGGAAAGGATTGC-3’。

Another aspect, this application provides the methods for detecting the recombinant nucleic acid segment comprising is mentioned using the application The primer of confession carries out PCR reaction, and the step of analyzing PCR product by template of testing gene group.Specifically, the method with Sample to be tested genomic DNA is template, carries out PCR amplification using aforementioned amplimer, is then obtained using aforementioned sequencing primer pair The amplified production obtained is sequenced, if sequencing result and sequence shown in SEQ ID NO:1 and/or 2 or its section are consistent or complementary, The then homologous recombination nucleic acid fragment in sample to be tested containing sequence shown in SEQ ID NO:1/ or 2.

It has been determined in sample to be tested by detection containing sequence or its area shown in SEQ ID NO:1 and/or SEQ ID NO:2 The recombinant nucleic acid segment of section can determine in sample to be tested comprising the recombinant nucleic acid segment with rice blast resistance function.

In addition, present invention also provides the kits of detection recombinant nucleic acid segment comprising primer as the aforementioned.

Further, present invention also provides screening the rice plant containing recombinant nucleic acid segment or seed method, Include the steps that detecting in the genome of rice plant to be measured and whether contains foregoing recombinant nucleic acid segment.

In one embodiment, it is detected using foregoing primer and whether is contained in the genome of rice plant to be measured Foregoing recombinant nucleic acid segment.In another embodiment, using the method for foregoing detection recombinant nucleic acid segment Whether contain foregoing recombinant nucleic acid segment in genome to detect rice plant to be measured.In yet another embodiment, It is detected using foregoing kit in the genome of rice plant to be measured and whether contains foregoing recombinant nucleic acid piece Section.

It yet still another aspect, this application provides what is screened by the method to contain recombinant nucleic acid disclosed in the present application The rice plant of segment or its seed.

Breeding provided by the present application based on full-length genome selection and use technology contains blast resistant gene group recombinant nucleic acid The method of the rice plant of segment has quick, accurate, stable advantage.It, can be only by target base only by the transformation of five generations Because group segment imports acceptor material, and the reply of background is realized simultaneously.The acceptor material of the application improvement be ' Y58S ' for Eurytopic photo-thermo-sensitive genetic male sterile line, rice blast resistance performance shortcoming.It, can be original excellent in reservation ' Y58S ' using the above method Its rice blast resistance is increased substantially in the case where point.Meanwhile recombinant nucleic acid segment provided by the present application and rice blast resistance are tight Close correlation can be used as the cultivation that Resistance resource is applied to other kinds.

Detailed description of the invention

Fig. 1 is CR012613 rice RICE60K full-length genome breeding chip test result in the embodiment of the present application 1；Wherein, Box indicated by abscissa number successively indicates 12 chromosomes of rice, and ordinate number is the physical location on rice genome [with megabasse (Mb) for unit], grey lines represent receptor parent ' Y58S ' genotype, and black lines represent donor parents ' paddy Plum No. 4 ' genotype, white line represent two parent genotypes unanimously i.e. without polymorphism section.No. 6 chromosome dark circles in figure Lines display block shown in point is the blast resistant gene group recombinant nucleic acid segment RecCR012613 imported.

Fig. 2 is qualification result in CR012613 rice blast resistance room in the embodiment of the present application 3；Blade as shown in the figure is successively Are as follows: (A) rice blast susceptible variety Lijiang xintuanheigu；(B) original kind ' Y58S '；(C) new lines CR012613 is improved；(D) rice Seasonal febrile diseases disease-resistant variety paddy plum No. 4.

Specific embodiment

Defined below and method is provided preferably to define the application and instruct this field general in the application practice Logical technical staff.Unless otherwise mentioned, term understands according to the common usage of person of ordinary skill in the relevant.

As used herein, " nucleotide sequence " includes being related to the deoxyribonucleotide or ribose core of single-stranded or double-stranded form Thuja acid polymer, and unless otherwise limitation, nucleotide sequence is write from left to right with 5 ' to 3 ' directions, including with natural nucleus The known analog (for example, peptide nucleic acid) of thuja acid fundamental property, the analog with as naturally occurring ucleotides side Formula hybridizes with single-chain nucleic acid.

