CN104285776B - The selection of male sterile line of maize - Google Patents
The selection of male sterile line of maize Download PDFInfo
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- CN104285776B CN104285776B CN201310751112.6A CN201310751112A CN104285776B CN 104285776 B CN104285776 B CN 104285776B CN 201310751112 A CN201310751112 A CN 201310751112A CN 104285776 B CN104285776 B CN 104285776B
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Abstract
The invention discloses the selection of male sterile line of maize.The invention provides a kind of selection of male sterile line of maize, for S Type Cytoplasmic Male-sterility in Maize system MD32CGMCC? No.8657 is donor, corn inbred line is acceptor, carries out backcross transformation, obtains the male sterile line of described corn inbred line.Experiment of the present invention proves, the present invention has following advantage: 1, utilize Molecular Marker Assisted Selection Technology and backcross transformation combine with technique, accelerates the transformation speed of male sterile line, only needs third backcross generation namely to obtain male sterile line S capital, capital 724 724.2, S type male sterile line MD32 sterility performance under multiple genetic background that the present invention selects is stablized thoroughly, in the corn hybrid seed male sterile production of hybrid seeds research reported, have no use.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of selection of male sterile line of maize.
Background technology
The selection and popularization of strong superior hybrid crosses is the important channel of improving corn yield.Utilize conventional producing method for seed preparing hybrid kind to need to carry out artificial emasculation to female parent, the not only labour of at substantial, increase seed produces cost, and exist due to emasculation not in time, thoroughly do not affect the potential risk of seed quality.The male sterile line production of hybrid seeds is utilized to be ensure that seed purity improves a kind of effective ways of corn yield.
As far back as the sixties in last century five, just start the research to the production of hybrid seeds of corn sterilization both at home and abroad.Cytoplasmic male sterility, owing to easily realizing the three series mating of male sterile line, maintainer and restorer, is the main path utilized in corn breeding.Cytoplasmic male sterile line can be divided into T, C and S type three major types.At the beginning of the sixties, T-shaped male sterile line introduces China.Owing to infecting corn southern leaf blight " T microspecies " specialization, the application of T-shaped male sterile line is severely limited.The seventies, China breeder introduces C type, S type male sterile line from external.Although C type male sterile line sterility is comparatively stable, thorough, but in special genotype background, show the postponement back mutation of fertility, and its restorer needs transformation again usually, the cycle is grown and complex steps, causes this type male sterile line to be difficult to popularization and application in practice.S type male sterile line is a group maximum in 3 types, there are some researches prove that the G. eurycarpa materials such as prosperous 7-2 are its natural strong restorers.But the fertility of S type male sterile line has larger difference because of the difference of genotype background, and under specific genetic background, fertility is highly stable.Male parent many genus Huang of domestic superior corn crossbreed replants matter, therefore for saving the transformation work of restorer, excavate and formulate new sterility thoroughly, the excellent S type male sterile line of inheritance stability, Comprehensive Traits is the key of quick sterilization production of hybrid seeds investigation and application.
The key realizing the mitochondrial DNA production of hybrid seeds is the seed selection of male sterile line, maintainer and restorer.The most frequently used method of seed selection male sterile line is backcross transformation method, namely making non-recurrent parent with stable male sterile line, select Elite inbred to make recurrent parent, carrying out many for backcrossing, be bred as the excellent sterile inbred line that sterility is stable, and former recurrent parent is the maintainer of this male sterile line.But only utilize traditional backcross transformation method, generally need backcross 5-6 generation, and the transformation cycle is longer, delayed the application of sterilization seeding technique on corn hybrid seed.Therefore, the seed selection speed how accelerating male sterile line is the problem that current male sterile seeding technique needs solution badly.
Summary of the invention
The object of this invention is to provide a kind of selection of male sterile line of maize.
Method provided by the invention, for be donor with S Type Cytoplasmic Male-sterility in Maize system MD32CGMCCNo.8657, corn inbred line for acceptor, carry out backcross transformation, obtain the male sterile line of described corn inbred line.
In said method, described corn inbred line is corn inbred line capital 724, and the male sterile obtained is the male sterile line S capital 724 in capital 724.
In said method, described in the number of times that backcrosses be 3 times.
In said method, described backcross transformation comprises the steps:
1) with S Type Cytoplasmic Male-sterility in Maize system MD32CGMCCNo.8657 be donor, corn inbred line capital 724 for acceptor, hybridization, obtain male sterile hybrid generation F
1;
2) with male sterile hybrid generation F
1for female parent, backcross with capital 724, obtain male sterile BC
1for colony;
3) with male sterile BC
1be female parent for individual plant, continue to backcross with capital 724, obtain male sterile BC
2for colony;
4) with male sterile BC
2be female parent for individual plant, continue to backcross with capital 724, obtain the male sterile line S capital 724 in capital 724.
