CN108012919A - Tri- gene pyramiding material of corn o2, o16 and wx is used for the method for quality breeding - Google Patents
Tri- gene pyramiding material of corn o2, o16 and wx is used for the method for quality breeding Download PDFInfo
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Abstract
The invention discloses a kind of method that tri- gene pyramiding material of corn o2, o16 and wx is used for quality breeding, its technical solution is to be hybridized conventional corn self-mating system and tri- gene pyramiding material of corn o2, o16 and wx, conventional corn self-mating system is recycled to be returned 2 times for recurrent parent, wherein BC1 BC2 from generation to generation seedling stage carry out molecular labeling assisting sifting, filter out the single plant of tri- gene pyramiding of o2, o16 and wx, and lysine and amylopectin content measure are carried out, obtain the fine quality self-mating system of the glutinous matter of high-lysine.The present invention is of great significance corn quality gene pyramiding breeding practice and theoretical research, and reference frame is provided for the improvement of corn quality character inheritance.
Description
Technical field
The invention belongs to molecular breeding technology field, is specially that a kind of tri- gene pyramiding material of corn o2, o16 and wx is used for
The method of quality breeding.
Background technology
Corn is main in the world feed and cereal crops.Quality is main breeding objective, directly affects people livestock and poultry
Healthy development and eating mouth feel.Quality of maize grain refers to nutritional ingredient contained in corn kernel, such as protein, fat
Fat, starch and various vitamins, the content of mineral element and quality etc..Wherein, the lysine in protein is that people livestock and poultry etc. are single
Stomach animal essential amino acid, amylopectin content and edible quality tight association in starch.Conventional corn lysine content
Relatively low (less than 0.3%), it is impossible to meet that the mankind eat and require (more than 0.5%) with the nutritional quality of food processing, can not expire
Requirement (0.6-0.8%) (FAO (Food and Agriculture Organization of the United Nation)/world health organisation recommendations) of the sufficient animal and fowl fodder to lysine content.To carry
The nutritional quality and edible quality of high corn, high-lysine corn (lysine content is high) and glutinous corn (amylopectin content
It is high) widely paid close attention to.
High-lysine corn can improve the nutritive value and feeding value of corn.Singleton and Jones (1935) is first
(1964) such as secondary discovery corn opaque-2 (o2) mutation, later Mertz find that o2 mutation can significantly improve corn kernel endosperm
In lysine and tryptophane.After Paez etc. (1969) has found the modifier of o2, Prasanna etc. (2001) utilizes o2
Modifier has cultivated the high-lysine corn of MAIZE WITH HARD ENDOSPERM --- high-quality protein maize (Quality Protein Maize,
QPM), lay a good foundation for the application of high-quality protein maize.O2 genes are located on the galianconism of the 7th chromosome of corn,
Pioneer companies and Missouri universities develop intragenic 3 SSR markers of o2, i.e. phi057, phi112 and umc1066,
Be successfully applied to corn high-lysine MAS (Marker Assisted Selection) (Babu et al, 2005；
Zhang et al,2010).Yang etc. (2005) utilizes one new high-lysine of Mu transposons insertional mutagenesis technology selection and breeding
Opaque-16 (o16) is mutated, its lysine content is up to 0.36%, and by the o16 assignments of genes gene mapping on the 8th chromosome long arm of corn
From the position within umc1141 molecular labelings 5cM between two molecular labeling of umc1141 and umc1121.Research has shown that, passes through height
Lysine gene pyramiding (o2 and o16), can improve the nutritional quality (Yang et al, 2005) of corn.
Waxy corn (waxy maize) Cooking Quality is excellent, gets consumer reception again.Waxy corn, most finds early in China
(Zeng Mengqian, 1987), amylopectin of its endosperm containing 95%-100%, Recessive genes mutation wx are responsible for controlling waxy corn seed
Endosperm is formed.Wx sites are located on the galianconism of the 9th chromosome, MaizeGDB websites have issued for for detect wx sites 3
SSR marker in a gene：Phi022, phi027 and phi061, are successfully used for MAS (Yang L et al, 2013).