In some embodiments, the nucleotide sequence of the application can be changed, is replaced with carrying out conserved amino acid It changes.In certain embodiments, can not be changed according to nucleotide sequence of the unifacial leaf codon preference to the application The replacement of amino acid sequence, such as the password with amino acid sequence can be encoded with the codon replacement of monocotyledon preference Son, without changing the encoded amino acid sequence of the nucleotide sequence.

Specifically, this application involves advanced optimize resulting nucleotide sequence to SEQ ID NO:1 or 2.This method More details are described in Murray etc. (1989) Nucleic Acids Res.17:477-498.Optimization nucleotide sequence can be used for Improve the expression of blast resistant gene group recombinant nucleic acid segment in rice.

In some embodiments, this application involves sequence shown in SEQ ID NO:1 or 2 or the variants of its section.Generally For, the variant of specific nucleotide sequence will have at least about 70% with the specific nucleotide sequence, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% Or 99.9% or higher sequence identity or above complementary series.Such variant sequence thereof includes one or more nucleic acid Addition, missing or the replacement of residue, so as to lead to the addition, removal or replacement of corresponding amino acid residue.Pass through ability Known alignment programs include that hybridization technique determines sequence identity in domain.The nucleotide sequence variants and sheet of embodiment The difference of the sequence of application may be as few as 1-15 nucleotide, as little as 1-10 (such as 6-10), and as little as 5, as little as 4,3,2 Or even 1 nucleotide.

The application further relate to comprising the sequence of specific site in sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or its Segment or its variant or its complementary series, for example, including sequence shown in SEQ ID NO:1 the 504th to 525 and the 697th to 721 The sequence or its segment of position nucleotide or its variant or its complementary series (include upstream homologous recombination area section boundary SNP/ The site Indel)；Sequence or its segment or its variant comprising the 514th to 706 nucleotide of sequence shown in SEQ ID NO:1 or Its complementary series (including upstream homologous recombination section and its boundary site SNP/Indel)；Include sequence shown in SEQ ID NO:1 The sequence or its segment of 504th to 525 and the 697th to 721 nucleotide or its variant or its complementary series, and it is following in It is any one or more: the 5th to 26, the 338th to 360 and the 730th to 754 nucleotide of sequence shown in SEQ ID NO:1 Sequence or its segment or its variant or its complementary series (include the upstream homologous recombination area site section boundary SNP/Indel, with And it is respectively derived from the site SNP/Indel of receptor fragments and/or donor fragment).Alternatively, including sequence shown in SEQ ID NO:2 The sequence or its segment or its variant or its complementary series for arranging the 181st to 203 and the 406th to 430 nucleotide are (comprising downstream The homologous recombination area site section boundary SNP/Indel)；Include the 192nd to 416 nucleotide of sequence shown in SEQ ID NO:2 Sequence or its segment or its variant or its complementary series (including downstream homologous recombination section and its boundary site SNP/Indel)； Sequence or its segment or its variant comprising the 181st to 203 and the 406th to 430 nucleotide of sequence shown in SEQ ID NO:2 Or its complementary series, and following any one or more of: sequence the 3rd to 21, the 143rd to 162 shown in SEQ ID NO:2 Position, the sequence of the 442nd to 465 and the 692nd to 713 nucleotide or its segment or its variant or its complementary series are (under including The site homologous recombination area section boundary SNP/Indel is swum, and is respectively derived from the SNP/ of donor fragment and/or receptor fragments The site Indel).

According to segment or section comprising above-mentioned specific site, corresponding SEQ ID NO:1 can be specifically identified And/or sequence shown in SEQ ID NO:2.Further, by identifying containing SEQ ID NO:1 and/or SEQ ID NO:2 institute The recombinant nucleic acid segment for showing sequence can determine in sample to be tested comprising the recombinant nucleic acid segment with rice blast resistance function.

As used herein, " rice " is any rice plant and including can be with all plant varieties of rice breeding.Such as It is used herein, " plant " or " plant ", including whole plant, plant cell, plant organ, plant protoplast, plant can be with Therefrom complete plant in regenerated plant cell tissue cultures, plant callus, vegetation bed and plant or plant part Cell, described plant part such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stem, root, the tip of a root, anther etc..

It can be adapted for the rice varieties of any required breeding in the present processes.That is, can be lacked any The excellent variety (i.e. Comprehensive Traits are preferable, it is contemplated that promising kind) of certain few beneficial traits is used as recurrent parent.With Another kind with beneficial traits lacking in this receptor is as donor parents, and provided beneficial traits are preferably aobvious Property Dominant gene.In the embodiment of the application, recurrent parent is used as using rice ' Y58S ', using having been found to have There is the rice ' Gu Mei 4 ' of good rice blast resistance as donor.

