CN108330203A - The method for identifying rice cytoplasmic type - Google Patents
The method for identifying rice cytoplasmic type Download PDFInfo
- Publication number
- CN108330203A CN108330203A CN201710034236.0A CN201710034236A CN108330203A CN 108330203 A CN108330203 A CN 108330203A CN 201710034236 A CN201710034236 A CN 201710034236A CN 108330203 A CN108330203 A CN 108330203A
- Authority
- CN
- China
- Prior art keywords
- rice
- type
- primer
- primer pair
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
This application discloses the methods of identification rice cytoplasmic type, this method is based on rice mitochondria genome hereditary variation, by analysing and comparing to mitochondrial genomes sequence, the shared special mitochondrial genomes sequence of the rice cytoplasmic type selected from Yebai, Indonesia's paddy field paddy type, ridge type, K-type and D types is filtered out, which contains the sites at least two InDel.Go out 2 pairs of primers for the same sites InDel optimization design, rice cytoplasmic type or identification of cell matter male sterile line and its hybrid rice variety are identified by Molecular Detection means.Method provided by the present application can quick, accurate, efficient, steadily identification rice cytoplasmic type, and related cytoplasmic male sterile rice and its hybrid rice variety.
Description
Technical field
The application belongs to rice molecular label Testing and appraisal field, in particular to a kind of Rapid identification rice cell
It is thin selected from Yebai, Indonesia's paddy field paddy type, ridge type, K-type, the rice of D types to be suitable for quick, Qualitative Identification for the method for matter type
Cytoplasm type.
Background technology
Cytoplasmic male sterility (cytoplasmic male sterility, CMS) is widely present in higher plant
Phenomenon is the result of cytoplasmic skeleton and nuclear genome interaction：When cytoplasm is sterile type and nucleus base
When because specific restoring gene being not present in group, show as cannot generating normal male gamete, and the development of female gamete and
The nutrient growth of plant is normal；When cytoplasm is to be educated there are when specific restoring gene in sterile type and nuclear genome
Property is restored, and shows as fertile.Studies have shown that functional caused by the variation of the CMS phenomenons and mitochondrial genomes of rice
The relationship between expression of obstacle or specific gene is close, while rice mitochondria belongs to matrilinear inheritance, thus can by mitochondrial markers
With the source of rice cytoplasmic of tracking or classify, (Luan Ji, Rice mtDNA genetic polymorphism and related gene expression are ground
Study carefully, 2012,66-90；And Fuji et al, BMC Genomics, 2010,11:209).
CMS is played an important role in terms of rice heterosis utilization, and hybrid rice is cultivated successfully in the 1970's, real
The China Xian Liao rice yield qualitative leap (Yuan Longping, " 21 Century Forum " proceedings in 2010,2010,374-376), wherein
Yebai (CMS-WA) cytoplasmic male sterility is most widely used in hybrid paddy rice field.Since wild-abortive type hybrid rice large area
Since popularization and application, the 1970s to the mid-80, breeders are found that more than 60 kind different cytoplasm sources successively
Cytoplasmic male sterile line type, including it is Yebai, packet bench-type, red lotus type, ridge type, D types, K-type, Indonesia's paddy field paddy type, short
Lose type and Yunnan type etc..According to abortion tissue signature, these cytoplasmic male sterilities can be divided into sporophyte type cytoplasmic male sterility
With gametophytic cytoplasmic male sterility two types (Zhu Yingguo, the biology of male sterility of rice, 2000).
Invention content
On the one hand, this application provides identification rice cytoplasmic type method, the method includes：I) using selected from
Under at least one primer pair expand the nucleic acid fragment in rice sample to be identified, wherein the first primer comprising forward direction to drawing
Object：5 '-TGGAGTGGAGGCATTGCTATTC-3 ' and reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3’；And/or
Second primer pair includes forward primer：5 '-ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-
CTAAATACAAAACAAGAGTC-3’；And ii) determine expanded nucleic acid fragment length, wherein when the first primer to expand
When the nucleic acid fragment length of increasing is 117bp and/or the nucleic acid fragment length of the second primer pair amplifies is 154bp, the rice sample
Product are cytoplasm type selected from the following：Yebai, Indonesia's paddy field paddy type, ridge type, K-type and D types.
On the other hand, this application provides identification cytoplasmic male sterile rice or its hybrid rice variety method,
The method includes：I) the nucleic acid piece in rice sample to be identified is expanded using at least one primer pair selected from the following
Section, wherein the first primer is to including forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ' and reverse primer：5’-
GTCTACGCTCGCTTTTGTTCTG-3’；And/or second primer pair include forward primer：5’-
ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And ii) determine and expanded
The length of the nucleic acid fragment of increasing, wherein when the first primer is 117bp and/or the second primer pair to the nucleic acid fragment length of amplification
The nucleic acid fragment length of amplification be 154bp when, the rice sample be cytoplasmic male sterile rice selected from the following or its
Hybrid rice variety：Yebai, Indonesia's paddy field paddy type, ridge type, K-type and D types.