Zhang Shukuan etc. utilizes traditional breeding technology binding molecule marker assisted selection technology in (2009), with 2 high-quality eggs
White maize self-mating system and 4 Waxy Corn Inbred Lines are material, and selection and breeding 18 high-quality protein Waxy Corn Inbred Lines, its lysine contain
Amount can reach more than 0.38%.Yang etc. (2013) has polymerize o16 and wx genes, obtains 13 familys, its lysine contains
Amount improves more than 16% than waxy corn system, and amylopectin content is similar to waxy corn system.Zhang etc. (2013) is by o2o16 bases
Because backcrossing is penetrated into wx systems, 3 parts of waxy corn systems containing o2 genes are obtained, its average lysine content is 0.555%；Obtain
3 parts of waxy corn systems containing o2o16 genes, its average lysine content is 0.616%, its amylopectin content and receptor parent
Waxy corn system QCL5019 is similar.As it can be seen that by corn o2 and o16 channel genes waxy corn system, the lysine of waxy corn can be improved
Content, obtains Cooking Quality and the good fresh edible maize breeding material of nutritional quality, this is not only to promoting fresh edible maize breeding
Develop it is significant, and to people while edible agreeable to the taste fresh edible maize the sufficient lysine nutrition of intake have it is real
Border meaning.
In short, these above-mentioned methods can improve the lysine content of waxy corn, improve the nutritional quality of waxy corn.But
Conventional corn lysine and amylopectin content are relatively low, and nutritional quality is poor, therefore, there is an urgent need to establish one quickly improve it is general
Logical corn nourishment quality method, effective means is provided for corn quality improvement and breeding etc..
The content of the invention
The object of the present invention is to provide a kind of tri- gene pyramiding material of corn o2, o16 and wx be used for quality breeding method,
To improve the nutritional quality of corn.
The technical scheme is that conventional corn self-mating system and tri- gene pyramiding material of corn o2, o16 and wx are carried out
Hybridization, recycles conventional corn self-mating system to be returned 2 times for recurrent parent, wherein BC1 BC2 carry out molecular labeling in seedling stage from generation to generation
Assisting sifting, filters out the single plant of tri- gene pyramiding of o2, o16 and wx, and carries out lysine and amylopectin content measure, obtains
The fine quality self-mating system of the glutinous matter of high-lysine.
Specifically, carry out in accordance with the following steps：
Step 1. chooses tri- gene pyramiding material QCL8002-11 of conventional corn selfing based material and corn o2, o16 and wx；
Step 2. is recurrent parent by conventional corn self-mating system and tri- gene pyramiding material of corn o2, o16 and wx is donor
Parent is hybridized, and obtains F1 generation seeds；
Step 3., using conventional corn self-mating system as male parent, is returned using the F1 generations plant of acquisition as female parent, is obtained
BC1F1 generation seeds；
Step 4. kind plants BC1F1 generation seeds, and seedling stage sampling, is using Marker-assisted selection genotype
The single plant of O2o2O16o16WXwx, using conventional corn self-mating system as male parent, is returned, and obtains BC2F1 generation seeds；
Step 5. kind plants BC2F1 generation seeds, and seedling stage sampling, is using Marker-assisted selection genotype
The single plant of O2o2O16o16WXwx, selfing, obtains BC2F2 generation seeds；
Step 6. kind plants BC2F2 generation seeds, and seedling stage sampling, is using Marker-assisted selection genotype
The single plant of o2o2o16o16wxwx, selfing, obtain BC2F3 generation seeds, then be selfed purifying can obtain Fineness gene polymerization
Self-mating system.
Wherein, the conventional corn self-mating system described in step 1 is common non-high-lysine, the selfing of non waxy corn
System, its genotype is O2O2O16O16WXWX；Tri- gene pyramiding material of o2, o16 and wx is QCL8002-11, and genotype is
o2o2o16o16wxwx.Preferably, conventional corn self-mating system is CML537 or CML539.
Further, the conventional corn self-mating system exists with tri- gene pyramiding material QCL8002-11 of o2, o16 and wx
Tri- gene locis of o2, o16 and wx have polymorphism.