In the selection of recombination plant provided herein, prospect choosing is carried out to recombination plant using molecular labeling It selects.The reliability of foreground selection depends primarily on tightness degree chain between label and target gene, to improve the accurate of selection Rate is general to carry out tracking selection to target gene with two adjacent labels of two sides simultaneously.

In the embodiment of the application, the foreground selection label of use includes favorable selection label and negative itemsets mark Note.In a particular embodiment, the positive foreground selection label of optimized Select to use is closely connected with target gene group segment The label Pi31 of lock, negative itemsets label is the label Pi2S67 positioned at target fragment upstream, and is located at target fragment downstream Label Pi2S122.

In the embodiment of the application, using above-mentioned foreground selection mark carry out homologous recombination detection when, side or The judgment criteria of unilateral homologous recombination is that Pi31 detects identical with ' Gu Mei 4 ' banding pattern, and Pi2S67 or Pi2S122 detection and ' Y58S ' identical banding pattern；The judgment criteria of two sides or bilateral homologous recombination is Pi31 detection banding pattern identical as ' Gu Mei 4 ', and Pi2S67 and Pi2S122 detects banding pattern identical as ' Y58S '.

In this application, any available chip can be used to carry out in breeding method provided herein Foreground selection.In preferred embodiments, it can be disclosed in Chinese patent application CN102747138A using the applicant Rice full-length genome breeding chip RICE6K, or the full base of rice disclosed in PCT international application WO/2014/121419 Because of a group breeding chip RICE60K.Full content in this two parts of application documents is incorporated herein by reference.

Following embodiment is merely to illustrate rather than limits the purpose of the application range.Unless otherwise specified, embodiment is pressed More solito experiment condition, such as Sambrook molecular cloning experiment 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 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 annotated referring to rice OryzasativaLcv.Nipponbare genome MSU/TIGR 6.1st edition (http://rice.plantbiology.msu.edu/).

Embodiment 1The recombination plant of breeding importing blast resistant gene group segment

Material used in the present embodiment is rice ' Y58S ' and rice ' Gu Mei 4 '.

Rice ' Gu Mei 4 ' has good rice blast resistance, and supposition may be the section pair Pi2 of No. 6 chromosome The rice blast resistance of the material plays key effect.

In the Breeding Process of recombination plant, foreground selection is carried out to recombination plant using molecular labeling, to used Foreground selection molecular labeling is screened.The 6.1st edition is annotated referring to rice OryzasativaLcv.Nipponbare genome MSU/TIGR, the 6th dye of downloading Colour solid 9,559,000 is to 10,990,000DNA sequence.The site SSR in above-mentioned sequence is carried out using SSRLocator software Scanning.Using 3.0 software of Primer Premier to the site the SSR design primer searched out, design primer 162 is right altogether.Pass through The method of PCR is screened polymorphism of the above-mentioned primer pair in ' Gu Mei 4 ' and ' Y58S ', is finally picked out in two parts of materials It is favorable selection label Pi31 and negative itemsets label respectively with the high foreground selection molecular labeling of polymorphism, amplification efficiency Pi2S67,Pi2S122.Specific primer information for the above-mentioned molecular labeling of PCR amplification is shown in Table 1.

1 foreground selection molecular labeling primer information of table

By rice ' Gu Mei 4 ' in genomic fragment where forementioned gene imported into rice ' Y58S ', detailed process It is as follows:

With ' Y58S ' for recurrent parent, ' Gu Mei 4 ' is that donor parents are hybridized, by obtained cenospecies and circulation Parent ' Y58S ' is returned, and obtains BC₁F₁Seed is marked after nursery using favorable selection label Pi31 and negative itemsets Pi2S67, Pi2S122 carry out recombination Single-plant selection, filter out 4 lists in target gene group DNA fragmentation side homologous recombination Strain, i.e. Pi31 detects banding pattern identical as ' Gu Mei 4 ', and Pi2S67 or Pi2S122 detects banding pattern identical as ' Y58S ', and utilizes Rice full-length genome breeding chip RICE6K (CN102747138A) carries out Foreground selection (Yu etc., Plant to it Biotechnology Journal.2014,12:28-37)。

Comparable chip is as a result, selection background replys best recombination list in the 4 unilateral homologous recombination single plants filtered out Strain (this generation background recovery value is more than 75%), is returned it again with recurrent parent ' Y58S ', obtains BC₂F₁Seed is educated It is detected using favorable selection label Pi31 after seedling, selects the recombination single plant containing target gene group segment, i.e. Pi31 Banding pattern identical as ' Gu Mei 4 ' is detected, Foreground selection is carried out to it using rice full-length genome breeding chip RICE6K.