In one embodiment, the primer pair used in the method also includes fluorophor.In another embodiment
In, the fluorophor is selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
On the other hand, this application provides the method for distinguishing the corresponding maintainer of cytoplasmic male sterile rice, institutes
The method of stating includes：I) nucleic acid fragment in rice sample to be identified is expanded using at least one primer pair selected from the following,
Wherein the first primer is to including forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ' and reverse primer：5’-
GTCTACGCTCGCTTTTGTTCTG-3’；And/or second primer pair include forward primer：5’-
ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And ii) determine and expanded
The length of the nucleic acid fragment of increasing, wherein when the first primer is 117bp and/or the second primer pair to the nucleic acid fragment length of amplification
When the nucleic acid fragment length of amplification is 154bp, the rice sample is cytoplasmic male sterile rice selected from the following：It loses open country
Type, Indonesia's paddy field paddy type, ridge type, K-type and D types.
In one embodiment, the primer pair used in the method also includes fluorophor.In another embodiment
In, the fluorophor is selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
Another aspect, this application provides distinguish cytoplasmic male sterile rice hybrid rice product with other types to hybridize
The method of rice varieties, the method includes：I) rice to be identified is expanded using at least one primer pair selected from the following
Nucleic acid fragment in sample, wherein the first primer is to including forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and it is anti-
To primer：5’-GTCTACGCTCGCTTTTGTTCTG-3’；And/or second primer pair include forward primer：5’-
ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And ii) determine and expanded
The length of the nucleic acid fragment of increasing, wherein when the first primer is 117bp and/or the second primer pair to the nucleic acid fragment length of amplification
When the nucleic acid fragment length of amplification is 154bp, the rice sample hybridizes for cytoplasmic male sterile rice selected from the following
Rice varieties：Yebai, Indonesia's paddy field paddy type, ridge type, K-type and D types.
In one embodiment, the primer pair used in the method also includes fluorophor.In another embodiment
In, the fluorophor is selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
In addition, present invention also provides the primer pair for identifying rice cytoplasmic type, it is selected from：The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ' and reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-
3’；And/or second primer pair：Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-
CTAAATACAAAACAAGAGTC-3’。
In one embodiment, the primer pair also includes fluorophor.In another embodiment, the fluorophor
Selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
Correspondingly, this application provides the kit for identifying rice cytoplasmic type, it includes the first primer pair and/
Or second primer pair, wherein the first primer is to including forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and reversely draw
Object：5’-GTCTACGCTCGCTTTTGTTCTG-3’；Second primer pair includes forward primer：5’-
ACCGAACTCACATTATTTGG-3 ' and reverse primer：5’-CTAAATACAAAACAAGAGTC-3’.
In one embodiment, the kit also includes：Amplification buffer, dNTP mixtures, archaeal dna polymerase,
ddH2O。
Further, present invention also provides primer pair or the following purposes of kit：I) it is used for whether identifying rice
Belong to Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D type cytoplasm types；Ii it) is used to identify Yebai or Indonesia's water
Field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice or its hybrid rice variety；Iii it) is used to differentiate Yebai
Or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice and corresponding maintainer；Iv it) is used to distinguish
Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D types hybrid rice variety and other types hybrid rice variety；Or v)
For distinguishing Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice or its hybrid rice
Kind and conventional rice kind.
As it can be seen that this application provides 2 pairs of InDel molecular labeling amplimers, using any of which to water to be detected
The nucleic acid fragment of rice sample carries out PCR amplification, can be stablized, the product of high specificity, can be sentenced according to product clip size
It is fixed whether to belong to Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D type rice cytoplasmic types, or whether belong to open country and lose
Type or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice and its hybrid rice variety, as a result surely
Fixed, reliable and high specificity.
Description of the drawings
Fig. 1 is mtCMSID01 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 323bp banding patterns；B is amplification gained 326bp banding patterns.
Fig. 2 is mtCMSID02 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 117bp banding patterns；B is amplification gained 120bp banding patterns.
Fig. 3 is mtCMSID03 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 164bp banding patterns；B is amplification gained 165bp banding patterns.
Fig. 4 is mtCMSID04 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 267bp banding patterns；B is amplification gained 268bp banding patterns.
Fig. 5 is mtCMSID05 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 247bp banding patterns；B is amplification gained 250bp banding patterns.
Fig. 6 is mtCMSID06 fluorescent primer ABI 3730XL DNA capillary electrophoresis detection result figures；Wherein A is amplification
Gained 154bp banding patterns；B is amplification gained 157bp banding patterns.
Specific implementation mode
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.
Insertion/deletion (insertion/deletion, InDel) is due to the DNA sequences at allele site
It is listed in the length polymorphism variation that the insertion/deletion of nucleotide fragments has occurred between Different Individual and generates.
