CN109644863A - Oat Recessive Male sterility molecular labeling and application - Google Patents
Oat Recessive Male sterility molecular labeling and application Download PDFInfo
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- 238000011835 investigation Methods 0.000 claims description 3
- 101100236238 Arabidopsis thaliana CAMS1 gene Proteins 0.000 claims description 2
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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- Genetics & Genomics (AREA)
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- Environmental Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the production method of oat recessive gms line RI2014 and the purposes of oat recessiveness line with genic sterile RI2014, and the molecule labelling method of oat recessive gms line RI2014 sterile gene, primer PCR, primer numbers CL1155, CL907 are carried out to RIS2014 oat recessive gms line RI2014, CL210, Uni2358, Uni2612, CL248, Uni2008 will control the assignment of genes gene mapping of RI2014S infertility line fertility character in chromosome 23.0Mb section.Mutant RI2014 disclosed by the invention itself has highly yielding ability, acts not only as sterile line and saves artificial emasculation to hybridize part cumbersome, and is also equipped with Parents function in breeding process.Use the molecule labelling method of oat recessive gms line RI2014 sterile gene disclosed by the invention, in the identification of plant, the genetic background of kind, screening target single plant, there is important application value in terms of breed improvement and yield forming and Clonal differentiation control.
Description
Technical field
The invention belongs to oat breeding technical field more particularly to a kind of oat recessive gms line gene molecule marker and
Using.
Background technique
Fertility is the important character of oat, is had great significance on oat industry.The research of Male sterility of oats is most
Earlier than the 1980s, China has found two kinds of oat infertility types: Avena stivai recessive cytoblast sterile and naked oats at present
Dominant genic male sterile, the external discovery about Male sterility of oats have not been reported.The recessive sterile classical genetics research of oat core
Show that sterile character is controlled by a pair of of recessive nuclear gene, which is CA male sterility (CAMS).CAMS is mainly used for swallow
The multiple uses such as preparation, circulation breeding, population improvement and the hybrid seeding of wheat cross combination.Since oat has complicated and Pang
Big genome (heterologous 6 times of bodies, divide A, C, D tri- groups, 6n=42), not yet progress gene order-checking, about oat gene group analysis
Positioning with important gene is carried out by molecular marking technique.
Male sterility is a kind of approach of a kind of generally existing biological phenomena and heterosis utilization in plant.
Male plant system infertility refers to a kind of male reproductive organ dysplasia or degenerates (mainly anther or pollen are degenerated), but female
The normal maternal oat material of sexual reproduction allelotaxis, since anther or pollen early development cause non-pollen type or without life extremely
Vigor pollen, it is solid to be unable to self-pollination, only could fertilization by foreign pollen.Avena self-pollinated plant is
Indefinite inflorescence is bloomed in 2-3d after general heading with heading with blooming.It blooms and is from top to base portion development, male and female when blooming
Stamen is mature simultaneously, and the heading of general single plant is bloomed to be completed within 1 week.It is following to start top when heading is bloomed at the top of single plant oat
Pistil And Stamen be also in the stage of development, and the stage of development more locating is more forward.Pollination is started out in flower flower glume
Start when putting or before open, so natural hybrization rate is extremely low, only a ten thousandth.Therefore this stealthy Genetic Sterility oat conduct
Genetic tool can generate a large amount of hybrid seeds by the method for artificial supplementary pollination, be the most successful hereditary work of hybridization oat
Tool.
Farther out, there is huge Breeding Potential, hybrid F for interspecific hybridization for affiliation between Avena stivai and naked oats1
Yield, quality and in terms of have very strong hybrid vigour.However, since the naked hybrid vigour of skin is in practical application
Very big difficulty is encountered, including a series of problems such as flowering asynchronism, setting percentage be low, wherein main is the naked hybrid F of skin1For setting percentage
Lower, the setting percentage of most hybrids is between 5%-50%.
Skin, naked oats interspecific hybridization, the Kernel trait of hybrid generation show as blending heredity.Plant fringe portion exists simultaneously
Naked grain shape and band batch two class seed of type, the precedence kissing of distribution and oat ear differentiation of the naked grain shape seed on fruit ear
It closes.The seed that the base portion little Hua of photosynthate more sufficient fringe top small ear and side shoot top small ear is generated is obtained, is formed naked
Grain shape seed, and the small ear multiform of fruit ear middle and lower part especially side shoot lower part is at band prisoner's type seed.From F2Start to selected list
Strain carries out isolation threshing, and selecting naked grain shape seed is, by for continuous single plant grain-by-grain seed selection, to eliminate full skin class as follow-on selection
Single plant more than type or band batch seed, selfing is homozygous, and until stablizing, character, which is stablized, generally needs the even longer time in 8 generations, takes into account target
General performance of the character on the single plant more than naked or bare particle grain stablizes heredity until selecting ideal strain and showing.
