CN106480179B - The molecular labeling of rice paddy seed fast-germination QTL qGS11 and its application - Google Patents
The molecular labeling of rice paddy seed fast-germination QTL qGS11 and its application Download PDFInfo
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Abstract
The invention discloses the molecular labeling of rice paddy seed fast-germination QTL qGS11 and its applications.PCR amplification is carried out using one or more pairs of pairs of oryza sativa genomic dnas in the primer of SSR marker of the present invention, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplifying the DNA fragmentation of corresponding size, the presence of control rice paddy seed fast-germination QTL qGS11 synergy allele is indicated.Through the invention isolate the molecule labelling method with close linkage with seed quick-speed germination gene qGS11, it is horizontal to can be predicted Seed Germination of Rice speed, allows rapid screening out high vigor rice varieties.
Description
Technical field
The invention belongs to seed science and technology application fields, are related to the molecule of rice paddy seed fast-germination QTL qGS11
Label and its application.
Background technique
In agricultural production, the abiotic stress such as low temperature and flood after planting can be often encountered, low vitality seed is sprouted slowly, out
Phenomena such as seedling rate is low and resistance is poor, causes field seedling to rot, is downright bad and weedy, eventually leads to Severe Reduction.Meanwhile with
Increasing labor shortage, Direct-seeding Rice promote it is more and more wider.Therefore, seed speed of germinating, the high vigor rice varieties of breeding are improved
It is of great significance.By excavation, the relevant gene/QTL of positional cloning rice paddy seed fast-germination, assisted using molecular labeling
The high vigor rice varieties of (MAS) breeding are selected, are an effectively methods.
Using different rice genetic group is constructed, having many scholars, to located multiple control seeds sproutings relevant
QTLs, but the main effect QTL qLTG3-1 that only one control rice paddy seed low temperature is sprouted is by successful clone.Utilize long-grained nonglutinous rice early period
It is related that kind IR28 to recombinant inbred lines (RILs) crowd surveillance that japonica rice variety high point rice constructs has arrived multiple seed sproutings
QTLs.On this basis, to be located at No. 11 chromosomes of rice on control seed speed of germinating QTL qGS11 carry out finely positioning,
Basis is identified to cultivate high vigor rice varieties using molecular marker-assisted selection method (MAS).
Summary of the invention
It is an object of the present invention to provide the molecule labelling methods of rice paddy seed fast-germination screening.Pass through detection and rice paddy seed
The chain molecular labeling of fast-germination gene loci can quickly filter out the rice varieties of fast-germination, improve rice paddy seed
Reliability, the validity of vigor screening；It can determine that whether there is or not control seed quick-speed germination channel genes into breeding lines, improve
The efficiency of selection of the high vigor character of rice accelerates breeding process.
The purpose of the present invention can be achieved through the following technical solutions:
The molecular labeling of rice paddy seed fast-germination QTL qGS11, the molecular labeling be selected from YB20, YB21,
Any one in RM26614；The molecular labeling YB20 upstream primer is YB20L:SEQ ID NO.1, and downstream primer is
YB20R:SEQ ID NO.2, amplified production size are 133bp；The molecular labeling YB21 upstream primer is YB21L:SEQ
ID NO.3, downstream primer are YB21R:SEQ ID NO.4, and amplified production size is 194bp；The molecular labeling RM26614
Upstream primer is RM26614L:SEQ ID NO.5, and downstream primer is RM26614R:SEQ ID NO.6, and amplified production size is
183bp。
The molecular labeling primer of rice paddy seed fast-germination QTL qGS11 of the present invention, the molecular labeling
YB20 upstream primer is YB20L:SEQ ID NO.1, and downstream primer is YB21R:SEQ ID NO.2, and amplified production size is
133bp；The molecular labeling YB21 upstream primer is YB21L:SEQ ID NO.3, and downstream primer is YB21R:SEQ ID
NO.4, amplified production size are 194bp；The molecular labeling RM26614 upstream primer is RM26614L:SEQ ID NO.5,
Downstream primer is RM26614R:SEQ ID NO.6, and amplified production size is 183bp.