PCR amplification is carried out according to the sequence design special primer of target site both sides, the length polymorphism of amplified fragments is
It is InDel labels, in whole gene group, the frequency of polymorphism of InDel is only second to SNP marker, is far above SSR marker.InDel
Labeled as codominant marker, there is preferable stability and more relatively rich state property, be widely used in high-resolution collection of illustrative plates structure
Build, association analysis, the assignment of genes gene mapping and objective trait evaluation of markers screening etc. fields.
More and more cytoplasmic male sterile rice mitochondrial genomes complete sequencing, pass through biological information credit
Analysis, can obtain a large amount of special DNA sequence dnas, these special DNA sequence dnas a certain cytoplasmic male sterile rice, certain
One type (sporophyte type cytoplasmic male sterility and gametophytic cytoplasmic male sterility) sterile line or certain several rice cell
There is uniqueness in matter male sterile line.
The application is by for the specific DNA sequence design primer screened, for identifying rice cytoplasmic type, a certain
Type (sporophyte type cytoplasmic male sterility and gametophytic cytoplasmic male sterility) sterile line or certain several rice cytoplasmic
Male sterile line and its Hybrid.
Specifically, present inventor is based on rice mitochondria genome hereditary variation, to mitochondrial genomes sequence
Row are analysed and compared, including：Nipponbare (accession number BA000029), WA-CMS (accession number JF281154), Lead
Rice (accession number AP011077), long-grained rice (accession number JF281153), Hassawi (accession number
JN861111), Hassawi hybrid rice (IR1112x Hassawi) (accession number JN861112).With WA-CMS mitochondrias
Genome sequence is classified as reference, carries out BLAST analyses and comparison with other mitochondrial genomes sequences respectively, filters out rice cytoplasmic
Male sterile line (including Yebai, Indonesia's paddy field paddy type, ridge type, K-type, D type cytoplasmic male sterile lines, belong to sporinite
Type cytoplasmic male sterility) shared special mitochondrial genomes sequence, which contains 4 insertion and deletions
(InDel) site, the position in Nipponbare mitochondrial genomes are ChrM respectively:204670 (1bp insertions), ChrM:
227762 (3bp missings), ChrM:286920 (3bp missings), ChrM:389371 (1bp insertions).
Since these sites InDel are to compare to obtain by sequence analysis, present inventor further passes through experiment
To verify its authenticity.In addition, because for designed by these sites InDel primer amplification effect and its resolving effect with
The structure of the sites InDel peripheral sequence is closely related, and present inventor is compared by many experiments, filters out optimal
Primer sequence.
Specifically, present inventor's optimal screening goes out for one of sites InDel (in Nipponbare line grains
Position in body genome is ChrM:286920 3bp missings) there are two pairs of specific primers：MtCMSID02 is (wherein,
MtCMSID02-F is 5 '-TGGAGTGGAGGCATTGCTATTC-3 ' and mtCMSID02-R is 5 '-
GTCTACGCTCGCTTTTGTTCTG-3’)；And mtCMSID06 (wherein, mtCMSID06-F 5 '-
ACCGAACTCACATTATTTGG-3 ' and mtCMSID06-R is 5 '-CTAAATACAAAACAAGAGTC-3 '), it can be accurate
Really, efficiently, stablize, reliably identify related rice cytoplasmic type, and related cytoplasmic male sterile rice and its
Cross combination.
Primer provided herein also may include applicable any fluorophor known in the art, to adapt to high-throughput inspection
Survey demand makes amplified production be detected suitable for fluorescent capillary electrophoresis tube.In a particular embodiment, it is glimmering to be selected from carboxyl for fluorophor
Light element, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine etc., such as 6-FAM, HEX etc..
Primer provided herein can be prepared into kit in order to use.Therefore, present invention also provides with
In the kit of identification cytoplasmic male sterile rice or its hybrid rice variety, it includes primer mtCMSID02 and/or
mtCMSID06。
Kit provided herein also may include other reagents required for carrying out PCR reactions, such as：Amplification is slow
Fliud flushing, dNTP mixtures, archaeal dna polymerase, ddH2O etc..
In a particular embodiment, the method for identification rice cytoplasmic type provided herein, utilizes the application institute
The primer pair mtCMSID02 and/or mtCMSID06 of offer expands rice sample to be identified.Specifically, work as primer pair
When the amplified fragments size of mtCMSID02 is 117bp and/or the amplified fragments size of primer pair mtCMSID06 is 154p, institute
It is Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D type rice cytoplasmic types to state rice sample；Work as primer pair
When the amplified fragments size of mtCMSID02 is 120bp and/or the amplified fragments size of primer pair mtCMSID06 is 157bp, institute
It is any one of Yebai, Indonesia's paddy field paddy type, ridge type, K-type, D type rice cytoplasmic types to state rice sample not.