Molecular labeling is the genetic marker based on inhereditary material inner nucleotide sequence variations, is DNA level
The direct reflection of genetic polymorphism.The molecular labeling of broad sense refers to heritable and detectable DNA sequence dna or protein, narrow sense
Molecular labeling refer to the characteristic DNA fragmentation that can reflect between bion or population the specificity of certain species diversity in genome.With
The development of molecular marking technique, the molecular labeling with the oat infertility linkage of characters is obtained, to the something lost of the identification of plant, kind
Pass background, screening target single plant, kind improvement, in terms of have important application value.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides the productions of oat recessive gms line RI2014
The molecule mark of the purposes and oat recessive gms line RI2014 sterile gene of method and oat recessive gms line RI2014
Note method, including the application in recurrent selection.
The present invention is implemented as follows: by the stealthy Genetic Sterility Avena stivai " CAMS " of original discovery and naked oats " 80066 "
Hybridization, cenospecies obtain F by a selfing generation2In generation, selects naked oats and naked oats " 80066 " backcrossing, a selfing generation of infertility
Obtain BC1F2, select infertility naked oats and naked oats " 80066 " continue backcrossing, selfing a generation obtain BC2F2, select infertility
Backcrossing is continued in naked oats and naked oats " 80066 ", a selfing generation obtains BC3F2, recombinant inbred lines is constructed by single seed descent,
F12The naked oats sterile line RI 2014 that generation screening obtains.
A kind of molecule labelling method of oat recessive gms line RI2014 sterile gene
(1) using oat varieties product swallow No. 2 as male parent, using obtained infertility single plant RI2014S as female parent, hybridization obtains F1
In generation, utilizes F1A generation selfing generation obtains the F of Fertility segregation2Group is shown: sterile single plant: fertile single plant by the investigation of field character
Segregation ratio is 1:3,
(2) F is extracted2Group's single plant total DNA takes the total DNA mixed in equal amounts of 10 plants of fertile single plants as fertile pond template, takes 10 plants
The total DNA mixed in equal amounts of sterile single plant is used as sterile pond template,
(3) 2073 pairs of marks of transcript profile sequencing exploitation are carried out using the near isogenic lines CAMS1 of Avena stivai recessive cytoblast sterile plant
It is denoted as carrying out PCR amplification for primer,
(4) resulting amplified production is detected with polyacrylamide gel, screening obtains polymorphism mark；With the polymorphism of acquisition
Labeled primer 7 is right, primer numbers CL1155, CL907, CL210, Uni2358, Uni2612, CL248, Uni2008,
(5) with F2Group's whole single plant is that template carries out PCR amplification, and products therefrom is detected using polyacrylamid gel, can
What is expanded is the single plant containing sterile gene, in conjunction with single plant phenotype, will control the base of RI2014 infertility line fertility character
Because being positioned in chromosome 23.0Mb section.
The DNA sequence dna of the primer are as follows:
CL1155. GACATTGTCAAGACCTGTAAGCC；GCTCATGCCCTAACATTACAACT；
CL907. CTCATGTGAATGGAAGCCTGTAT；AACAAAGACTTCGGTGCATTCT；
CL210. TCTTGGAAGGTCTGTTCTCTGAC；AAGAAGATAGAAGCCAAATCCGA；
Uni2358. TGAGAACACAATTGCTCAAAATG；CTGAAGTTTGGAGAACCAGAAGT；
Uni2612. CTTCCTCTTCATGCCATTGTTAT； CTCCATGGCAACAAACACTTAAC；
CL248. GGAATGCCATAACATGTACACAA；CCGACAGGATACACTGAGCG；
Uni2008. CTCTTCTTCCTCTGCTCCTTCTT；CTTTCTGAGCAAGCACCATTAGT.
Number is CL1155, CL907, CL210, Uni2358, Uni2612, the primers DNA sequences of CL248, Uni2008
First half is forward primer, and the latter half of DNA sequence dna is reverse primer.