Application of the molecular labeling of the present invention in the rice varieties of screening fast-germination.
Application of the molecular labeling primer of the present invention in the rice varieties of screening fast-germination.
Rice paddy seed fast-germination molecule screening technique, steps are as follows:
(1) rice leaf is taken, genomic DNA is extracted.
(2) PCR expansion is carried out to the genomic DNA using any 1 pair or 1 pair or more molecular labeling primer in table 1
Increase, pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplifying the DNA piece of corresponding size
Section indicates that qGS11 synergy allele exists.
Table 1
Wherein, the PCR reaction system: volume is 25 microlitres, wherein 2.5 microlitres of 10 × buffer, 25mMMgCl2
1.5 microlitres, 4pmol/ microlitres 2.5 microlitres of primer pair, 2 microlitres of 2.5mM dNTPs, 5 units/0.2 microlitre of microlitre Taq enzyme, template
20 nanogram of DNA, adds water to 25 microlitres.Response procedures are 95 DEG C of initial denaturation 5min of DNA；95 DEG C of initial denaturation 30s, 50 DEG C of annealing
30s, 72 DEG C of extensions are opened up 30s, are recycled 35 times；Last 72 DEG C of extensions 10min.
Upstream and downstream primer amplification rice variety IR28 or IR28 and japonica rice filial generation with SSR molecular marker YB20
Genomic DNA, if it is possible to amplify the amplified fragments of 133bp, then indicate that the long-grained nonglutinous rice IR28 synergy allele of qGS11 is deposited
Otherwise 128bp is being amplified, then is showing that synergy allele does not import.In 5125 BC5F2In segregating population, matched with exchange
Subnumber calculates, this label is isolated with seed quick-speed germination gene qGS11, single to mark efficiency of selection up to 99.9%.
Upstream and downstream primer amplification rice variety IR28 or IR28 and japonica rice filial generation with SSR molecular marker YB21
Genomic DNA, if it is possible to amplify the amplified fragments of 194bp, then indicate that the long-grained nonglutinous rice IR28 synergy allele of qGS11 is deposited
Otherwise 178bp is being amplified, then is showing that synergy allele does not import.In 5125 BC5F2In segregating population, matched with exchange
Subnumber calculates, this label is isolated with seed quick-speed germination gene qGS11, single to mark efficiency of selection up to 100%.
After being hybridized with upstream and downstream primer amplification the rice variety IR28 or IR28 of SSR molecular marker RM26614 with japonica rice
The genomic DNA in generation, if it is possible to amplify the amplified fragments of 183bp, then indicate the long-grained nonglutinous rice IR28 synergy equipotential base of qGS11
Because existing, 200bp is otherwise amplified, then shows that synergy allele does not import.In 5125 BC5F2In segregating population, with friendship
Subnumber calculating is changed, what this label was isolated with seed quick-speed germination gene qGS11, it is single to mark efficiency of selection up to 99.8%.
Above-mentioned 3 molecular labelings are to the efficiency of selection of qGS11 99% or more, and wherein SSR marker YB21 and qGS11 is divided into
From selection accuracy rate is high, up to 100%.Above-mentioned 3 labels can select a use, can also arbitrarily select 2 pairs or 2 with subscript
Note is used in combination.Any 2 molecular labelings combination selection, efficiency of selection also reach 100% in 3 labels.
Beneficial effect
The molecule labelling method of rice paddy seed fast-germination screening provided by the present invention, has the advantage that
(1) molecular labeling developed through the invention for the first time from rice variety IR28 on No. 11 chromosome long arms of rice
Navigate to control seed speed of germinating QTL qGS11, and isolated or the SSR marker YB20 of height close linkage,
YB21 and RM26614.
