CN106148543A - A kind of SSR primer mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. to and application - Google Patents
A kind of SSR primer mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. to and application Download PDFInfo
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- CN106148543A CN106148543A CN201610753276.6A CN201610753276A CN106148543A CN 106148543 A CN106148543 A CN 106148543A CN 201610753276 A CN201610753276 A CN 201610753276A CN 106148543 A CN106148543 A CN 106148543A
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- 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
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Abstract
The invention belongs to plant molecular genetics field, relate to SSR primer mutually chain with setting percentage on No. 5 chromosomes of a kind of Oryza sativa L. to and application in breeding rice kind.SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of a kind of Oryza sativa L., the forward primer of described primer pair is the sequence shown in Seq ID NO.1, and the downstream primer of described primer pair is the sequence shown in Seq ID NO.2.The SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. that the present invention provides, is the chain SSR marker relevant to setting percentage QTL, it is possible to more precise Identification height setting percentage material and the specific band of low setting percentage material in 34 parts of Core Germplasms；The prediction of setting percentage in early days can be carried out and to whether containing and the judgement of setting percentage related gene in breeding material；The QTL that setting percentage is relevant whether can be contained with Rapid identification breeding material.With low cost, to identify simple, directivity is clear and definite.
Description
Technical field
The invention belongs to plant molecular genetics field, relate on No. 5 chromosomes of a kind of Oryza sativa L. mutually chain with setting percentage
SSR primer to and application in breeding rice kind.
Background technology
SSR marker is also called simple sequence repeat, is one of the most the most frequently used microsatellite marker.Core
Structure be by 2-6 nucleotide be repeat unit string joint group become up to tens nucleotide sequences, be the most all conservative relatively
Strong unique sequence, its both sides are relatively to guard and special single-copy sequence.According to special conservative sequence, can manually close
Becoming primer to carry out PCR amplification, due to the variation quantitatively of single microsatellite locus repetitive, individual amplified production is being grown
Change on degree just produces the polymorphism of length, and each amplification site just represents the pair of alleles in this site.Therefore,
PCR primer creates the polymorphism of length.SSR marker is set up on round pcr, owing to having codominance, rich content, polymorphic
The feature that property is high, is therefore widely used on vegeto-animal genetic breeding research.
Seed-Setting Percentage in Rice is the important factor affecting yield, utilizes SSR and all kinds of labelling, and colony carries out Seed-Setting Percentage in Rice
Position the most in the ascendant with the work of clone.Chen Qing congruence utilizes the long-grained nonglutinous rice material of low setting percentage and the long-grained nonglutinous rice product of high setting percentage
Kind of T226 is that parent builds RIL, the setting percentage of RIL carries out genotype-by-environment interaction analysis, QTL fixed
Position is analyzed, found that 17 QTLs relevant to setting percentage, wherein most QTL is all only in 1 or 2 specific environment
Find, and be positioned at the interval qSSR3-1 of the 3rd chromosome MRG5959-MRG2180, unanimously detect in 6 environment altogether, contribution
Rate is maximum (15.6-35.7%) respectively in each environment, the allele of its potentiation derives from parent T226 (Chen Qingquan,
2007).Zhou Yong etc. utilize chromosome Single Segment Substitution Lines in Rice to position Seed-Setting Percentage in Rice QTLs, with Guanglu ai 4 and Japonica
85 chromosome Single Segment Substitution Lines in Rices are research material, by one factor analysis of variance and multiple comparisons, to relevant to setting percentage
QTLs detect.Found that 9 setting percentage QTLs, it is distributed on 7 chromosomes in 12 chromosomes of Oryza sativa L. (week
Bravely, 2013).High clouds etc. utilize chromosome Single Segment Substitution Lines in Rice to position Seed-Setting Percentage in Rice QTL, raise rice No. 6 for being subject to rice variety
Body, japonica rice variety Japonica be donor build a set of chromosome segment substitution line be material, utilize multiple regression analysis side
Method, in conjunction with Bin collection of illustrative plates, identifies to the setting percentage QTL on substitution line, result shows, 14 control Seed-Setting Percentage in Rice character
QTL, lay respectively at 3-11 chromosome.The QTL of contribution rate maximum is qSSR5.1, and contribution rate is 49.76%, is positioned in the 5th
On chromosome in 731482bp interval (high cloud, 2014).