In a particular embodiment, identification cytoplasmic male sterile rice provided herein or its hybrid rice product
The method of kind, rice sample to be identified is expanded using primer pair mtCMSID02 and/or mtCMSID06 provided herein
Product.Specifically, when the amplification piece that the amplified fragments size of primer pair mtCMSID02 is 117bp and/or primer pair mtCMSID06
When Duan great little is 154p, the rice sample is that Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D types rice cytoplasmic are male
Property sterile line or its hybrid rice variety；When the amplified fragments size of primer pair mtCMSID02 is 120bp and/or primer pair
When the amplified fragments size of mtCMSID06 is 157bp, the rice sample is not Yebai, Indonesia's paddy field paddy type, ridge type, K
Any one of type, D types cytoplasmic male sterile rice and its cross combination.
In another embodiment, differentiation Yebai provided herein or Indonesia's paddy field paddy type or ridge type or K
The method of type or D types cytoplasmic male sterile rice and corresponding maintainer utilizes primer pair provided herein
MtCMSID02 and/or mtCMSID06 expands rice sample to be distinguished.Specifically, when the amplification of primer pair mtCMSID02
When clip size is 117bp and/or the amplified fragments size of primer pair mtCMSID0+ is 154bp, the rice sample is open country
Lose type or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice or its hybrid rice variety；Work as primer
When amplified fragments size to mtCMSID02 is 120bp and/or the amplified fragments size of primer pair mtCMSID06 is 157bp,
The rice sample is Yebai, Indonesia's paddy field paddy type, ridge type, K-type, the corresponding holding of D type cytoplasmic male sterile rices
System.
In another specific embodiment, differentiation Yebai provided herein or Indonesia's paddy field paddy type or ridge type or K
The method of type or D type hybrid rice product and other types hybrid rice variety, utilizes primer pair provided herein
MtCMSID02 and/or mtCMSID06 expands rice sample to be distinguished.Specifically, when the amplification of primer pair mtCMSID02
When clip size is 117bp and/or the amplified fragments size of primer pair mtCMSID06 is 154bp, the rice sample is open country
Lose type or Indonesia's paddy field paddy type or ridge type or K-type or D type hybrid rice varieties；When the amplified fragments of primer pair mtCMSID02 are big
When small is 120bp and/or the amplified fragments size of primer pair mtCMSID06 is 157bp, the rice sample is other types
Hybrid rice variety.
Following embodiment is merely to illustrate and the purpose of unrestricted 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.
Embodiment 1The exploitation of InDel labels
In NCBI (https://www.ncbi.nlm.nih.gov/) in download published mitochondrial genomes sequence,
Including：Nipponbare (accession number BA000029), WA-CMS (accession number JF281154), Lead rice (accession number
AP011077), long-grained rice (accession number JF281153), Hassawi (accession number JN861111), Hassawi
Hybrid rice (IR1112 x Hassawi) (accession number JN861112).
Using WA-CMS mitochondrial genomes sequence as reference, BLAST points are carried out with other mitochondrial genomes sequences respectively
Analysis compares, and finds 4 sites InDel altogether, is the insertion point of 2 1bp, the deletion segment of 2 3bp respectively.
Position of the above-mentioned difference site in Nipponbare mitochondrial genomes is that 1bp is inserted into ChrM respectively:204670、
3bp lacks ChrM:227762,3bp lacks ChrM:286920,1bp is inserted into ChrM:389371.
It is directed to above-mentioned each sites InDel respectively using Primer Premier 5 and designs multipair PCR amplification primer, primer
Length design parameter is 20 ± 2bp, and G/C content is not less than 45%, Tm values between 55 DEG C to 60 DEG C.
Multipair primer is devised for above-mentioned 4 sites InDel respectively and carried out PCR amplification verification, herein only with wherein
The representative primer of four couples for, be respectively designated as mtCMSID01, mtCMSID02, mtCMSID03 and mtCMSID04, sequence
Row are as follows：
MtCMSID01 (3bp missings)：
mtCMSID01-F：5 '-ACGGTAAATCGCACAGGTT-3 ',
mtCMSID01-R：5’-CAGTAGATAGTAGAGAGGAGG-3’；
MtCMSID02 (3bp missings)：
mtCMSID02-F：5 '-TGGAGTGGAGGCATTGCTATTC-3 ',
mtCMSID02-R：5’-GTCTACGCTCGCTTTTGTTCTG-3’；
MtCMSID03 (1bp insertions)：
mtCMSID03-F：5 '-TAATGGGTGGCATAGCCCGTAG-3 ',
mtCMSID03-R：5’-TGAGTCGCTCGTTCAACTGTCC-3’；
MtCMSID04 (1bp insertions)：
mtCMSID04-F：5 '-CTTTACTTCGGCTGTGGTCAT-3 ',
mtCMSID04-R：5’-GGTTTCTTAATCGGTGTCTATC-3’.
In order to adapt to high-throughput detection demand, amplified production is set to be detected suitable for fluorescent capillary electrophoresis tube, in above-mentioned primer
Middle addition fluorophor.
In 5 ' the end addition 6-FAM fluorescent bases of sense primer mtCMSID01-F, mtCMSID03-F and mtCMSID04-F
Group, synthesize mtCMSID01-F-FAM, mtCMSID03-F-FAM and mtCMSID04-F-FAM primer, the primer still can with it is corresponding
Downstream primer be combined.