Further, the molecular labeling described in step (4) is glutinous gene internal labeling phi061, high-lysine gene (o2)
Internal labeling phi112, the chain mark umc1141 of high-lysine gene (o16).Here phi061 mark, phi112 mark and
The sequence reference of umc1141 markswww.maizegdb.orgThe data of upper announcement, can find primer sequence, and primer sequence has south
Jing Jinsirui biotechnologies company synthesizes.
Further, BC1F1 generation populations size should be determined according to being actually needed, to the screening acquisition 1 in step (4)
Pnca gene type is the single plant of O2o2O16o16WXwx, should detect no less than 8 single plants in theory (computational methods are 2 × 2 × 2).
During atual detection, it should determine that BC1F1 generation populations plant size as needed.BC2F1 generation populations size should basis
It is actually needed and determines, to the single plant that 1 pnca gene type of screening acquisition is O2o2O16o16WXwx in step (5), in theory should
No less than 8 single plants of detection (computational methods are 2 × 2 × 2).During atual detection, it should determine BC2F1 from generation to generation as needed
Size is planted by colony.BC2F2 generation populations size should be determined according to being actually needed, to 1 plant of base of screening acquisition in step (6)
Because of the single plant that type is o2o2o16o16wxwx, no less than 64 single plants should be detected in theory (computational methods are 4 × 4 × 4).In reality
In the detection process of border, it should determine that BC2F2 generation populations plant size as needed.
The present invention takes above technical scheme, it is possible to achieve tri- gene of o2, o16 and wx penetrates into conventional corn selfing at the same time
In system, compared with single-gene penetrates into, drastically increase work efficiency, and utilize this research method, can also isolate containing
The material (o2o2o16o16wxwx, o2o2o16o16, o2o2wxwx, o16o16wxwx) of different genes polymeric type and contain list
The material (o2o2, o16o16, wxwx) of gene, enriches breeding resources, and basic material is chosen extensively, and conventional corn is selfed
The selection of system does not limit, and can be the conventional corn self-mating system in temperate zone, subtropical zone and the torrid zone.The material quality character formulated out
Stabilization does not separate.Successfully selected containing different genes polymeric type material (o2o2o16o16wxwx,
O2o2o16o16, o2o2wxwx, o16o16wxwx) and be corn product containing monogenic material (o2o2, o16o16, wxwx)
Kind breeding provides material foundation.
Brief description of the drawings
Fig. 1 is used for quality breeding technology path schematic diagram for tri- gene pyramiding material of corn o2, o16 and wx；
Fig. 2 is that corn opaque2 gene internal labelings phi112 marks the electrophoretogram in parent and progeny population；
Fig. 3 is that corn wx gene internal labelings phi061 marks the electrophoretogram in parent and progeny population；
Fig. 4 is that corn opaque16 gene linkages mark umc1141 to mark the electrophoretogram in parent and progeny population.
Embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment.The numerical value that is directed to,
Number or ratio are mass figures, mass fraction or mass ratio such as without mark.
DNA extractions, PCR amplification and the electrophoresis detection used in embodiment, include the following steps：
DNA is extracted：Behind corn seed plantation field, when emerging to 51 heart of leaf, centrifuge tube (0.5mLEppendorf is utilized
Pipe) lid take blade (about 10mg)；Stainless shot (specification is added in each centrifuge tube：A diameter of 2.5mm) 3；Sample will be taken
Centrifuge tube be placed on every a hole in 96 hole depth orifice plates；100uL 2%CTAB extracting solutions are added in centrifuge tube, on grind away instrument
1300rpm shakes 1-3min；By ground sample, place in 65 DEG C of water-baths or baking oven and heat 30min, during which on every 10min
It is lower to overturn several times；After cooling down room temperature, 100uL working solution (chloroforms are added：Isoamyl alcohol=24:1 mixed liquor), shake up 3-
5min, centrifuges 10min；Supernatant in centrifuge tube is transferred in 96 hole PCR plates；Add 7.5uLNaCl (5moL/L) and
The absolute ethyl alcohol of 100uL precoolings, covers silica gel lid, turns upside down several times, static until DNA precipitations, after of short duration centrifugation, are opened
Silica gel lid, upset PCR plate outwell whole liquid；Add 75% ethanol 150uL and rinse precipitation 1-2 times；After of short duration centrifugation, silicon is opened
Jiao Gai, upset PCR plate outwells whole liquid, room temperature, slant setting, until DNA is dried；Add 150uL ddH2O dissolvings, this liquid
Body is corn gene group DNA working solution.