Make female parent with the sterile single plant in sterile line RI 2014S, it is miscellaneous to make male parent pollination respectively with Avena stivai and naked oats
It hands over, obtains F1, observe F1Setting percentage；
New varieties, Shen are cultivated as the intermediate materials of recurrent selection using with the sterile line for being accredited as stealthy male nuclear sterile gene
It asks someone to provide application method of the Male sterility of oats system RI2014 in oat recurrent breeding, the specific steps of which are as follows:
1) the excellent breeding parent material of character number 1,2,3,4 respectively will be accredited as, parent material 1 is hybridized to obtain with 2
F1A, hybridize parent material 3 to obtain F with 41B；
2) with F obtained in step 1)1AAnd F1BMake male parent respectively, is hybridized with RI 2014S sterile line as female parent, wherein F1A
Hybridize to obtain F with 2014 sterile line of RI1C, F is obtained by being selfed a generation2C；F1BHybridize to obtain F with RI 2014S sterile line1D, lead to
It crosses a selfing generation and obtains F2D;
3) with F2CIn sterile single plant as maternal, with F1BF is obtained for paternal hybrid1, by F1A selfing generation obtains F2, test F2
Fertility, obtain heterozygosis strain, strain self progeny screens to heterozygosis, the moderate heterozygosis strain of plant height, florescence is obtained, to miscellaneous
In conjunction strain selfing 8-9 generation, obtains the stable kind of performance.
It is also possible that with F2DIn sterile single plant as maternal, with F1AF is obtained for paternal hybrid1, by F1A selfing generation obtains
F2, test F2Fertility, obtain heterozygosis strain, strain self progeny screens to heterozygosis, obtains plant height, florescence moderate miscellaneous
Strain is closed, the stable kind of performance is obtained to heterozygosis strain selfing 8-9 generation.
Select F2CAnd F2DThe excellent single plant conducive to breeding of middle phenotype utilizes above-mentioned oat recessive gms line RI2014
The molecule labelling method identification sterile single plant therein of sterile gene and fertile single plant.
The beneficial effects of the present invention are: utilizing the stealthy Genetic Sterility Avena stivai " CAMS " of original discovery and naked oats
" 80066 " hybridization building recombinant inbred lines, screens the self-mating system of fertility feature, cultivates the sterile line RI2014 of screening, the infertility
The intermediate materials advantage that system can be used for oat recurrent breeding is: 1) sterility of male sterile line is allogene control, no
Light is not long under normal growing conditions and temperature influences for fertility, is oat ideal recurrent selection intermediate materials.2) sterile line
Its genome of RI2014 is the mixture of Avena stivai and naked oats, is hybridized with skin, naked oats, and cenospecies is fertile.3) sterile
Be Nms2014 small ear upward, have highly yielding ability, the parent of excellent variety can be, remove from artificial emasculation hybridization process, improve it is miscellaneous
Seed production efficiency is handed over, manpower is saved, seed costs is reduced, while providing hybrid seeding efficiency, seed purity, guarantees seed production quality
One effectively approach.4) male sterility gene CAMS of the invention is a new male sterility gene, can be convenient and efficient
Identify Sterile plants and fertile plants in oat crossbreeding in seedling stage, the prior art is overcome to need to identify until plant heading stage
Fertility and the problem of expend a large amount of human and material resources and time.5) present invention includes CL1155, CL907, CL210, Uni2358,
Uni2612, CL248 and Uni2008, seven molecular labelings can position and mark Male sterility of oats with the oat linkage of characters
Gene C AMS connects genomic dna sequence and oat fertile gene, in the identification of plant, the genetic background of kind, sieve
Target single plant is selected, has important application value in terms of breed improvement and yield forming and Clonal differentiation control.
Detailed description of the invention
Genetic linkage map between Fig. 1 RI2014 sterile gene and molecular marked compound of the present invention；
Fig. 2 is the building flow chart of mutant RI2014 of the invention；
Fig. 3 is applicating flow chart of the mutant RI2014 of the invention separated in oat recurrent breeding.
Specific embodiment
In order to be more clearly understood that technical solution of the present invention, the following further describes the present invention with reference to the drawings.
Embodiment 1: building recombinant inbred lines obtains sterile line.