In 5 ' the end addition 6-HEX fluorophors of sense primer mtCMSID02-F, mtCMSID02-F-HEX primers are synthesized,
The primer can be still combined with mtCMSID02-R.
According to previous designs process：
When mtCMSID01 amplified fragments size be 323bp either mtCMSID02 amplified fragments size be 117bp or
MtCMSID03 amplified fragments size is 165bp mtCMSID04 amplified fragments size when being 268bp, which is
Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile rice and its cross combination；
When mtCMSID01 amplified fragments size be 326bp either mtCMSID02 amplified fragments size be 120bp or
MtCMSID03 amplified fragments size is 164bp mtCMSID04 amplified fragments size when being 267bp, and the rice material is not
It is any one of Yebai, Indonesia's paddy field paddy type, ridge type, K-type, D types cytoplasmic male sterile rice and its cross combination.
To new germplasm source type sterile line peasants who dig gold 2A, WA type CMS system depth 95A and its corresponding maintainer depth 95B, make
It is detected with above-mentioned primer, examines the parting effect of primer, concrete operations as follows.
Single seed or about 1cm linear leafs are taken, FOREGENE plant leaf blade Direct PCR kits (Plant Leaf are used
Direct PCR Kit, Chengdu Fu Ji Bioisystech Co., Ltd) it is detected.
It is 10 μ l that PCR, which reacts total volume, including：DNA extracting solutions, 2 μ l；2xLeaf PCR EasyTMMix, 5 μ l；It is positive
And reverse primer, each 0.1 μ l (10nmol/L)；ddH2O, 2.8 μ l.
PCR response procedures are：94℃3min；94 DEG C of 10sec, 57 DEG C of 20sec, 72 DEG C of 30sec, 38 cycles；72℃
5min；25℃1sec.
It is not added with the primer pair amplifies products therefrom of fluorophor for using, electrophoresis is carried out using polyacrylamide gel
Analysis.
For using the primer pair amplifies products therefrom of addition fluorophor to take 1 μ l and 8.95 μ l after 200 times of dilution
HiDi, 0.05 μ l Liz 500 are mixed, and in carrying out Capillary Electrophoresis on ABI 3730XL DNA analysis instrument, are as a result used
GeneMapper softwares are read.
MtCMSID01 fluorescent primers expand banding pattern as shown in Figure 1, mtCMSID02 fluorescent primers amplification banding pattern such as Fig. 2 institutes
Show, mtCMSID03 fluorescent primers expand banding pattern as shown in figure 3, mtCMSID04 fluorescent primers amplification banding pattern is as shown in Figure 4.Each
In figure, upper abscissa indicates stripe size；Lower abscissa " al " represents allelotype, and numerical value indicates the corresponding band in top
Size；Y value indicates peak value；Gray area is the sections Bin of setting.
The results show that in new germplasm source type sterile line peasants who dig gold 2A and depth 95B, mtCMSID01, mtCMSID02,
MtCMSID03 and mtCMSID04 amplified production sizes are followed successively by 326bp, 120bp, 164bp and 267bp；Yebai male not
Educate and be in deep 95A, mtCMSID01, mtCMSID02, mtCMSID03 and mtCMSID04 amplified production size be followed successively by 323bp,
117bp, 165bp and 268bp；It is consistent with expection, shows that the sites InDel being directed to are true and reliable.
In addition, mtCMSID01 and mtCMSID02 amplified productions read and identify through system, it is single main peak, is easy to sentence
Not, as a result more acurrate reliable.There is the peaks stutter in mtCMSID03 and mtCMSID04 amplified productions, because site itself only has
1bp differences, system be easy to cause deviation when reading, need manual correction, be unsuitable for detecting on a large scale, therefore give up.
At Indel identical as mtCMSID01, the primer that another pair can accurately and reliably be differentiated is had also obtained
MtCMSID05, sequence are：
mtCMSID05-F：5’-ATTACCAACGGGTAGTGACAA-3’
mtCMSID05-R：5’-TACCATAATCCAAAGATTGAGTAC-3’；
When mtCMSID05 amplified fragments sizes are 247bp, which is Yebai or Indonesia's paddy field paddy type or ridge
Type or K-type or D types cytoplasmic male sterile rice and its cross combination；When mtCMSID05 amplified fragments sizes are 250bp
When, which is not Yebai, Indonesia's paddy field paddy type, ridge type, K-type, D types cytoplasmic male sterile rice and its hybridization
Any one of combination.
At Indel identical as mtCMSID02, the primer that another pair can accurately and reliably be differentiated is had also obtained
MtCMSID06, sequence are：
mtCMSID06-F：5’-ACCGAACTCACATTATTTGG-3’
mtCMSID06-R：5’-CTAAATACAAAACAAGAGTC-3’；
When mtCMSID06 amplified fragments sizes are 154bp, which is Yebai or Indonesia's paddy field paddy type or ridge
Type or K-type or D types cytoplasmic male sterile rice and its cross combination；When mtCMSID06 amplified fragments sizes are 157bp
When, which is not Yebai, Indonesia's paddy field paddy type, ridge type, K-type, D types cytoplasmic male sterile rice and its hybridization
Any one of combination.