PCR amplification：Reaction system (cumulative volume 10ul)：The MgCL2 1ul of 10*Buffer 1ul, 25mM, glycerine 1ul,
Taq enzyme 0.075ul, the ddH2O 2.975ul of the dNTPs 0.75ul, 5U/uL of the primer each 0.6ul, 2mM of 5uM.
Amplification program：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 30s, 30 circulate,
72 DEG C of extension 10min, 4 DEG C of preservations.
Electrophoresis detection：3 μ L PCR products are taken, the electrophoresis in 6% denaturing polyacrylamide, through silver staining, rinses, colour developing
Deng observing and recording result of the test using film illominator.
Embodiment 1：
1st, test material：(genotype is corn o2, o16 and wx gene pyramiding system QCL8002-11
O2o2o16o16wxwx), conventional corn self-mating system CML537 (genotype O2O2O16O16WXWX).
2nd, winter in 2012, in Hainan, Le Dong plants parent, conventional corn self-mating system CML537 and gene pyramiding system
QCL8002-11 (o2o2o16o16wxwx), each 1 row, is hybridized in florescence, obtains F1 generation seeds；
3rd, 2013 spring, plants F1 generation seeds, 1 row, conventional corn self-mating system CML537 is utilized in florescence in Kweiyang
Backcrossing, obtains BC1F1 generation seeds；
4th, winter in 2013, in Hainan, Le Dong plants BC1F1,29 rows, 3 meters of railway carriage or compartments, totally 306 single plants, seedling stage sampling, extraction
Genomic DNA, single plant of the genotype for O2o2O16o16WXwx is selected using molecular labeling phi061, phi112 and umc1141,
As a result 33 single plants be have selected altogether, be returned in florescence using conventional corn self-mating system CML537, obtain BC2F1 generation seeds；
5th, 2014 spring, BC2F1 is planted in Kweiyang, 20 rows, 3 meters of railway carriage or compartments, totally 222 single plants, seedling stage sampling, extracts genome
DNA, selects single plant of the genotype for O2o2O16o16WXwx, as a result altogether using molecular labeling phi061, phi112 and umc1141
29 single plants are have selected, are selfed in florescence, obtain BC2F2 generation seeds；
6th, 2014 winters Hainan plantation BC2F2 colonies, totally 26 row, 3 meters of railway carriage or compartments, totally 295 single plants,
Seedling stage samples, and is using Marker-assisted selection genotype
o2o2o16o16wxwx、o2o2o16o16、o2o2wxwx、o16o16wxwx、o2o2、o16o16、wxwx
Single plant, selfing, obtains BC2F3 generation seeds, then is selfed purifying and can obtain containing different genes polymeric type
Material (o2o2o16o16wxwx, o2o2o16o16, o2o2wxwx, o16o16wxwx) and containing monogenic material (o2o2,
O16o16, wxwx), provide material foundation for corn variety breeding.
Embodiment 2：
1st, test material：(genotype is corn o2, o16 and wx gene pyramiding system QCL8002-11
O2o2o16o16wxwx), conventional corn self-mating system CML530 (genotype O2O2O16O16WXWX).