It selects background to reply preferable single plant (this generation background recovery value is more than 87.5%), makes itself and recurrent parent ' Y58S ' is returned again, and BC is obtained₃F₁Seed, after nursery using favorable selection label Pi31 and negative indicia Pi2S67, Pi2S122 carries out the screening of target gene group segment other side homologous recombination segment to the seed of harvest, obtains 3 in target patch The single plant of section two sides recombination, i.e. Pi31 detect banding pattern identical as ' Gu Mei 4 ', and Pi2S67 and Pi2S122 detection and ' Y58S ' Identical banding pattern.

Single plant is exchanged to above-mentioned 3 bilaterals using rice full-length genome breeding chip RICE60K (WO/2014/121419) Background and target fragment selection (Chen etc., Molecular Plant.2014,7:541-553) are carried out, importing target is screened Segment is smaller, and the target single plant one that background has been replied (this generation background recovery value is more than 93.75%).

The individual plant selfing chosen is primary, obtain BC₃F₂, it is detected using favorable selection label Pi31 after nursery, The single plant containing target gene group segment is selected, i.e. Pi31 is detected banding pattern identical as ' Gu Mei 4 ', educated using rice full-length genome Kind chip RICE60K carries out Foreground selection to it.

It is final to obtain target fragment homozygosis, and background replys strain one of (background recovery value is more than 99%), is named as CR012613.Chip test result is shown in Fig. 1.

Embodiment 2The determination of homologous recombination segment after importing rice blast resistance gene group segment

In order to determine the rice blast resistance gene group clip size of importing, the homozygous single plant of ' Y58S ' introgressed segment is carried out The sequencings of target gene group segment two sides homologous recombination segments.By blast resistant gene group recombinant nucleic acid contained by CR012613 Segment is named as RecCR012613.

Primarily determine that the upstream RecCR012613 is homologous heavy by rice full-length genome breeding chip RICE60K testing result Pack section is located between label R0610178008AC and R0610183178CT, and downstream homologous recombination segment is located at label Between F0610739800TC and R0610747083GT.

Meanwhile full genome is carried out to ' Y58S ', ' Gu Mei 4 ' and tri- samples of CR012613 using Miseq sequencing technologies Group sequencing.Library foundation is carried out using TruSeq Nano DNA LT Kit (illumina) kit, uses Library Quantification Kit-Universal (KAPA Biosystems) kit is quantified, and MiSeq is used V2Reagent Kit (illumina) kit carries out sequencing reaction.It is examined using the desk-top sequenator of Miseq (illumina) It surveys.Specific steps and method are referring to each kit and sequenator operation instructions.

It is according to aforementioned SNP chip and Miseq sequencing result, the upstream RecCR012613 homologous recombination segment is further true It is scheduled on the section 10179514bp to 10183000bp of the 6th chromosome, downstream homologous recombination segment is located in the 6th chromosome The section 10744036bp to 10746823bp.

On this basis, the 6.1st edition is annotated referring to rice OryzasativaLcv.Nipponbare genome MSU/TIGR, downloads respective segments DNA sequence Column.Using the amplification of 5.0 software design of Primer Premier and sequencing primer, design requirement is that long 22nt of primer or so, GC contain Measure 40-60% and no mispairing.

With receptor parent ' Y58S ' and donor parents ' Gu Mei 4 ' it is control, to RecCR012613 upstream and downstream homologous recombination Segment designs amplimer, is expanded using high fidelity enzyme KOD FX Neo (TOYOBO), is sought using two-step method or three-step approach Look for best amplification condition, it is ensured that amplified production is shown as single bright band in agarose gel electrophoresis detection.Filter out 1 pair Primer is used for the amplification of upstream homologous recombination segment, reaction condition are as follows: 94 DEG C of 2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C 150sec, 37 circulations；20℃1min.Filter out amplification of 1 pair of primer for downstream homologous recombination segment, reaction condition Are as follows: 94 DEG C of 2min；98 DEG C of 10sec, 61 DEG C of 30sec, 68 DEG C of 150sec, 37 circulations；20℃1min.