(2) the rice paddy seed fast-germination QTL qGS11 of Molecular mapping, identification method are quickly simple through the invention
Just, efficiency of selection is high.Only need to detect the amplified band feature of these labels, so that it may judge seed speed of germinating gene
QGS11 whether there is, to predict that rice seed germination speed is horizontal, the high vigor rice of screening that can be quick, purposive
Kind or strain, for improveing Rice Seed Vigor.These labels are isolated with fast-germination gene qGS11 or height closely connects
Lock, single that efficiency of selection is marked to reach 99%-100%, any double labelling selection rate is up to 100%.
(3) molecular marker assisted selection breeding objective is clear, and save the cost is not affected by environment.In traditional breeding way
In, first have to collect seed quick-speed germination parent and backbone parent carry out it is a series of hybridize, be returned, and will be until harvesting seed
After broken suspend mode, sprout phenotypic evaluation to offspring to select single plant.Traditional breeding way is affected by environment big, reliability
It is low.Through the invention isolate the molecule labelling method with close linkage with seed quick-speed germination gene qGS11, can be predicted water
Rice sprouts speed, can just identify the single plant of fast-germination in Their Seed Germinating Period, eliminate other plant, can effectively control
Breeding Scale processed improves breeding efficiency, allows rapid screening out high vigor rice varieties.Meanwhile in rice breeding informative population,
The single plant that seed quick-speed germination gene qGS11 can be provided with Rapid identification greatly improves efficiency of selection, shortens the breeding time limit,
For the high vigor breed improvement of rice and breeding service.
Detailed description of the invention
Fig. 1: two parent high point rice and IR28 4d sprout phenotypic map
Fig. 2: qGS11 just positions figure on No. 11 chromosomes
Fig. 3: qGS11 on No. 11 chromosomes finely positioning figure
Fig. 4: the molecular labeling polymorphism electrophoretic band newly developed with qGS11 close linkage
Specific embodiment
Embodiment 1
(1) materials and methods:
1. material: the high vigor rice varieties IR28 of parent and low vitality kind high point rice, and using IR28 as recurrent parent structure
Build BC1F2And BC5F2Group.
2. extracting individual DNA with SDS method.
3. molecular markers development: new primer is designed with OryzasativaLcv.Nipponbare and 9311 for reference sequences, with parent IR28 and high point rice
For template, PCR amplification is carried out, screens polymorphism mark.
4.PCR reaction system: volume is 25 microlitres, wherein 2.5 microlitres of 10 × buffer, 25mM MgCl21.5 microlitres,
4pmol/ microlitres 2.5 microlitres of primer pair, 2 microlitres of 2.5mM dNTPs, 5 units/0.2 microlitre of microlitre Taq enzyme, template DNA 20 is received
Gram, add water to 25 microlitres.Response procedures are 95 DEG C of initial denaturation 5min of DNA；95 DEG C of initial denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C are prolonged
30s is stretched, is recycled 35 times；Last 72 DEG C of extensions 10min.PCR amplification is carried out on biometre amplification instrument, amplified production exists
8% non-denaturing polyacrylamide gel (has 7.6g acrylamide and the double acryloyls of 0.4g methene in polyacrylamide solution containing 100ml
Amine) on be separated by electrophoresis.
5. the first site seed quick-speed germination QTL: parent and BC1F2Segregating population is all planted in Agricultural University Of Nanjing Jiangpu
Experiment station samples and harvests mature seed, and impurity elimination, seed dormancy is abolished in 50 DEG C of drying, then is stored in -20 DEG C of refrigerators, in case
It is used for a long time；Each single plant takes 30 seeds, and 3 repetitions carry out the identification of seed speed of germinating at 25 DEG C, completes rice paddy seed
Speed of germinating QTL Primary Location.
6. software operation: software used is QTL IciMapping 4.1, and minimum LOD value is set as 2.5, and paces 1 obtain
Linkage map carries out QTL positioning analysis (Fig. 2).