2nd, winter in 2010, in Hainan, Le Dong plants parent, conventional corn self-mating system CML530 and gene pyramiding system
QCL8002-11 (o2o2o16o16wxwx), each 1 row, is hybridized in florescence, obtains F1 generation seeds；
3rd, 2011 spring, plants F1 generation seeds, 1 row, conventional corn self-mating system CML530 is utilized in florescence in Kweiyang
Backcrossing, obtains BC1F1 generation seeds；
4th, winter in 2011, in Hainan, Le Dong plants BC1F1, and 50 rows, 3 meters of railway carriage or compartments, totally 1010 single plants, seedling stage sampling, carries
Genomic DNA is taken, list of the genotype for O2o2O16o16WXwx is selected using molecular labeling phi061, phi112 and umc1141
Strain, as a result have selected 32 single plants altogether, with reference to Analysis of Genetic Background, 19 lists of selection background recovery rate high (more than 75%)
Strain, is returned in florescence using conventional corn self-mating system CML530, obtains BC2F1 generation seeds；
5th, spring in 2012, BC2F1 is planted in Kweiyang, 115 rows, 3 meters of railway carriage or compartments, totally 1213 single plants, seedling stage sampling, extracts base
Because of a group DNA, single plant of the genotype for O2o2O16o16WXwx, knot are selected using molecular labeling phi061, phi112 and umc1141
Fruit have selected 130 single plants altogether, is selfed in florescence, obtain BC2F2 generation seeds；According to seed phenotype, opaque seed is selected and remain
Grain is retained.
6th, spring in 2013, in Kweiyang plantation BC2F2 colonies, 50 rows, 3 meters of railway carriage or compartments, totally 411 single plants, select different genes
Type type (o2o2o16o16wxwx, o2o2o16o16, o2o2wxwx, o16o16wxwx, o2o2, wxwx, o16o16), is as a result selected
Select 5 plants of o2o2o16o16wxwx genotype, 2 plants of o2o2o16o16 genotype, 2 plants of o2o2wxwx genotype, o16o16wxwx
2 plants of genotype, 1 plant of o2o2 genotype, 1 plant of wxwx genotype, 1 plant of o16o16 genotype.Selfing obtains BC2F3.It is selfed again pure
Change can obtain containing different genes polymeric type material (o2o2o16o16wxwx, o2o2o16o16, o2o2wxwx,
O16o16wxwx) and containing monogenic material (o2o2, o16o16, wxwx), material foundation is provided for corn variety breeding.Obtain
What is obtained detects containing different genes polymeric material through Ministry of Agriculture's cereal and quality of item supervision and inspection center (Beijing), examines
As a result such as following table.
The seed lysine content of 1 part different genes polymeric type material of table
(Ministry of Agriculture's cereal and quality of item supervision and inspection center inspection result, 2015)
Successfully QCL8002-11 (o2o2o16o16wxwx) Quality Gene is imported into using the method in embodiment general
In logical corn inbred line, acquisition contains different genes polymeric material through in Ministry of Agriculture's cereal and quality of item supervision, inspection and testing
The heart (Beijing) detects, and tri- gene pyramiding material lysine content of corn o2, o16 and wx reaches 0.7%, two base of corn o2 and o16
Because polymeric material lysine content reaches more than 0.63%, two gene pyramiding material lysine content of corn o2 and wx reaches
More than 0.61%, two gene pyramiding material lysine content of corn o16 and wx reaches more than 0.39%.Experiment shows, according to reality
The method operation in example is applied, can be real by tri- channel genes of o2, o16 and wx in QCL8002-11 into conventional corn self-mating system
Now to the quality trait genetic improvement of conventional corn self-mating system.
Certainly, the above is the concrete application example of the present invention, and the present invention also has other embodiments, all using equivalent
The technical solution that replacement or equivalent transformation are formed, all falls within protection domain of the presently claimed invention.
Claims (5)
1. a kind of tri- gene pyramiding material of corn o2, o16 and wx is used for the method for quality breeding, it is characterised in that：Will be common beautiful
Rice self-mating system is hybridized with tri- gene pyramiding material of corn o2, o16 and wx, and it is recurrent parent to recycle conventional corn self-mating system
Backcrossing 2 times, wherein BC1 BC2 from generation to generation seedling stage carry out molecular labeling assisting sifting, filter out tri- gene pyramiding of o2, o16 and wx
Single plant, and carry out lysine and amylopectin content measure, obtain the glutinous matter of high-lysine fine quality self-mating system.