Technology path according to Fig.2, by the stealthy Genetic Sterility Avena stivai " CAMS " of original discovery and naked oats
" 80066 " hybridization obtains F1, utilize F1It is selfed a generation and obtains F2Generation, from F2In generation, chooses naked oats and naked oats " 80066 " of infertility
Backcrossing obtains BC1F1, it is selfed a generation and obtains BC1F2, from BC1F2The naked oats of selection infertility continue to be returned with naked oats " 80066 "
Obtain BC2F1, it is selfed a generation and obtains BC2F2, from BC2F2The naked oats of middle selection infertility and naked oats " 80066 " continue to be returned
To BC3F1, obtain in single plant fringe be entirely naked oats material, selfing a generation obtain BC3F2, utilize BC3F2In single plant fringe all
It is that the fertile individual plant selfings of heterozygosis of naked oats obtains BC3F3, BC3F3In each family select the selfing of seed, pass through single seed descent
Recombinant inbred lines is constructed, in F12The naked oats sterile line RI2014 that generation screening obtains.
2073 pairs of labels that RI2014S is developed with transcript profile, winter in 2014 innovate in Chinese Taiyuan Shanxi academy of agricultural sciences
In greenhouse, female parent is made with RI2014 infertility single plant RI2014S, with three naked oats kinds: Bai Yan 2, Jin Yan 8 and dam oats 1
Number make male parent is hybridized respectively, with three Avena stivai kinds: cover swallow No. 1, dam swallow No. 6 and swallow No. 4 are made male parent and hybridized respectively,
Obtain F1Seed, by obtained F1Seed sowing was tested in Chinese Taiyuan Shanxi academy of agricultural sciences innovation greenhouse in sowing in 2015,
Harvest seed, investigate setting percentage, obtain the result that it is as shown in the table, it was demonstrated that the sterile single plant RI2014S of sterile line RI2014 with not
The F that same Avena stivai, naked oats hybridize1It is fertile.
The F that the sterile single plant RI2014S of table sterile line RI2014 of the invention hybridizes from different naked oats Avena stivais1
Setting percentage
。
Embodiment 2: target group
The sterile single plant RI2014S of mutant RI2014 is hybridized with oat varieties " product swallow No. 2 ", obtains F from a selfing generation2
Segregating population, for carrying out the positioning of male sterility gene.Shown by the investigation of field character: sterile single plant: fertile single plant
Segregation ratio is 1:3, and sterile single plant phenotype goes out that stamen development is abnormal or stamen is degenerated completely, is detailed in other testimonial materials photograph
Piece.
Embodiment 3: Parent and F2For the extraction of genes of individuals group DNA
Male parent is stamen and the normotrophic single plant of gynoecium, and female parent is that Stamen development is normal, the abnormal single plant of stamen development.
Extract Parent, 173 F of target group respectively with RNA isolation kit2The genomic DNA of individual, specific method
It is as follows.
(1) it by 1.0 grams of fresh blades, shreds in the mortar for being put into and liquid nitrogen being added, after liquid nitrogen grinding, is used after ground
Small spoon scoops in centrifuge tube cooling in liquid nitrogen in advance, and the name of ground sample is marked on centrifuge tube, by centrifuge tube
It has been put in the ice chest of liquid nitrogen.
(2) the sample of required grinding it is all ground after, extracted by the specification step in kit.
DNA method for extracting is extracted according to RNA isolation kit, and described kit can be bought by market and be obtained, and the invention patent is real
It applies the plant gene DNA that example uses TIANGEN Biotech (Beijing) Co., Ltd. to produce and extracts box (DP350-03) reagent
Box；
1) experiment before mercaptoethanol is added in the GP1 of preheating, make its final concentration of 0.1%；In buffer GD and rinsing liquid PW
Dehydrated alcohol is added；
2) ground powder is quickly transferred in the centrifuge tube for being pre-loaded with 65 DEG C of 700 ul preheating buffer GP1, rapidly
After being mixed by inversion, centrifuge tube is placed on 65 DEG C of 20 min of water-bath, reverse centrifuge tube is during water-bath to mix sample for several times；
3) 700 μ l chloroforms are added, mix well, 12,000 rpm (~ 13,400 × g) are centrifuged 5 min；
4) carefully upper strata aqueous phase obtained by previous step is transferred in a new centrifuge tube, 700 μ l buffer GP2 is added, sufficiently
It mixes；
5) liquid of mixing is transferred in adsorption column CB3,12,000 rpm (~ 13,400 × g) are centrifuged 30 sec, discard useless
Liquid.(absorption column volume is 700 μ l or so, and centrifugation is added in graded)；
6) 500 μ l buffer GD(be added into adsorption column CB3 please first checked whether before and dehydrated alcohol has been added), 12,
000 rpm (~ 13,400 × g) is centrifuged 30 sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe；
7) 600 μ l rinsing liquid PW(be added into adsorption column CB3 please first checked whether before and dehydrated alcohol has been added), 12,
000 rpm (~ 13,400 × g) is centrifuged 30 sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe；
8) repetitive operation step 7.