7. linked marker verifying with section determine: to contain the site qGS11 background family similar with IR28 be returned,
Selfing obtains BC5F2The offspring of each single plant of segregating population carries out seed speed of germinating Characters Identification under normal condition, and combines
Its genotype constructs rice genetic map, verifies chain molecular labeling, reduces positioning section to effectively determine.
(2) result and analysis
In conjunction with BC1F2Seed sprouts phenotype to segregating population under normal operation, is with 4d seed planting percent and germination index
Index finds to be located at the QTL necessary being for controlling seed speed of germinating on No. 11 chromosomes, is located at label using software analysis
Between RM3428 and RM26614.
To BC5F2The individual phenotypic evaluations of 5125 plants of separation of segregating population and labeled analysis, in molecular labeling YB20 and
11 exchange strains are obtained between RM26614.Label YB21 and qGS11 is isolated, and does not exchange strain, and instruction sheet is marked to qGS11's
Efficiency of selection is up to 100%；Mark YB21 and RM13443 and qGS11 exchange rate 1% hereinafter, single label imitates the selection of qGS11
Rate is up to 99% or more (Fig. 3).
By detection and the chain molecular labeling in rice seed germination rate gene site, Rice Seed Vigor can be predicted
Level can determine that whether there is or not control rice seed germination rate genes to import in breeding lines, improve rice breeding efficiency of selection,
Accelerate Breeding progress.Using being isolated with qGS11 or YB20, YB21 and RM26614 of close linkage carry out single label selection, choosing
Selecting efficiency can reach 99%-100%, and any double labelling combination efficiency of selection reaches 100%, can be used for molecular labeling auxiliary choosing
Select breeding.
Embodiment 2
(1) materials and methods:
1. material: rice varieties IR28 and high point rice
2. extracting individual DNA with SDS method.
3. label: YB20, YB21 and RM26614.
4.PCR reaction system: volume is 25 microlitres, wherein 2.5 microlitres of 10 × buffer, 25mM MgCl21.5 microlitres,
4pmol/ microlitres 2.5 microlitres of primer pair, 2 microlitres of 2.5mM dNTPs, 5 units/0.2 microlitre of microlitre Taq enzyme, template DNA 20 is received
Gram, add water to 25 microlitres.Response procedures are 95 DEG C of initial denaturation 5min of DNA；95 DEG C of initial denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C are prolonged
30s is stretched, is recycled 35 times；Last 72 DEG C of extensions 10min.PCR amplification is carried out on biometre amplification instrument, amplified production exists
8% non-denaturing polyacrylamide gel (has 7.6g acrylamide and the double acryloyls of 0.4g methene in polyacrylamide solution containing 100ml
Amine) on be separated by electrophoresis.
(2) result and analysis
The genomic DNA of rice variety IR28 or high point rice, water are expanded with the upstream and downstream primer of SSR molecular marker YB20
Rice varieties IR28 can amplify the amplified fragments of 133bp, indicate that there are qGS11 synergy equipotential bases in rice varieties IR28
Cause, high point rice amplify 128bp, show that there is no qGS11 synergy allele in the rice of high point.With SSR molecular marker YB21's
Upstream and downstream primer expands the genomic DNA of rice variety IR28 or high point rice, and rice varieties IR28 can amplify 194bp's
Amplified fragments indicate that, there are qGS11 synergy allele in rice varieties IR28, high point rice amplifies 178bp, shows high point
QGS11 synergy allele is not present in rice.Rice variety IR28 is expanded with the upstream and downstream primer of SSR molecular marker RM26614
Or the genomic DNA of high point rice, rice varieties IR28 can amplify the amplified fragments of 183bp, indicate rice varieties
There are qGS11 synergy allele high point rice to amplify 200bp in IR28, shows that there is no qGS11 synergy equipotentials in the rice of high point
(Fig. 4, corresponding two swimming lanes of every kind of molecular labeling primer in figure, the swimming lane on the left side correspond to high point rice, the swimming lane correspondence on the left side to gene
Rice varieties IR28).