At present, studies in China concentrates on QTL location and gene clone's aspect to the research of setting percentage, assists at molecular marker
Selection and use and less to the detection of setting percentage related locus in breeding material.
The relevant QTL of substantial amounts of Seed-Setting Percentage in Rice orientates the breeding of Oryza sativa L. height setting percentage as and has established theoretical basis, but
Most QTL are to assemble colony based on two kinds of setting percentage materials that there were significant differences to study, and identify obtaining in progeny population.
Only reside within the stage of location.They have the disadvantages that
(1) only having polymorphism in two parent materials, the SSR marker relevant to setting percentage screened is in particular cluster
Body has dependency relation.
(2) SSR marker relevant to setting percentage in target group, not yet detects in breeding material, there is not yet
The rice germplasm of different setting percentages is identified and contacts by setting percentage SSR primer of being correlated with.The highest hence with rate.
Summary of the invention
In order to solve in rice breeding the early stage test problems to breeding material setting percentage, and solve relevant to setting percentage
The aggregation problem of QTL, the present invention provides SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of a kind of Oryza sativa L., is used for detecting
Whether this QTL that in breeding material, setting percentage is relevant exists.Can quickly judge whether plant carries high setting percentage gene.Utilize
Conventional art judges that Seed-Setting Percentage in Rice to wait until solid rear perusal, if difference is little, calculates after seed maturity to be waited until,
Cycle is longer.Utilize molecular marker assisted selection, can directly judge whether containing high setting percentage QTL in seedling stage, quick and convenient.
To achieve these goals, present invention employs techniques below scheme:
The invention discloses SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of a kind of Oryza sativa L., described primer pair
Forward primer be the sequence shown in Seq ID NO.1, the downstream primer of described primer pair is the sequence shown in Seq ID NO.2
Row.
Present invention also offers SSR primer mutually chain with setting percentage on No. 5 chromosomes of described Oryza sativa L. to for screening
Whether Oryza sativa L. carries the method for high setting percentage gene, including step: with oryza sativa genomic dna to be detected as template, with right
Require that the SSR primer described in 1, to carrying out PCR amplification, contrasts with the band of the Japonica of low setting percentage, thus identifies material
Whether material contains the related gene of high setting percentage.
Present invention also offers SSR primer mutually chain with setting percentage on No. 5 chromosomes of described Oryza sativa L. to auxiliary in Oryza sativa L.
Help the purposes in breeding.
Compared with prior art, the present invention possesses following beneficial effect:
(1) SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. that the present invention provides, be and setting percentage QTL
Relevant chain SSR marker, it is possible to more precise Identification height setting percentage material and low setting percentage material in 34 parts of Core Germplasms
Specific band.The prediction of setting percentage in early days can be carried out and to whether containing and setting percentage related gene in breeding material
Judgement.The QTL that setting percentage is relevant whether can be contained with Rapid identification breeding material.With low cost, to identify simple, directivity is bright
Really.
(2) mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. that the present invention provides SSR primer pair and setting percentage height phase
Closing, be a QTL relevant to setting percentage, contribution rate is 24.6%, be one new do not reported relevant to setting percentage
QTL。
(3) SSR primer mutually chain with setting percentage on No. 5 chromosomes of Oryza sativa L. that the present invention provides is not at 34 parts of setting percentages
Same Core Germplasms detects, it is possible to effectively detect specific band and the specific band of low setting percentage of high setting percentage, the highest
Imitate and determine whether rice material contains setting percentage and be correlated with QTL or whether contain high setting percentage gene at rice plant Early seedling stage.
Therefore, utilize the present invention, can rapidly and efficiently identify whether rice breed contains setting percentage and be correlated with QTL.
Accompanying drawing explanation
SSR primer mutually chain with setting percentage on No. 5 chromosomes of the Oryza sativa L. that Fig. 1 is the present invention is in 34 parts of core Oryza sativa L.