Using preceding method, to new germplasm source type sterile line peasants who dig gold 2A, WA type CMS system depth 95A and its corresponding guarantor
It is deep 95B to hold, and is detected using mtCMSID05 and mtCMSID06 primers, and the parting effect of primer is examined.
MtCMSID05 fluorescent primers expand banding pattern as shown in figure 5, mtCMSID06 fluorescent primers amplification banding pattern such as Fig. 6 institutes
Show.In the various figures, upper abscissa indicates stripe size；Lower abscissa " al " represents allelotype, and numerical value indicates top pair
The stripe size answered；Y value indicates peak value；Gray area is the sections Bin of setting.
The results show that in new germplasm source type sterile line peasants who dig gold 2A and depth 95B, mtCMSID05 and mtCMSID06 amplified productions
Size is respectively 250bp and 157bp；In Yebai male sterile line depth 95A, mtCMSID05 and mtCMSID06 amplified productions
Size is respectively 247bp and 154bp；With expection be consistent, for the sites InDel it is true and reliable.MtCMSID05 and
MtCMSID06 amplified productions read and identify through system, are single main peak, are easy to differentiate, as a result accurately and reliably.
Finally, mtCMSID01, mtCMSID05, mtCMSID02 and mtCMSID06 are selected from designed multipair primer
For identifying Different Types of Rice cytoplasmic male sterile line and its cross combination.
Embodiment 2Utilize InDel Marker Identification Different Types of Rice cytoplasmic male sterile lines
To the sterile line and each material of maintainer listed by table 1, the inspection of genotype is carried out in the same manner as in example 1
It surveys.
Each material genotype call results are referring to Tables 1 and 2.
The result shows that mtCMSID01, mtCMSID05, mtCMSID02 and mtCMSID06 can be by Yebai or Indonesia paddy fields
Paddy type or ridge type or K-type or D types cytoplasmic male sterile rice are mutually distinguished with other types sterile line.
Because maintainer is different from sterile line cytoplasmic skeleton, nuclear genome is identical, so mtCMSID01,
MtCMSID05, mtCMSID02 and mtCMSID06 can also be by Yebai or Indonesia's paddy field paddy types or ridge type or K-type or D type water
The corresponding maintainer of rice cytoplasmic male sterile line distinguishes.
Table 1 utilizes mtCMSID01 and mtCMSID05 detection different type cytoplasmic male sterile lines and the result of maintainer
Table 2 utilizes mtCMSID02 and mtCMSID06 detection different type cytoplasmic male sterile lines and the result of maintainer
Embodiment 3Utilize InDel Marker Identification different type hybrid rice varieties
Ternary hybrid rice by cytoplasmic male sterile line with from corresponding restorer combo, in table 3 " cytoplasm type "
For the corresponding male sterility set type of series of three-series hybrid rice kind, " rich two excellent No. 4 " are double-linear hybrid rice kind.
Multiple hybrid rice varieties are carried out with the detection of genotype in the same manner as in example 1, as a result such as 3 institute of table
Show.The result shows that mtCMSID01, mtCMSID05, mtCMSID02 and mtCMSID06 can accurately distinguish Yebai or print
Buddhist nun paddy field paddy type or ridge type or K-type or D types hybrid rice variety and other types hybrid rice variety.
The testing result of more than 3 a hybrid rice variety of table
Embodiment 4Utilize InDel Marker Identification conventional rice kinds
30 parts of conventional rice kinds are carried out with the detection of genotype in the same manner as in example 1, as a result such as 4 He of table
Shown in table 5.
The result shows that in the conventional rice kind detected, the amplified band of mtCMSID01 is 326bp,
The amplified band of mtCMSID05 is 250bp, and the amplified band of mtCMSID02 is 120bp, the amplified band of mtCMSID06
It is 157bp, is different from Yebai or Indonesia's paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile line or its is miscellaneous
Hand over rice varieties.
As it can be seen that mtCMSID01, mtCMSID05, mtCMSID02 and mtCMSID06 can be accurately by Yebai or Indonesia
Paddy field paddy type or ridge type or K-type or D types cytoplasmic male sterile line or its hybrid rice variety are mutually distinguished with conventional rice kind.
Table 4 detects the result of 30 parts of conventional rice kinds using mtCMSID01 and mtCMSID05
Table 5 detects the result of 30 parts of conventional rice kinds using mtCMSID02 and mtCMSID06
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, these modifications or improvements on the basis of without departing from the application spirit, belong to this application claims range.