Claims (8)
1. the molecular labeling of rice paddy seed fast-germination QTL qGS11, it is characterised in that the molecular labeling be selected from YB20,
Any one in YB21, RM26614；The upstream primer of the molecular labeling YB20 is RMYB20L:SEQ ID NO.1, under
Trip primer is RMTB20R:SEQ ID NO.2, and amplified production size is 133bp；The upstream primer of the molecular labeling YB21
For YB21L:SEQ ID NO.3, downstream primer is YB21R:SEQ ID NO.4, and amplified production size is 194bp；Point
The upstream primer of son label RM26614 is RM26614L:SEQ ID NO.5, and downstream primer is RM26614R:SEQ ID NO.6,
Amplified production size is 183bp.
2. expanding the molecular labeling primer of rice paddy seed fast-germination QTL qGS11 described in claim 1, it is characterised in that institute
The upstream primer of the molecular labeling YB20 stated is RMYB20L:SEQ ID NO.1, and downstream primer is RMTB20R:SEQ ID
NO.2, amplified production size are 133bp；The upstream primer of the molecular labeling YB21 is YB21L:SEQ ID NO.3, downstream
Primer is YB21R:SEQ ID NO.4, and amplified production size is 194bp；The upstream primer of the molecular labeling RM26614 is
RM26614L:SEQ ID NO.5, downstream primer are RM26614R:SEQ ID NO.6, and amplified production size is 183bp.
3. application of the molecular labeling primer as claimed in claim 2 in the rice varieties of screening fast-germination.
4. a kind of method for quickly identifying qGS11 synergy allele presence or absence, characterized by comprising the steps of:
(1) rice leaf is taken, genomic DNA is extracted；
(2) using any pair in molecular labeling primer as claimed in claim 2, it is two or three pairs of to the genome
DNA carries out PCR amplification, and pcr amplification product carries out electrophoresis detection on 8% non-denaturing polyacrylamide gel, if amplifying correspondence
The DNA fragmentation of size indicates the presence of seed quick-speed germination QTL qGS11 synergy allele.
5. according to the method described in claim 4, it is characterized in that the PCR reacts: volume is 25 microlitres, wherein 10 ×
2.5 microlitres of buffer, 25mM MgCl2 1.5 microlitres, 4pmol/ microlitres 2.5 microlitres of primer pair, 2 microlitres of 2.5mM dNTPs, 5
Unit/0.2 microlitre of microlitre Taq enzyme, 20 nanogram of template DNA add water to 25 microlitres；Response procedures are 95 DEG C of initial denaturations of DNA
5min；95 DEG C of initial denaturations 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s are recycled 35 times；Last 72 DEG C of extensions 10min.
6. according to the method described in claim 4, it is characterized in that the upstream and downstream primer of SSR molecular marker YB20 expands long-grained nonglutinous rice product
The genomic DNA of kind IR28 or IR28 and japonica rice filial generation, if it is possible to which the amplified fragments for amplifying 133bp then indicate
QGS11 long-grained nonglutinous rice IR28 synergy allele exist, otherwise amplify 128bp, then show that synergy allele does not import.
7. according to the method described in claim 4, it is characterized in that expanding long-grained nonglutinous rice with the upstream and downstream primer of SSR molecular marker YB21
The genomic DNA of kind IR28 or IR28 and japonica rice filial generation, if it is possible to which the amplified fragments for amplifying 194bp are then marked
Will qGS11 long-grained nonglutinous rice IR28 synergy allele exist, otherwise amplify 178bp, then show that synergy allele is not led
Enter.
8. according to the method described in claim 4, it is characterized in that being expanded with the upstream and downstream primer of SSR molecular marker RM26614
The genomic DNA of rice variety IR28 or IR28 and japonica rice filial generation, if it is possible to the amplified fragments of 183bp are amplified,
It then indicates that the long-grained nonglutinous rice IR28 synergy allele of qGS11 exists, otherwise amplifies 200bp, then show that synergy allele does not have
There is importing.
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