In addition, being sequenced using amplified production as template using Sanger PCR sequencing PCR, upstream homologous recombination segment is set altogether 7 sequencing primers are counted, effect is sequenced according to practical, picks out 2 sequencings for the site SNP or Indel.It is homologous to downstream heavy Group segment designs 6 sequencing primers altogether, and effect is sequenced according to practical, picks out 2 sequencings for the site SNP or Indel.Tool The amplimer and sequencing primer sequence of body are shown in Table 2.

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

The upstream RecCR012613 homologous recombination sequencing fragment length is 764bp (SEQ ID NO:1).1-514bp is receptor The genomic segment of ' Y58S ', with donor ' Gu Mei 4 ' compared with, there are 3 SNP.This 555bp section of 515-705bp is same Source recombinates section.706-764bp is donor ' Gu Mei 4 ' genomic fragment, compared with receptor ' Y58S ', there are 2 SNP, and 1 Indel。

The downstream RecCR012613 homologous recombination sequencing fragment length is 715bp (SEQ ID NO:2).1-192bp is donor The genomic segment of ' Gu Mei 4 ', compared with receptor ' Y58S ', there are 3 SNP.This 223bp section of 193-415bp is same Source recombinates section.416-715bp is receptor ' Y58S ' genomic fragment, with donor ' Gu Mei 4 ' compared with, there are 2 SNP, 1 Indel。

CR012613, ' Y58S ' and ' Gu Mei 4 ' SNP or Indel Site discrepancy is shown in Table 3.In table " position " are as follows: upstream is same The position of source recombinant fragment SNP or Indel Site discrepancy for SEQ ID NO:1, downstream homologous recombination segment SNP or The position of Indel Site discrepancy is for SEQ ID NO:2.

Table 3CR012613, ' Y58S ' and ' Gu Mei 4 ' SNP or Indel Site discrepancy

Embodiment 3' Y58S ' imports the Resistance Identification after blast resistant gene group segment

It is anti-to the new lines CR012613, recurrent parent ' Y58S ', rice blast of the application breeding in order to identify resistance effect Sick kind paddy plum No. 4 (as positive controls) and rice blast susceptible variety Lijiang xintuanheigu (as negative control) carry out Indoor plantation is identified with the following method after being cultivated to the 3-4 leaf phase:

Choose 2015 from Yichang separation M15Bb-1-1, M15Bb-1-2, M15Bb-2-1, M15Bb-3-1, M15Bb-4-1, M15Bb-5-1, M15Bb-6-1 totally 7 bacterial strains as inoculating strain.Bacterial strain is saved using -20 DEG C of sorghum grain method, Using it is preceding by the sorghum grain of preservation take out to potato dextrose medium (PDA) plate activation (PDA: peeled potatoes 200g, Glucose 20g, agar powder 15g, distilled water are settled to 1L), 28 DEG C take after illumination cultivation 5 days the fresh mycelia block of diameter 5mm to turn Be connected in sorghum grain culture medium (sorghum grain 500g be added 1.5L distilled water, filter off liquid after boiling to boiling, pull sorghum grain out dress Enter 250ml triangular flask, 100ml/ bottles, moist heat sterilization 20 minutes), 10 pieces/bottle, sorghum grain is shaken daily after connecing bacterium 2 days it is scattered, 28 DEG C Dark culturing to mycelia covers with sorghum grain.Then sorghum grain is spread out on sterile gauze, sterile damp gauze is covered, 25 DEG C, RH >=95% is cultivated under 12h illumination condition 4-5 days and is generated to a large amount of spores, washed down with sterile water (containing 0.02% polysorbas20) Spore, adjustment concentration to 5 × 10⁵A/ml.

With above-mentioned conidial suspension spray inoculation CR012613, ' Y58S ', Gu Mei 4 and Lijiang xintuanheigu, connect Three repetitions of kind.It is inoculated with transparent cover on back cover, 28 DEG C of dark culturings for 24 hours, then investigated after 5 days by 16h illumination cultivation.