9) adsorption column CB3 is put back in collecting pipe, 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, outwell waste liquid.It will inhale
Attached column CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
10) adsorption column CB3 is transferred in a clean centrifuge tube, 50-200 μ l is vacantly added dropwise to the intermediate position of adsorbed film
Elution buffer TE, is placed at room temperature for 2-5 min, and 12,000 rpm (~ 13,400 × g) are centrifuged 2 min, by solution be collected into from
In heart pipe；
Buffer GP1(Buffer GP1)
Buffer GP2(Buffer GP2)
Buffer GD(Buffer GD)
Rinsing liquid PW(Buffer PW)
Elution buffer TE(Buffer TE)
Adsorption column CB3(Spin Columns CB3)
Collecting pipe (2 ml) (2 ml of Collection Tubes).
(3) genomic DNA is detected with 0.8% Ago-Gel.
(4) by obtained Parent and F2The genomic DNA in generation is template, with molecular labeling amplimer to expanding
Increase.
CL1155. GACATTGTCAAGACCTGTAAGCC；GCTCATGCCCTAACATTACAACT；
CL907. CTCATGTGAATGGAAGCCTGTAT；AACAAAGACTTCGGTGCATTCT；
CL210. TCTTGGAAGGTCTGTTCTCTGAC；AAGAAGATAGAAGCCAAATCCGA；
Uni2358. TGAGAACACAATTGCTCAAAATG; CTGAAGTTTGGAGAACCAGAAGT；
Uni2612. CTTCCTCTTCATGCCATTGTTAT; CTCCATGGCAACAAACACTTAAC；
CL248. GGAATGCCATAACATGTACACAA; CCGACAGGATACACTGAGCG；
Uni2008. CTCTTCTTCCTCTGCTCCTTCTT; CTTTCTGAGCAAGCACCATTAGT。
Embodiment 4: the preparation of molecular labeling
With the Parent or F extracted in embodiment 32The genomic DNA in generation is template, with molecular labeling amplimer pair
(CL1155, CL907, CL210, Uni2358, Uni2612, CL248 and Uni2008) carries out PCR amplification.
PCR reaction system is as follows:
0.8 μ L of forward primer
0.8 μ L of reverse primer
1 μ L of DNA profiling
ddH2O 2.4µL
MIX 5µL
PCR response procedures are as follows:
94 DEG C of initial denaturation 120s；94 DEG C of denaturation 30s, 57 DEG C of annealing 45s, 72 DEG C of extension 40s run 35 circulations；Last 72 DEG C
Extend 600s.Pcr amplification product can be saved at 4 DEG C.
PCR product electrophoresis detection: polyacrylamide gel electrophoresis according to glue and records.
The validity and polymorphism of primer: refer to whether there is amplified production in this validity, polymorphism refers to that Parent is shown in
The clip size of amplified production is variant.
The screening of primer: Parent and F is carried out according to following screening criteria1There is amplified production, and Parent expands
Volume increase object has clearly banding pattern and position is variant, F1The heterozygosis banding pattern for showing as Parent banding pattern, that is, have male parent and female parent
Two bands of banding pattern.
The selection result filters out 7 pairs of primers from 2073 pairs of primers of design according to appeal screening criteria.
Genetic map construction
Have the molecular labeling of polymorphism to F with 7 pairs of primers of exploitation2173 individuals of group carry out PCR detection, used
Template is F obtained2173 individual genomic DNAs of group.
Agarose gel electrophoresis is carried out to PCR product, obtains molecular labeling primer to the result of 173 individual amplifications.
Whole electrophoresis results are subjected to data statistic analysis, the specific method is as follows: by F2Group's single plant amplified band is father
This type is denoted as a, and amplified band is that maternal type is denoted as b, and amplified band is denoted as h, band pattern containing male parent type and maternal type simultaneously
Paste or missing be denoted as-, be equivalent to shortage of data, finally obtain F2Gene of the 173 individual XX of group to primer amplification
Type data.For example, be a with 173 individual data that pair of primers obtains, a, h, b ... totally 173 data are used
173 individual data that second pair of primer obtains are b, a, h, b ... totally 173 data ... totally 173 numbers
According to totally 7 pairs of primers count respectively, and gained is both F2The genotype data of group.