In said method, in step 2) and step 3) between, also comprise the steps: from described male sterile BC
1for choosing in colony and the male sterile BC of described corn inbred line capital 724 genetic similarty at 92.5-95%
1for individual plant;
In said method, described from described male sterile BC
1for choosing in colony and the male sterile BC of described corn capital 724 genetic similarty at 92.5-95%
1method for individual plant comprises the steps: with 40 pairs of SSR core primers respectively to male sterile BC
1carrying out pcr amplification for individual plant and corn inbred line capital 724, obtaining SSR bands of a spectrum, by comparing BC
1for the SSR collection of illustrative plates in individual plant and corn inbred line capital 724, calculate genetic similarty;
Described genetic similarty computing formula: [1-(difference number of alleles/(2 × more total number of sites))] × 100%
Described difference number of alleles is to increase the described male sterile BC obtained with SSR core primers
1for the allelomorph number that the SSR bands of a spectrum banding pattern in individual plant SSR bands of a spectrum banding pattern and described corn inbred line capital 724 is inconsistent; Described more total number of sites is 40.
In above-mentioned 40 pairs of SSR core primers, the sequence of each primer is respectively in sequence table shown in sequence 1-80, specifically sees the table 2 of embodiment.
In said method, between step 3) and step 4), also comprise the steps: from described male sterile BC
2for choosing in colony and the male sterile BC of described corn inbred line capital 724 genetic similarty at 98.75%-100%
2for individual plant.
Described from described male sterile BC
2for choosing in colony and the male sterile BC of described corn inbred line capital 724 genetic similarty at 98.75%-100%
2method for individual plant comprises the steps: with primer A respectively to male sterile BC
2carrying out pcr amplification for individual plant and corn inbred line capital 724, obtaining SSR bands of a spectrum, by comparing BC
2for the SSR collection of illustrative plates in individual plant and corn inbred line capital 724, calculate genetic similarty;
Described primer A is BC
2for the maternal BC that individual plant is corresponding
1dai Yujing 724 compares the different SSR primer of amplification banding pattern;
Described genetic similarty computing formula: [1-(difference number of alleles/(2 × more total number of sites))] × 100%
Described difference number of alleles is to increase the described male sterile BC obtained with primer A
2for the allelomorph number that the SSR bands of a spectrum banding pattern in individual plant SSR bands of a spectrum banding pattern and described corn inbred line capital 724 is inconsistent;
Described more total number of sites is 40.
The application of S Type Cytoplasmic Male-sterility in Maize system MD32CGMCCNo.8657 in selecting and breeding corn male sterile line is also the scope of protection of the invention.
Corn male sterile line MD32 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 25th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.8657, and this plant Classification And Nomenclature is corn (Zeamays).
Experiment of the present invention proves, the present invention has following advantage:
1, utilize Molecular Marker Assisted Selection Technology and backcross transformation combine with technique, accelerate the transformation speed of male sterile line, only need third backcross generation namely to obtain male sterile line S capital, capital 724 724.
2, S type cytoplasmic male sterile line MD32 sterility performance under multiple genetic background that the present invention selects is stablized thoroughly, in the corn hybrid seed male sterile production of hybrid seeds research reported, have no use.
Embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The seed selection in embodiment 1, male sterile line S capital 724
One, the determination of male sterile line donor MD32 cytoplasmic male sterility type
1, corn male sterile line MD32
Corn male sterile line MD32 Breeding Process: utilize the colony of X system (based on X1132) built to carry out " tall and big tight " choosing system, to build new choosing be basic population with drawing crossbreed outward therefrom to select excellent S5 system of high generation.In self progeny, find sterile strain, tassel flower pesticide does not expose, and without pollen, selects the close sisters' strain of same head progeny row phenotype to pollinate in pairs to it.Filial generation all shows thoroughly sterile, and plant phenotype also tends to basically identical.Then be male sterile line of maize MD32 by this material designation.
Corn male sterile line MD32 sterility is thorough: flower pesticide does not expose, without pollen;
Corn male sterile line MD32 sterility is stablized: under multiple genetic background, and sterility performance thoroughly;
Corn male sterile line MD32 Comprehensive Traits is excellent: seed bud gesture is vigorous, and power of emerging is strong, and seedling leaf sheath look purple, blade is roomy, and leaf look dark green, and plant type half is compact, fruit ear cartridge type, and grain type half Hard grain type, comprehensive resistance is good.
Corn male sterile line MD32 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 25th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.8657, and this plant Classification And Nomenclature is corn (Zeamays).