Investigation standard is 0 grade (highly resistance, HR): not having symptom；1 grade (anti-, R): the brown scab of very little；2 grades (in resist, 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): being about the oval scab of 1-3cm, central canescence, edge brown；5 grades (height sense, HS): long And wide big oval scab, scab fusion is in blocks, until blade is withered.Wherein 0-2 grades is disease-resistant, and 3-5 grade are susceptible.Inoculation knot Fruit is shown in Table 4 and Fig. 2.

Table 4 is inoculated with the Resistant expression after rice blast fungus

Although above having made detailed description to the application with a general description of the specific embodiments, On the basis of the application, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, without departing from the application spirit on the basis of these modifications or improvements, belong to this application claims range.

Sequence table

<110>Chinese subset rolls into a ball Co., Ltd

<120>rice genome recombinant nucleic acid segment RecCR012613 and its detection method

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 764

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(514)

<223>' Y58S' genomic segment is derived from

<220>

<221> misc_recomb

<222> (515)..(705)

<223>homologous recombination section

<220>

<221> gene

<222> (706)..(764)

<223>' Gu Mei 4 ' genomic segment is derived from

<400> 1

ttttagccat aggacgaaga tctaacaact cagattattt ggtaacaaaa tatcaaacat 60

taaaacagtc caacaagtgt atcattatca tgagaaaaaa accaagaaat accgttgata 120

agcgcccaat tccatgaaat gccattgata agcgcccaat tccaagaaat agcattgaca 180

ggtgtgagtt ccatgaaatg ccattgtaca agcaattttg tcctaaaaat gccatcagca 240

tcgttagtcc caagtgggag ttgtaggccc agctcacata gccgactgcg ttttcaaacc 300

tcagaaaatc ggatcctcct cattaaatca gatcgaatca ttctcgattc taaaattggt 360

tgatgacttt tgatacactt gatctactat ttctgttttt atcaaaatct gagtgaatcc 420

ataaagctcg agataaaaac gactacattt aaaaggagat tagatctggt aaattaaagc 480

agaattgatt ggaaaaatca tgggaaaaat gataacgggt agtgaggaat cgatttttac 540

aatcaattgg agaaggtaac gcatgaagag gcagatcaac gtggcgacgg tgcgggtccg 600

gccagcgaca aaaaataaaa ccgaatccat accaaagtgt gtaaaagagc aaatttaaaa 660

aattgcaacg aaaaacgagc ggatgaaaaa aaaaaaggaa aaaggtgacg aaaaaaaacg 720

gcgaaaaaat aaaccggaaa aaacaaaaaa acaaagaaaa aaac 764

<210> 2

<211> 715

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(192)

<223>' Gu Mei 4 ' genomic segment is derived from

<220>

<221> misc_recomb

<222> (193)..(415)

<223>homologous recombination section

<220>

<221> gene

<222> (416)..(715)