With 3.0 software of MapMaker, genetic linkage maps drafting is carried out, genetic linkage map is obtained.The heredity obtained from this
It can determine the position of 7 pairs of primers and the genetic distance with oat fertile gene on linkage map.
The assignment of genes gene mapping
It is similar with male parent type character to be denoted as a according to 173 individual fertility phenotypes, it is similar with maternal character to be denoted as b.?
The phenotypic data individual to 173, by 173 individual phenotypic datas and 173 individual genotype datas before obtaining
It is compared, similar Gao Ze represents the label and fertility character close linkage, and fertile gene is located in genetic linkage maps
On.
Pollen activity detection inside anther:
F2In single plant, fertile single plant has pollen in early flowering season, full-bloom stage, latter stage of blooming；Sterile single plant is in early flowering season, full blossom
Phase, the latter stage of blooming are without pollen.Scanning electron microscopic observation anther tabletting shows and (is detailed in other testimonial material photos), F2Sterile plant flower
Medicine is shrivelled, and the pollen grain of surface corrugationless, inner elliptical shape is seldom, there is mature flower powder, but its form deformity in individual anther
It is inactive;Fertile plant anther is full, and surface folding is more, and inside is full of by oval pollen, fertile pollen ellipse, and activity is good
It is good.
The F of sterile plant and different cultivars test crosslGeneration, fertility restorer；Fertile plant and infertility after the plant selfing of recovery fertility
Strain is separated in 3:1；Sterile plant and the F for restoring fertilitylBackcrossing, BC1Fertility is 1:1 separation.Illustrate that sterile character is by a pair of recessive
Karyogene control.
Embodiment 5: the application of sterile line RI2014
Sterile line of the invention hybridizes with Avena stivai, naked oats and the higher F of setting percentage can be obtained1, offspring F1、F2In not the same year
Part is in crop field or chamber planting, F1The fertility performance of plant is all fertile, F2No matter in greenhouse or crop field and different year, group
In occur some sterile plants always.Sterile line infertility character of the invention is stablized, and the factors such as temperature and illumination are unable to shadow in environment
Its Sterility variation is rung, temperature, photosensitive infertility are not belonging to, there is highly yielding ability, the step of when hybridization can remove " artificial emasculation " from, seedling stage
Identification can use the label that the present invention develops and carry out assisted Selection, therefore the sterile line obtains Optimality to oat hybridization is provided
Shape provides critically important intermediate materials.
The technology path of this implementation can be found in Fig. 3, and Fig. 3 is applying including sterile line RI2014 of the present invention for applicant's design
In the general technical process of oat breeding: selection Avena stivai and each two key variety (these of naked oats good breeding parent first
A bit " key variety " be in production with merit quality, be all it is available, for those skilled in the art
For according to general standard be selected as " key variety " of this example not suffering, it is clear to describe, applicant by these
" key variety " number is " key variety 1,2,3,4 " respectively, is to refer to different kinds, but be all derived from high-quality and high
The material of product kind), number key variety 1,2,3,4, the wherein pairing of key variety 1 and 2 uses, and the pairing of key variety 3 and 4 makes
With, according to conventional hybridization method respectively obtain Avena stivai hybridization F1(or be F1A), naked oats kind F1(or be F1B)；With F1A
And F1BRespectively as male parent, using sterile line single plant RI2014S of the invention as female parent, with F1AHybridize with RI2014S, obtains
F1C, with F1BHybridize with RI2014S, obtains F1D；A selfing generation obtains F respectively again later2CAnd F2D；Select F2CAnd F2DMiddle phenotype
The excellent single plant conducive to breeding identifies sterile single plant therein and fertile single plant using the labeled primer that the present invention develops, point
Not with F2CIn sterile single plant be female parent, use F2DIn the pollen of fertile single plant pollinate, while with F2DIn sterile single plant
For female parent, F is used2CIn the pollen of fertile single plant pollinate, obtain new F1N；By new F1NSelfing, from F2In generation, starts, real
Conventional selection breeding (method that conventional selection breeding method is report) is applied, the fertile single plant (selection for selecting economical character excellent
The method of the excellent fertile single plant of economical character is the method for report), by being continuously selfed and selecting (the continuous side being selfed and select
Method is the method for report), finally obtain that the florescence is stable, plant type is moderate, the stable oat new lines of yield high yield.
Another: RI2014 described in the present invention is group's general name with oat sterile gene, and RI2014S has oat
The appellation of sterile gene single plant is numbered.