Sequence table
<110>Chinese subset rolls into a ball Co., Ltd
<120>The method for identifying rice cytoplasmic type
<130> 16C12756CN
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
acggtaaatc gcacaggtt 19
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
cagtagatag tagagaggag g 21
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<400> 3
tggagtggag gcattgctat tc 22
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
gtctacgctc gcttttgttc tg 22
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
taatgggtgg catagcccgt ag 22
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence
<400> 6
tgagtcgctc gttcaactgt cc 22
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
ctttacttcg gctgtggtca t 21
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
ggtttcttaa tcggtgtcta tc 22
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
attaccaacg ggtagtgaca a 21
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence
<400> 10
taccataatc caaagattga gtac 24
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<400> 11
accgaactca cattatttgg 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
ctaaatacaa aacaagagtc 20
Claims (10)
1. the method for identifying rice cytoplasmic type comprising：
The nucleic acid fragment in rice sample to be identified is expanded using at least one primer pair selected from the following：
The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and
Reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3’；And/or
Second primer pair：
Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ', and
Reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And
Determine the length of expanded nucleic acid fragment,
Wherein, long for the nucleic acid fragment of 117bp and/or the second primer pair amplifies to the nucleic acid fragment length of amplification when the first primer
When degree is 154bp, the rice sample is cytoplasm type selected from the following：Yebai, Indonesia's paddy field paddy type, ridge type, K-type and
D types.
2. identifying cytoplasmic male sterile rice or the method for its hybrid rice variety comprising:
The nucleic acid fragment in rice sample to be identified is expanded using at least one primer pair selected from the following：
The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and
Reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3’；And/or
Second primer pair：
Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ', and
Reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And
Determine the length of expanded nucleic acid fragment,
Wherein, long for the nucleic acid fragment of 117bp and/or the second primer pair amplifies to the nucleic acid fragment length of amplification when the first primer
When degree is 154bp, the rice sample is cytoplasmic male sterile rice selected from the following or its hybrid rice variety：It loses open country
Type, Indonesia's paddy field paddy type, ridge type, K-type and D types.
3. the method for distinguishing the corresponding maintainer of cytoplasmic male sterile rice comprising：
The nucleic acid fragment in rice sample to be distinguished is expanded using at least one primer pair selected from the following：
The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and
Reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3；And/or
Second primer pair：
Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ', and
Reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And
Determine the length of expanded nucleic acid fragment,
Wherein, long for the nucleic acid fragment of 117bp and/or the second primer pair amplifies to the nucleic acid fragment length of amplification when the first primer
When degree is 154bp, the rice sample is cytoplasmic male sterile rice selected from the following：Yebai, Indonesia's paddy field paddy type,
Ridge type, K-type and D types.
4. the method for distinguishing cytoplasmic male sterile rice hybrid rice variety and other types hybrid rice variety, packet
It includes：
The nucleic acid fragment in rice sample to be distinguished is expanded using at least one primer pair selected from the following：
The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and
Reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3；And/or
Second primer pair：
Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ', and
Reverse primer：5’-CTAAATACAAAACAAGAGTC-3’；And
Determine the length of expanded nucleic acid fragment,
Wherein, it is 117bp to the nucleic acid fragment length of amplification when the first primer and/or the amplified fragments size of the second primer pair is
When 154bp, the rice sample is cytoplasmic male sterile rice hybrid rice variety selected from the following：Yebai, Indonesia
Paddy field paddy type, ridge type, K-type and D types.
5. method according to any one of claims 1 to 4, wherein the primer pair also includes fluorophor；Optionally, institute
It states fluorophor and is selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
6. method according to any one of claims 1 to 4, wherein the rice sample is selected from seed and blade.
7. the primer pair for identifying rice cytoplasmic type, is selected from：
The first primer pair：
Forward primer：5 '-TGGAGTGGAGGCATTGCTATTC-3 ', and
Reverse primer：5’-GTCTACGCTCGCTTTTGTTCTG-3’；And/or
Second primer pair：
Forward primer：5 '-ACCGAACTCACATTATTTGG-3 ', and
Reverse primer：5’-CTAAATACAAAACAAGAGTC-3’.
8. primer pair as claimed in claim 7, wherein the primer pair also includes fluorophor；Optionally, the fluorescent base
Group is selected from Fluoresceincarboxylic acid, chlordene fluorescein, carboxyl tetramethylrhodamine and carboxy-X-rhodamine.