<223>' Y58S' genomic segment is derived from

<400> 2

ccggcttgat ctatgcggct ccgtcgcccc agcgtgccat cctcaagctc gagctcttct 60

ctgcccgccg tcgcccctcg ctagcttgac ttcgtccttc acctacgccc gcacgtcgtc 120

cctgagctag agctcctctc cgcgcgccgt tgtcgcacct cgctcgacct cgatctcttg 180

tagcactcac acgccatact tgggctcaag ctcctctcca ccgtcctggc tcaacctcaa 240

tctccacggc ttcgtcgatg cccgcgcgcc atctcggagc tcaagctcct ctccatcgct 300

atcgcctccg cgactcgact tctactgtac ctggacatga gctcctcttt gcttgcgtcg 360

cttgagatag ttgggggaag tcgggaagag atcgccggcg tggtagaaga ggagttagcg 420

gactcgctgg tgccttccca accgaggtgt tccctttttt ttccctcagc cacactgaca 480

agtgggccac tggtacaaag tgatacattt tggtaataaa atctggtgta aagtgaaagt 540

attgagttaa tggtggttca atgtgaaacc atggttaaaa aacatggacc aatatgcaat 600

ttacaaattt atgaaatttt tttactagag ataggtatag atatgaaata tattcttacc 660

acatctcaag tcgatactta acattatttg ggagaaaaaa aaacacattt tggat 715

<210> 3

<211> 23

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 3

ctcttgccaa acatgccagg gta 23

<210> 4

<211> 23

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 4

ctcacgttca cattcgattc tcc 23

<210> 5

<211> 19

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 5

gccatcagca tcgttagtc 19

<210> 6

<211> 23

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 6

ctagaccaca aagaccacga cag 23

<210> 7

<211> 23

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 7

tgtgctcact gggtaggatg tct 23

<210> 8

<211> 19

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 8

acgtcgtccc tgagctaga 19

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 9

atccaaaccc gttgttgcac 20

<210> 10

<211> 20

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 10

cggcaattgc cacgatgata 20

<210> 11

<211> 20

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 11

ccgatgcaag aacaagctaa 20

<210> 12

<211> 20

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 12

ccaccacatc accagtgttt 20

<210> 13

<211> 20

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 13

gacttgaaaa ccagtgcgtg 20

<210> 14

<211> 22

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 14

cctacctaat ggaaaggatt gc 22

Claims (8)

1. rice genome recombinant nucleic acid segment, it includes:

- the first recombinant nucleic acid segment, is selected from:

I) sequence or its segment comprising the 504th to 525 and the 697th to 721 nucleotide of sequence shown in SEQ ID NO:1 or Its variant or its complementary series；

Ii sequence or its segment or its variant) comprising the 514th to 706 nucleotide of sequence shown in SEQ ID NO:1 or its mutually Complementary series；

Iii sequence or its segment) comprising the 504th to 525 and the 697th to 721 nucleotide of sequence shown in SEQ ID NO:1 Or sequence the 5th to 26 shown in its variant or its complementary series and SEQ ID NO:1, the 338th to 360 and the 730th to At least one of 754 nucleotide sequence or its segment or its variant or its complementary series；Or

Iv) comprising sequence or its segment or its variant or its complementary series shown in SEQ ID NO:1；And/or

- the second recombinant nucleic acid segment, is selected from:

V) sequence or its segment comprising the 181st to 203 and the 406th to 430 nucleotide of sequence shown in SEQ ID NO:2 or Its variant or its complementary series；

Vi sequence or its segment or its variant) comprising the 192nd to 416 nucleotide of sequence shown in SEQ ID NO:2 or its mutually Complementary series；

Vii sequence or its segment) comprising the 181st to 203 and the 406th to 430 nucleotide of sequence shown in SEQ ID NO:2 Or sequence the 3rd to 21, the 143rd to 162, the 442nd to 465 shown in its variant or its complementary series and SEQ ID NO:2 At least one of position and the 692nd to 713 nucleotide sequence or its segment or its variant or its complementary series；Or

Viii) comprising sequence or its segment or its variant or its complementary series shown in SEQ ID NO:2.

2. the primer of recombinant nucleic acid segment described in claim 1 is detected, wherein the primer includes:

The primer for detecting the first recombinant nucleic acid segment, is selected from:

(I) primer of the sequence of the 504th to 525 nucleotide of sequence shown in specific recognition SEQ ID NO:1, and specificity are known The primer of the sequence of the 697th to 721 nucleotide of sequence shown in other SEQ ID NO:1；

(II) primer of the sequence of the 514th to 706 nucleotide of sequence shown in specific recognition SEQ ID NO:1；

(III) primer of the sequence of the 504th to 525 nucleotide of sequence shown in specific recognition SEQ ID NO:1, and specificity Identify the primer and specific recognition SEQ ID of the sequence of the 697th to 721 nucleotide of sequence shown in SEQ ID NO:1 The primer of at least one of sequence the 5th to 26 shown in NO:1, the 338th to 360 and the 730th to 754 nucleotide sequence；Or

(IV) primer of sequence shown in specific recognition SEQ ID NO:1；And/or

The primer for detecting the second recombinant nucleic acid segment, is selected from:

(V) primer of the sequence of the 181st to 203 nucleotide of sequence shown in specific recognition SEQ ID NO:2, and specificity are known The primer of the sequence of the 406th to 430 nucleotide of sequence shown in other SEQ ID NO:2；

(VI) primer of the sequence of the 192nd to 416 nucleotide of sequence shown in specific recognition SEQ ID NO:2；

(VII) primer of the sequence of the 181st to 203 nucleotide of sequence shown in specific recognition SEQ ID NO:2, and specificity Identify the primer and specific recognition SEQ ID of the sequence of the 406th to 430 nucleotide of sequence shown in SEQ ID NO:2 At least one of sequence the 3rd to 21 shown in NO:2, the 143rd to 162, the 442nd to 465 and the 692nd to 713 nucleotide The primer of sequence；Or

(VIII) primer of sequence shown in specific recognition SEQ ID NO:2.