2, the determination of male sterile line MD32 cytoplasmic male sterility type
The DNA extracting corn male sterile line MD32 seedling, as template, carries out pcr amplification with the primer as following table 1, obtains amplified production on Agarose gel, 90V electrophoresis 1 hour 30 minutes.Corn MD32 cytoplasmic sterility type is differentiated according to amplified fragments collection of illustrative plates.
As a result, utilize C, the DNA of T-shaped primer pair MD32 is when carrying out pcr amplification, all amplified production do not detected; Utilize S type primer pair its when detecting, PCR primer clip size is 885bp.Therefore determine that MD32 is S type cytoplasmic male sterile line.
Table 1 is the primer of qualification kytoplasm type
| Kytoplasm type | Primer sequence |
| C-1 | TGAAAGGGTGGTGGAATA |
| C-2 | GAGCCAAAGTAATGAGAAAA |
| S-1 | GATGCTATGCTAAGCGAGAT |
| S-2 | CCGCTAACCCACTCTTCT |
| T-1 | GTCGTGTCCTGGTAGCCT |
| T-2 | CCTCCTTCATTCCGTTGT |
Two, the seed selection in male sterile line of maize S capital 724
1, the acquisition of male sterile hybrid generation F1
The First Year summer, with male sterile line of maize MD32(non-recurrent parent) do donor, capital 724(Corn Rearch Center, Beijing Farming & Forestry Research Academy, kind power application notification number is CNA007811E) (recurrent parent) be acceptor hybridization assembly F
1generation, results F
1the seed in generation.
Winter in the same year Hainan first generation, plantation F
1the seed in generation, obtains 127 strain F
1for milpa.
127 strain F
1all do not expose for milpa flower pesticide, without pollen, sterility performance thoroughly.
2, backcross a BC
1the acquisition in generation
1) backcross
With F
1do female parent for plant, continue to backcross with recurrent parent capital 724, results BC
1for the seed of colony.
Winter in the same year Hainan second generation, plantation BC
1for the seed of colony, obtain BC
1for colony.
BC
1all do not expose for colony's flower pesticide, without pollen, sterility performance thoroughly.
2) molecular marker screening
From 2000 strain BC of plantation
1for selecting 616 BC that male flower is completely sterile, tree characteristics (plant height, Ear height, plant type, tassel branch number and Ear Characters) is close with capital 724 in colony
1for individual plant listing mark; Extract 616 BC
1for the DNA of single-strain blade as template, 40 pairs of SSR core primers (table 2) are utilized to carry out pcr amplification to each strain respectively; With capital 724 for contrast.Often pair of corresponding site of SSR core primers; There are 2 allelomorph in each site.
Table 2 is 40 pairs of SSR core primers
Each BC
1obtain pcr amplification collection of illustrative plates corresponding to 40 sites for individual plant, the pcr amplification collection of illustrative plates corresponding with 40 sites that capital 724 obtains compares, and the inconsistent allelomorph number of pcr amplification collection of illustrative plates bands of a spectrum is difference number of alleles; More total number of sites is 40.
Genetic similarty computing formula: [1-(difference number of alleles/(2 × more total number of sites))] × 100%
According to amplification, choose the female parent that front 30 individual plant minimum with capital 724 difference number of alleles backcrosses as next round, and calculate through genetic similarty, the genetic similarty between front 30 individual plants and capital 724 is all between 92.5-95%.
3, backcross quadratic B C
2the acquisition in generation
1) backcross
With above-mentioned 2 choose and the BC of capital 724 genetic similarty between 92.5-95%
1do female parent for individual plant, continue to backcross with recurrent parent capital 724, results BC
2for the seed of colony.
Summer next year, plantation BC
2for the seed of colony, by head progeny row field planting, each head progeny row plants 50 strains, amounts to 1500 strains.
2) molecular marker screening
Select completely sterile, the tree characteristics of male flower and close 300 BC in capital 724
2for individual plant listing mark, extract 300 BC respectively
2for the DNA of single-strain blade as template, utilize BC
2for the maternal BC that individual plant is corresponding
1dai Yujing 724 increase the different SSR primer pair of banding pattern its carry out pcr amplification; With capital 724 for contrast.
According to the method described above, calculate genetic similarty, wherein, difference number of alleles is male sterile BC
2for the allelomorph number that the SSR bands of a spectrum banding pattern in individual plant SSR bands of a spectrum banding pattern and described corn inbred line capital 724 is inconsistent, BC
2for the individual plant SSR bands of a spectrum of colony for using BC
2for the maternal BC that individual plant is corresponding
1the SSR primer that Dai Yujing 724 compares amplification banding pattern inconsistent carries out the bands of a spectrum obtained that increase; More total number of sites is 40.
Choosing genetic similarty is minimum front 30 individual plants of 98.75%-100%(and capital 724 difference number of alleles) female parent that backcrosses as next round.