Plant the denaturing polyacrylamide gel electrophoresis test strip of amplified production in matter；
Explanation about reference:
1-DOZIANCLOUA::IRGC 56877-1；2-STG 556011::IRGC 4059-1；3-RATHAL::IRGC
31524-1；4-CANELLA DE FERRO；5-VARY MADINIKA 3566::GERVEX 8448-C1；6-KOSATA::
IRGC 31454-1；7-BATUKURU WEE::IRGC 67614-1；8-SULTANI；9-KU 115；10-KOGONI 91-1::
C1；11-ORYZICA SABANA 10::IRGC 117018-1；12-PIN TAWNG::IRGC 40673-1；13-IAC
165::GERVEX 8508-C1；14-WAS 198-B-3-1-3::C1；15-ANAYANSI::IRGC 77474-1；16-IR
5::IRGC 10321-1；17-PERUM KARUPPAN::IRGC 15524-1；18-SALUMPIKIT::IRTP 4777-C1；
19-E 5168::IRGC 68021-1；20-Japonica；21-TSIPALA FOTSY::IRGC 69973-1；22-YE TI
ZHAN::IRGC 68296-1；23-PICONEGRO::IRGC 117022-1；24-NCS 130::IRGC 51879-1；25-
YAKADA::IRGC 51096-1；26-GBUAPU 1::IRGC 63265-1；27-MANDRIRAVINA::IRGC 69960-1；
28-BAKUNG(H)::IRGC 60220-1；29-ZALCHA::IRGC 62190-1；30-PAWHTUN::IRGC 33562-1；
31-RATHKANDIRAM::IRGC 36507-1；32-PURBIA(KALANSAR)::IRGC 59189-1；33-SUDUWEE::
IRGC 8972-1；34-IR 31917-45-3-2::IRGC 78132-1.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
In following example use instrument, reagent, test kit all can be by being commercially available；RM593 is described in embodiment
Molecular marker.
Embodiment 1: primer screening
It is loaded in www.genomene.org under SSR primer；RM593SSR primer sequence is as shown in table 1.The sequence of SSR in table 6
Row itself come from disclosed Internet resources.
Table 1 RM593 sequence
Embodiment 2: CTAB method extracts the STb gene of Oryza sativa L., and step is as follows:
(1) each material weighs the fresh blade of 0.4-0.5g, adds liquid nitrogen and grinds rapidly, then turn in mortar
Enter in the sterile centrifugation tube of 2.0mLA；Dry substance is the most first pulverized with destructor, and takes powder；
(2) often pipe adds extract with CTAB buffer (PH8.0) 700 μ L, 65 DEG C of water-bath 45min of preheating 65 DEG C, every
5min takes out and shakes up 1 time；
(3) adding soft the mixing of 700 μ L chloroforms/isoamyl alcohol (24:1) to shake 8 minutes, then 12000r/min is centrifuged 10min；
(4) take in the sterile centrifugation tube that supernatant proceeds to another 1.5mL, add the isopropanol of 600 μ L pre-coolings, reverse mixed
Even, it is placed in-20 DEG C of more than refrigerator freezing 30min；
(5) centrifuge tube is centrifuged 10min with 12000r/min, takes precipitation；
(6) dehydrated alcohol rinses 1 time, after being centrifuged, carefully removes ethanol；
(7) add aquesterilisa after drying or air-dry to dissolve, make final concentration reach 20ng/ μ L, standby.
The formula pH=8.0 of table 2 extract with CTAB liquid (1000mL)
Table 3 PCR reaction system
Then using the ETC-811PCR amplification instrument of east KingMax new company, 15 μ L PCR reaction systems, amplification program is:
Denaturation, 95 DEG C, 5min；
Degeneration, 95 DEG C, 40 seconds；
Annealing, 55 DEG C, 30 seconds；
Extend, 72 DEG C, 50 seconds；
Repeat 2-4 step, 35 circulations；
72 DEG C extend 10min, product 4 DEG C preservation.
PCR primer adds 2% bromophenol blue 3 μ L, 95 DEG C of degeneration 5min, and the polyacrylamide gel electrophoresis of 8%, with reference to Panaud
The aobvious technology of quickly dye improved Deng (1996) silver staining and coloration method dyes and reads tape, according to the big small records of PCR primer
Labelling.
8% acrylamide denatured sol solution is configured by the acrylamide mother solution (4 DEG C of preservations) of 40%, formula such as following table
4, table 5 and table 6.
Table 4 40% acrylamide mother solution
Table 55 × tbe buffer liquid
The acrylamide denatured solution of table 6 8%
Electrophoresis step:
Add 1 × TBE electrophoretic buffer 700ml, insert 48 hole combs, point sample 1 μ L, invariable power 80W, electrophoresis 45min.