The above description is only a preferred embodiment of the present invention, thus it is all according to the configuration described in the scope of the patent application of the present invention,
The equivalent change or modification that feature and principle are done, is included in the scope of the patent application of the present invention.
Sequence table
<110>Crop Variety Resource Inst., Shanxi Prov. Academy of Agriculture Sciences
<120>oat Recessive Male sterility molecular labeling and application
<130> 2018001
<141> 2019-01-11
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gacattgtca agacctgtaa gcc 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gctcatgccc taacattaca act 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctcatgtgaa tggaagcctg tat 23
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aacaaagact tcggtgcatt ct 22
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tcttggaagg tctgttctct gac 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aagaagatag aagccaaatc cga 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tgagaacaca attgctcaaa atg 23
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ctgaagtttg gagaaccaga agt 23
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cttcctcttc atgccattgt tat 23
<210> 10
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctccatggca acaaacactt aac 23
<210> 11
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ggaatgccat aacatgtaca caa 23
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ccgacaggat acactgagcg 20
<210> 13
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ctcttcttcc tctgctcctt ctt 23
<210> 14
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ctttctgagc aagcaccatt agt 23
Claims (7)
1. a kind of production method of oat recessive gms line RI2014, it is characterised in that: the following steps are included:
The stealthy Genetic Sterility Avena stivai " CAMS " of original discovery and naked oats " 80066 " are hybridized, cenospecies passes through a selfing generation
Obtain F2In generation, selects the naked oats of infertility and naked oats " 80066 " backcrossing, a selfing generation to obtain BC1F2, select the naked swallow of infertility
Backcrossing is continued in wheat and naked oats " 80066 ", a selfing generation obtains BC2F2, select infertility naked oats and naked oats " 80066 " after
Continuous backcrossing, a selfing generation obtain BC3F2, recombinant inbred lines is constructed by single seed descent, in F12The naked oats that generation screening obtains are not
Educating is RI 2014.
2. the purposes of oat recessive gms line RI2014 described in claim 1 a kind of, which is characterized in that oat recessiveness core is not
Educating is F that RI2014 hybridizes from different Avena stivais, naked oats1It is fertile.
3. a kind of molecule labelling method of oat recessive gms line RI2014 sterile gene, it is characterised in that: be with number
CL1155, CL907, CL210, Uni2358, Uni2612, CL248, Uni20087 carry out PCR amplification, gained are produced to primer
Object is detected using polyacrylamid gel, and be able to carry out amplification is that the single plant containing sterile gene will be controlled in conjunction with single plant phenotype
The assignment of genes gene mapping of RI2014S infertility line fertility character processed in chromosome 23.0Mb section, the number be CL1155 just
To primer sequence as shown in SEQ ID NO:1 in sequence table, the reverse primer sequences that the number is CL1155 such as sequence table
Shown in middle SEQ ID NO:2, the forward primer sequence that the number is CL907 is as shown in SEQ ID NO:3 in sequence table, institute
The reverse primer sequences that the number stated is CL907 as shown in SEQ ID NO:4 in sequence table, the number be CL210 just
To primer sequence as shown in SEQ ID NO:5 in sequence table, the number is in the reverse primer sequences such as sequence table of CL210
Shown in SEQ ID NO:6, the forward primer sequence that the number is Uni2358 is as shown in SEQ ID NO:7 in sequence table, institute
For the reverse primer sequences that the number stated is Uni2358 as shown in SEQ ID NO:8 in sequence table, the number is Uni2612
Forward primer sequence as shown in SEQ ID NO:9 in sequence table, the reverse primer sequences that the number is Uni2612 such as sequence
In list shown in SEQ ID NO:10, SEQ ID NO:11 in the forward primer sequence such as sequence table that the number is CL248
Shown, as shown in SEQ ID NO:12 in sequence table, the number is the reverse primer sequences that the number is CL248
For the forward primer sequence of Uni20087 as shown in SEQ ID NO:13 in sequence table, the number is reversely drawing for Uni20087
Object sequence is as shown in SEQ ID NO:14 in sequence table.