2. tri- gene pyramiding material of corn o2, o16 and wx according to claim 1 is used for the method for quality breeding, it is special
Sign is to carry out in accordance with the following steps：
Step 1. chooses tri- gene pyramiding material QCL8002-11 of conventional corn selfing based material and corn o2, o16 and wx；
Step 2. is recurrent parent by conventional corn self-mating system and tri- gene pyramiding material of corn o2, o16 and wx is donor parents
Hybridized, obtain F1 generation seeds；
Step 3., using conventional corn self-mating system as male parent, is returned using the F1 generations plant of acquisition as female parent, obtains BC1F1
Generation seed；
Step 4. kind plants BC1F1 generation seeds, and seedling stage sampling, is O2o2O16o16WXwx's using Marker-assisted selection genotype
Single plant, using conventional corn self-mating system as male parent, is returned, and obtains BC2F1 generation seeds；
Step 5. kind plants BC2F1 generation seeds, and seedling stage sampling, is O2o2O16o16WXwx's using Marker-assisted selection genotype
Single plant, selfing, obtains BC2F2 generation seeds；
Step 6. kind plants BC2F2 generation seeds, and seedling stage sampling, is o2o2o16o16wxwx's using Marker-assisted selection genotype
Single plant, selfing, obtains BC2F3 generation seeds, then is selfed the self-mating system for purifying and can obtaining Fineness gene polymerization.
3. tri- gene pyramiding material of corn o2, o16 and wx according to claim 2 is used for the method for quality breeding, it is special
Sign is：Conventional corn self-mating system described in step 1 is common non-high-lysine, non waxy corn inbred line, its base
Because type is O2O2O16O16WXWX；Tri- gene pyramiding material of o2, o16 and wx is QCL8002-11, and genotype is
o2o2o16o16wxwx。
4. the tri- gene pyramiding material of corn o2, o16 and wx according to Claims 2 or 3 is used for the method for quality breeding, its
It is characterized in that：The conventional corn self-mating system is with tri- gene pyramiding material QCL8002-11 of o2, o16 and wx in o2, o16 and wx
Three gene locis have polymorphism.
5. the tri- gene pyramiding material of corn o2, o16 and wx according to Claims 2 or 3 is used for the method for quality breeding, its
It is characterized in that：The single plant that the 1 pnca gene type that obtains is O2o2O16o16WXwx is screened in step 4, should detect no less than 8 lists
Strain；The single plant that the 1 pnca gene type that obtains is O2o2O16o16WXwx is screened in steps of 5, should detect no less than 8 single plants；Will
Screening obtains the single plant that 1 pnca gene type is o2o2o16o16wxwx in step 6, should detect no less than 64 single plants.
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| CN110157834A (en) * | 2019-07-01 | 2019-08-23 | 北京市农林科学院 | SNP site OE2-5 relevant to corn high-lysine character and its application |
| CN110199871A (en) * | 2019-07-01 | 2019-09-06 | 北京市农林科学院 | A kind of selection of high-lysine corn self-mating system |
| CN111512956A (en) * | 2020-04-22 | 2020-08-11 | 四川省农业科学院水稻高粱研究所 | Method for improving yield and quality of fresh corn |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157834A (en) * | 2019-07-01 | 2019-08-23 | 北京市农林科学院 | SNP site OE2-5 relevant to corn high-lysine character and its application |
| CN110199871A (en) * | 2019-07-01 | 2019-09-06 | 北京市农林科学院 | A kind of selection of high-lysine corn self-mating system |
| CN110199871B (en) * | 2019-07-01 | 2020-12-29 | 北京市农林科学院 | Breeding method of high-lysine maize inbred line |
| CN110157834B (en) * | 2019-07-01 | 2022-04-19 | 北京市农林科学院 | SNP site OE2-5 related to high lysine character of corn and application thereof |
| CN111512956A (en) * | 2020-04-22 | 2020-08-11 | 四川省农业科学院水稻高粱研究所 | Method for improving yield and quality of fresh corn |
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