Colouring method (express method):
After amplified production separates with 8% polyacrylamide denaturing gel electrophoresis, colouring method and step are as follows:
Dyeing 8-12min (1.5g Han silver nitrate, ethanol 100mL in 1200mL dye liquor)；
Deionized water rinses rapidly the 5-10 second；
Development 10min (1300mL shows the 11g Han sodium hydroxide in liquid, the formaldehyde 6mL of 37%)；
Take out after deionized water rinsing 3-5min and dry.
7 34 parts of Core Germplasms information of table and setting percentage
As shown in table 7, utilize SSR primer that the QTL that setting percentage is relevant is detected, one and setting percentage height detected
Relevant QTL is positioned on No. 5 chromosomes at 31.4cM, and relevant SSR primer is RM593, and this QTL contribution rate is 24.6%.Right
34 parts of rice varieties being all from national genebank carry out Seed-Setting Percentage in Rice qualification by the method in example, and carry out SSR marker
Analyze.Interpretation of result, what the genotype of Core Germplasms was the same with Japonica is designated as A, and genotype is as high setting percentage parent
Be designated as B.Setting percentage kind more than 95% has 14 parts, and wherein 13 parts is the banding pattern of high setting percentage, accounts for whole high setting percentage
Planting the 92.85% of matter, setting percentage kind below 80% has 17 parts, and wherein 9 parts is Japonica banding pattern (with reference to Fig. 1), accounts for
The 52.94% of whole low setting percentage kind matter.
In sum, on No. 5 chromosomes of the Oryza sativa L. of the present invention, the SSR primer mutually chain with setting percentage can be relatively to RM593
Distinguish the kind that under same environmental condition, setting percentage height is extreme significantly.
The aforementioned description to the specific illustrative embodiment of the present invention illustrates that and the purpose of illustration.These describe
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, can much change
And change.The purpose selected exemplary embodiment and describe is to explain that the certain principles of the present invention and reality thereof should
With so that those skilled in the art be capable of and utilize the present invention various different exemplary and
Various different selections and change.The scope of the present invention is intended to be limited by claims and equivalents thereof.
Claims (3)
1. SSR primer pair mutually chain with setting percentage on No. 5 chromosomes of an Oryza sativa L., it is characterised in that described primer pair
Forward primer is the sequence shown in Seq ID NO.1, and the downstream primer of described primer pair is the sequence shown in Seq ID NO.2.
2. SSR primer mutually chain with setting percentage on No. 5 chromosomes of the Oryza sativa L. described in claim 1 is to being used for whether screening Oryza sativa L.
The method carrying high setting percentage gene, it is characterised in that include step: with oryza sativa genomic dna to be detected as template, with power
Profit requires that the SSR primer described in 1, to carrying out PCR amplification, contrasts with the band of the Japonica of low setting percentage, thus identifies
Whether material contains the related gene of high setting percentage.
3. SSR primer mutually chain with setting percentage on No. 5 chromosomes of the Oryza sativa L. described in claim 1 is in Oryza sativa L. assistant breeding
Purposes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880671A (en) * | 2010-05-27 | 2010-11-10 | 华中农业大学 | Cloning and application of major gene GS5 capable of controlling width and weight of rice grain |
| CN102010464A (en) * | 2010-08-26 | 2011-04-13 | 浙江大学 | Rice phosphorus absorption and transfer regulator gene OsPHF1 and application thereof |
-
2016
- 2016-08-29 CN CN201610753276.6A patent/CN106148543A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880671A (en) * | 2010-05-27 | 2010-11-10 | 华中农业大学 | Cloning and application of major gene GS5 capable of controlling width and weight of rice grain |
| CN102010464A (en) * | 2010-08-26 | 2011-04-13 | 浙江大学 | Rice phosphorus absorption and transfer regulator gene OsPHF1 and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| SUSAN R. MCCOUCH,ET AL: "Development and Mapping of 2240 New SSR Markers for Rice", 《DNA RESEARCH》 * |
| 潘英华等: "利用野栽杂交分离群体定位水稻结实率QTLs", 《西南农业学报》 * |
| 鄢宝: "稻米粒重和品质性状的遗传分析", 《中国优秀硕士学位论文全文数据库》 * |
| 韩龙植等: "不同生长环境下水稻结实率数量性状位点的检测", 《作物学报》 * |
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Application publication date: 20161123 |