4. a kind of molecule labelling method of oat recessive gms line RI2014 sterile gene according to claim 3,
It is characterized in that: specifically includes the following steps:
(1) using oat varieties product swallow No. 2 as male parent, using obtained infertility single plant RI2014S as female parent, hybridization obtains F1
In generation, utilizes F1A generation selfing generation obtains the F of Fertility segregation2Group is shown: sterile single plant: fertile list by the investigation of field character
Strain segregation ratio is 1:3,
(2) F is extracted2Group's single plant total DNA takes the total DNA mixed in equal amounts of 10 plants of fertile single plants as fertile pond template, takes 10 plants
The total DNA mixed in equal amounts of sterile single plant is used as sterile pond template,
(3) 2073 pairs of marks of transcript profile sequencing exploitation are carried out using the near isogenic lines CAMS1 of Avena stivai recessive cytoblast sterile plant
It is denoted as carrying out PCR amplification for primer,
(4) resulting amplified production is detected with polyacrylamide gel, screening obtains polymorphism mark；With the polymorphism of acquisition
Labeled primer 7 is right, primer numbers CL1155, CL907, CL210, Uni2358, Uni2612, CL248, Uni2008,
(5) with F2Group's whole single plant is that template carries out PCR amplification, products therefrom is detected using polyacrylamid gel, Neng Goujin
Row amplification is the single plant containing sterile gene, in conjunction with single plant phenotype, will control the gene of RI2014 infertility line fertility character
It is positioned in chromosome 23.0Mb section.
5. a kind of application method of Male sterility of oats system RI2014 in oat recurrent breeding, which is characterized in that its specific step
It is rapid as follows:
(1) the excellent breeding parent material of character number 1,2,3,4 respectively will be accredited as, parent material 1 is hybridized to obtain with 2
F1A, hybridize parent material 3 to obtain F with 41B；
(2) with F obtained in step (1)1AAnd F1BMake male parent respectively, is hybridized with 2014 sterile line of RI as female parent, wherein
F1AHybridize to obtain F with RI 2014S infertility single plant1C, F is obtained by being selfed a generation2C；F1BHybridize with RI 2014S infertility single plant
To F1D, F is obtained by being selfed a generation2D;
(3) with F2CIn sterile single plant as maternal, with F1BF is obtained for paternal hybrid1, by F1A selfing generation obtains F2, test F2
Fertility, obtain heterozygosis strain, strain self progeny screens to heterozygosis, the moderate heterozygosis strain of plant height, florescence is obtained, to miscellaneous
In conjunction strain selfing 8-9 generation, obtains the stable kind of performance.
6. a kind of application method of the Male sterility of oats system RI2014 according to claim 5 in oat recurrent breeding,
It is characterized in that, the step (3) is with F2DIn sterile single plant as maternal, with F1AF is obtained for paternal hybrid1, by F1From
A generation is handed over to obtain F2, test F2Fertility, obtain heterozygosis strain, strain self progeny screens to heterozygosis, obtains plant height, flower
Phase moderate heterozygosis strain obtains the stable kind of performance to heterozygosis strain selfing 8-9 generation.
7. a kind of application side of the Male sterility of oats system RI2014 according to claim 5 or 6 in oat recurrent breeding
Method, which is characterized in that selection F2CAnd F2DThe middle phenotype excellent single plant conducive to breeding utilizes claim when selecting sterile single plant
The molecule labelling method identification sterile single plant therein of oat recessive gms line RI2014 sterile gene described in 3 and fertile list
Strain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116411120A (en) * | 2023-03-30 | 2023-07-11 | 四川农业大学 | KASP molecular marker coseparated with oat nude gene N1 and application thereof |
| CN118956902A (en) * | 2024-10-17 | 2024-11-15 | 中国农业科学院北京畜牧兽医研究所 | A gene and molecular marker related to plant height traits in oats |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103688845A (en) * | 2013-11-27 | 2014-04-02 | 山西省农业科学院农作物品种资源研究所 | Breeding method for 50% male sterile line of oat |
| CN103688844A (en) * | 2013-11-27 | 2014-04-02 | 山西省农业科学院农作物品种资源研究所 | Recurrent breeding method for oat by using recessive genic male sterile strain |
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2019
- 2019-01-11 CN CN201910026024.7A patent/CN109644863A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103688845A (en) * | 2013-11-27 | 2014-04-02 | 山西省农业科学院农作物品种资源研究所 | Breeding method for 50% male sterile line of oat |
| CN103688844A (en) * | 2013-11-27 | 2014-04-02 | 山西省农业科学院农作物品种资源研究所 | Recurrent breeding method for oat by using recessive genic male sterile strain |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116411120A (en) * | 2023-03-30 | 2023-07-11 | 四川农业大学 | KASP molecular marker coseparated with oat nude gene N1 and application thereof |
| CN118956902A (en) * | 2024-10-17 | 2024-11-15 | 中国农业科学院北京畜牧兽医研究所 | A gene and molecular marker related to plant height traits in oats |
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