4, backcross the acquisition in 3 male sterile line S capital 724
With above-mentioned 3 choose and the BC of capital 724 genetic similarty between 98.75%-100%
2do female parent for individual plant, continue to backcross with recurrent parent capital 724, results BC
3for colony, be male sterile line S capital 724.
Male sterile line S capital 724 flower pesticide does not expose, and without pollen, sterility performance thoroughly.
Three, the comparing of conventional transformation and the inventive method
Conventional transformation is substantially identical with the method for above-mentioned two, and unlike not carrying out molecular marker screening, result backcrossed for 6 generations, could obtain the male sterile line (genetic background response rate is shown in Table 3) that genetic background is returned to recurrent parent.
Calculate genetic background response rate G (g)=[L+X (g)]/2L; Wherein, g refers to backcross generations number, and G (g) refers to the genetic background response rate in g generation; X (g) refers at the g that backcrosses for the molecular labeling quantity showing as receptor parent banding pattern; L refer to participate in analyze molecular labeling quantity.
Table 3 is the background response rate of the inventive method and conventional breeding
As seen from the above, method of the present invention utilizes molecular labeling auxiliary background to select to combine with backcross transformation, and namely 3 generations that only needed to backcross obtain the male sterile line that genetic background is returned to recurrent parent.
Claims (7)
1. the selection of a male sterile line of maize, it is characterized in that: take deposit number as the S Type Cytoplasmic Male-sterility in Maize system MD32 of CGMCCNo.8657 be donor, corn inbred line is acceptor, carry out backcross transformation, obtain the male sterile line of described corn inbred line.
2. method according to claim 1, is characterized in that: described corn inbred line is corn inbred line capital 724, and the male sterile obtained is the male sterile line S capital 724 in capital 724.
3. method according to claim 1 and 2, is characterized in that: described in the number of times that backcrosses be 3 times.
4. method according to claim 2, is characterized in that: described backcross transformation comprises the steps:
1) with male sterile line of maize MD32CGMCCNo.8657 be donor, corn inbred line capital 724 for acceptor, hybridization, obtain male sterile hybrid generation F
1;
2) with male sterile hybrid generation F
1for female parent, backcross with capital 724, obtain male sterile BC
1for colony;
3) with male sterile BC
1be female parent for individual plant, continue to backcross with capital 724, obtain male sterile BC
2for colony;
4) with male sterile BC
2be female parent for individual plant, continue to backcross with capital 724, obtain the male sterile line S capital 724 in capital 724.
5. method according to claim 4, is characterized in that: in step 2) and step 3) between, also comprise the steps: from described male sterile BC
1for choosing in colony and the male sterile BC of described corn capital 724 genetic similarty at 92.5-95%
1for individual plant.
6. method according to claim 5, is characterized in that: in step 3) and step 4) between, also comprise the steps: from described male sterile BC
2for choosing in colony and the male sterile BC of described corn capital 724 genetic similarty at 98.75%-100%
2for individual plant.
7. deposit number is the application of S Type Cytoplasmic Male-sterility in Maize system MD32 in selecting and breeding corn male sterile line of CGMCCNo.8657.
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| CN104823659A (en) * | 2015-05-04 | 2015-08-12 | 河北省农林科学院粮油作物研究所 | Method for obtaining Jidou No.17 backcross introgression population |
| CN104823660A (en) * | 2015-05-04 | 2015-08-12 | 河北省农林科学院粮油作物研究所 | Method for obtaining Wuxing No.1 backcross introgression population |
| CN104839015B (en) * | 2015-06-10 | 2017-04-26 | 浙江新安化工集团股份有限公司 | Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation |
| CN106319037A (en) * | 2015-07-10 | 2017-01-11 | 沈阳化工大学 | SSR molecular labeling kit for detecting purity of maize variety Liangyu 88# |
| CN105132420B (en) * | 2015-09-23 | 2018-02-13 | 北京市农林科学院 | A kind of primer set for identifying corn variety purity and application |
| CN108220473B (en) * | 2018-02-06 | 2021-10-01 | 北京市农林科学院 | Identification of maize S-type cytoplasmic male sterile material by using chloroplast InDel marker |
| CN111575398B (en) * | 2020-05-28 | 2022-05-03 | 北京市农林科学院 | Molecular marker of male sterility related gene of No. 4 chromosome of corn and application of molecular marker |
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| CN1736166A (en) * | 2005-08-22 | 2006-02-22 | 河北冀南种业有限公司 | Sterile corn hybrid seed production method |
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| CN1736166A (en) * | 2005-08-22 | 2006-02-22 | 河北冀南种业有限公司 | Sterile corn hybrid seed production method |
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