3. the primer of recombinant nucleic acid segment described in claim 1 is detected, wherein the primer is selected from:

(I) primer pair of the first recombinant nucleic acid segment is expanded

5 '-CTCTTGCCAAACATGCCAGGGTA-3 ',

5'-CTCACGTTCACATTCGATTCTCC-3'；And

(II) primer of the first recombinant nucleic acid segment is sequenced

5'-CTCTTGCCAAACATGCCAGGGTA-3'；

5'-GCCATCAGCATCGTTAGTC-3'；And/or optionally,

(III) primer pair of the second recombinant nucleic acid segment is expanded

5 '-CTAGACCACAAAGACCACGACAG-3 ',

5'-TGTGCTCACTGGGTAGGATGTCT-3'；And

(IV) primer of the second recombinant nucleic acid segment is sequenced

5'-CTAGACCACAAAGACCACGACAG-3'；

5’-ACGTCGTCCCTGAGCTAGA-3’。

4. breeding contains the method for the rice plant of recombinant nucleic acid segment described in claim 1, wherein the recombinant nucleic acid piece Section has the function of rice blast resistance, and the described method comprises the following steps:

1) by recurrent parent rice ' Y58S ' and donor rice ' Gu Mei No. 4 numbers ' hybridize, by obtained cenospecies and circulation Parent is returned, and obtains first backcross generation, marks Pi31 and negative itemsets to mark Pi2S67, Pi2S122 couple using favorable selection Its unilateral homologous recombination segment screening for carrying out blast resistant gene group segment, and using rice full-length genome breeding chip to it Carry out Foreground selection；

2) selection background is replied preferable recombination single plant and is returned again with recurrent parent, obtains second backcross generation, utilizes forward direction Selected marker Pi31 detects it, selects the recombination single plant containing blast resistant gene group segment, then complete using rice Genomic breeding chip carries out Foreground selection to it；

3) the recombination single plant that selection background has been replied is returned again with recurrent parent, is obtained third backcross generation, is utilized forward direction The other side that selected marker Pi31 and negative itemsets label Pi2S67, Pi2S122 carry out blast resistant gene group segment to it is same The screening of source recombinant fragment, and Foreground selection is carried out to it using rice full-length genome breeding chip；And

4) selection introgressed segment is small, and the recombination single plant that background has been replied, and the recombination individual plant selfing chosen is primary, is selfed Kind, it is detected using favorable selection label Pi31, and background choosing is carried out to it using rice full-length genome breeding chip It selects, the final rice plant for obtaining the recombinant nucleic acid segment of group containing homozygous gene and background and replying.

5. method as claimed in claim 4, the expansion used when wherein carrying out foreground selection to recombination plant using molecular labeling It is as follows to increase primer:

The primer pair of amplifier molecule label Pi31 comprising:

Forward primer: 5 '-ATCCAAACCCGTTGTTGCAC-3 ',

Reverse primer: 5 '-CGGCAATTGCCACGATGATA-3 '；

The primer pair of amplifier molecule label Pi2S67 comprising:

Forward primer: 5 '-CCGATGCAAGAACAAGCTAA-3 ',

Reverse primer: 5 '-CCACCACATCACCAGTGTTT-3 '；And

The primer pair of amplifier molecule label Pi2S122 comprising:

Forward primer: 5 '-GACTTGAAAACCAGTGCGTG-3 ',

Reverse primer: 5 '-CCTACCTAATGGAAAGGATTGC-3 '.

6. the method for detecting recombinant nucleic acid segment described in claim 1 comprising primer described in claim 2 or 3 is used, PCR reaction, and the step of analyzing PCR product are carried out by template of testing gene group.

7. detecting the kit of recombinant nucleic acid segment described in claim 1 comprising primer described in claim 2 or 3.

8. screening containing recombinant nucleic acid segment described in claim 1 rice plant or seed method comprising detection to Survey the step of whether containing recombinant nucleic acid segment described in claim 1 in the genome of rice plant or seed；

Preferably, using primer described in claim 2 or 3, perhaps using method of claim 6 or using power Benefit require 7 described in kit detected.