9. the kit for identifying rice cytoplasmic type, it includes the primer pairs described in claim 7 or 8；Optionally, institute
Stating kit also includes：Amplification buffer, dNTP mixtures, archaeal dna polymerase and ddH2O。
10. primer pair as claimed in claim 7 or 8 or kit as claimed in claim 9, are used for：
I) rice cytoplasmic type is identified；
Ii cytoplasmic male sterile rice or its hybrid rice variety) are identified；
Iii) differentiate the corresponding maintainer of cytoplasmic male sterile rice；
Iv cytoplasmic male sterile rice hybrid rice variety and other types hybrid rice variety) are distinguished；Or
V) cytoplasmic male sterile rice or its hybrid rice variety and conventional rice kind are distinguished；
Wherein, the rice cytoplasmic type or cytoplasmic male sterile rice are selected from：Yebai, Indonesia's paddy field paddy type,
Ridge type, K-type and D types.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710034236.0A CN108330203B (en) | 2017-01-18 | 2017-01-18 | Method for identifying rice cytoplasm type |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710034236.0A CN108330203B (en) | 2017-01-18 | 2017-01-18 | Method for identifying rice cytoplasm type |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108330203A true CN108330203A (en) | 2018-07-27 |
| CN108330203B CN108330203B (en) | 2021-07-13 |
Family
ID=62921662
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710034236.0A Active CN108330203B (en) | 2017-01-18 | 2017-01-18 | Method for identifying rice cytoplasm type |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108330203B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865937A (en) * | 2014-03-13 | 2014-06-18 | 华南农业大学 | Cytoplasmic male sterility restorer gene in rice and application thereof |
| CN104789648A (en) * | 2014-12-25 | 2015-07-22 | 中国种子集团有限公司 | Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof |
| CN105755140A (en) * | 2016-04-15 | 2016-07-13 | 中国农业科学院棉花研究所 | InDel marker for cotton cytoplasm male sterility restoring lines and method for identifying molecules by aid of InDel marker |
| WO2016125143A1 (en) * | 2015-02-02 | 2016-08-11 | Danziger Innovations Ltd. | Mdmv based vector for gene expression and silencing |
-
2017
- 2017-01-18 CN CN201710034236.0A patent/CN108330203B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865937A (en) * | 2014-03-13 | 2014-06-18 | 华南农业大学 | Cytoplasmic male sterility restorer gene in rice and application thereof |
| CN104789648A (en) * | 2014-12-25 | 2015-07-22 | 中国种子集团有限公司 | Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof |
| WO2016125143A1 (en) * | 2015-02-02 | 2016-08-11 | Danziger Innovations Ltd. | Mdmv based vector for gene expression and silencing |
| CN105755140A (en) * | 2016-04-15 | 2016-07-13 | 中国农业科学院棉花研究所 | InDel marker for cotton cytoplasm male sterility restoring lines and method for identifying molecules by aid of InDel marker |
Non-Patent Citations (2)
| Title |
|---|
| SOTA FUJII 等: "Discovery of global genomic re-organization based on comparison of two newly sequenced rice mitochondrial genomes with cytoplasmic male sterility-related genes", 《BMC GENOMICS》 * |
| 栾霁: "水稻线粒体DNA遗传多态性及相关基因表达的研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108330203B (en) | 2021-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107619857B (en) | Method for detecting CNV (CNV) marker of beef cattle KLF8 gene and application of CNV marker | |
| CN112626257B (en) | SNP molecular marker for detecting purity of sunflower variety and application thereof | |
| CN110257547B (en) | Corn core SNP marker developed based on KASP technology and application thereof | |
| US20210285063A1 (en) | Genome-wide maize snp array and use thereof | |
| CN108048595A (en) | Indel molecular labelings and its application with pumpkin photoperiod insensitive character close linkage | |
| CN110872633B (en) | Method for identifying purity of Jingke 968 corn hybrid based on SNP marker | |
| CN110724758B (en) | Method for identifying purity of Jingnongke 728 corn hybrid based on SNP marker | |
| CN110042171A (en) | Identify the method and related molecular marker of Yield Traits of Wheat | |
| CN110777216B (en) | Method for identifying purity of Jingke waxy 2000 corn hybrid based on SNP marker | |
| CN110541041B (en) | SNP marker related to Chinese domestic horse dwarf trait and application thereof | |
| CN115094156A (en) | Development and application of KASP marker of rice high-temperature-resistant gene TT1 | |
| CN113249509B (en) | Identification primer and identification method for interspecific hybrid progeny of populus jaborandi and populus microphylla | |
| CN117106967A (en) | Functional KASP molecular marker of rice blast resistance gene and application thereof | |
| CN116024376A (en) | Corn root hair related InDel marker identification primer and application thereof | |
| CN111549172B (en) | Watermelon leaf posterior green gene linkage site and CAPS marker | |
| CN114606335A (en) | Development and application of KASP molecular marker of sugarcane mosaic virus disease resistance gene of corn | |
| CN108330203A (en) | The method for identifying rice cytoplasmic type | |
| CN108330202A (en) | The method for identifying rice cytoplasmic type | |
| CN103589797A (en) | SNP (single nucleotide polymorphism) genotyping method and application thereof | |
| JP7166638B2 (en) | Polymorphism detection method | |
| CN108486263A (en) | Differentiate corn male sterility T-type cytoplasm type using chloroplastic marker | |
| CN108676906A (en) | The DCIPThe chloroplast of maize sites genome SSR and the application in cultivar identification | |
| CN110616256B (en) | Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application | |
| CN116814841B (en) | Primer group for identifying rice black brown glume gene HK4, and method and application thereof | |
| CN108427866B (en) | Crop inbred line group identification method based on molecular marker technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |