CN102511372A

CN102511372A - Corn inbred line assistant seed selection method by using IDP molecular marker

Info

Publication number: CN102511372A
Application number: CN2011103070318A
Authority: CN
Inventors: 赵晋锋; 王国英; 余爱丽; 郭二虎; 刘永忠; 杜艳伟; 王高鸿; 常海霞
Original assignee: MILLET RESEARCH INSTITUTE SHANXI ACADEMY OF AGRICULTURAL SCIENCE
Current assignee: MILLET RESEARCH INSTITUTE SHANXI ACADEMY OF AGRICULTURAL SCIENCE
Priority date: 2011-10-12
Filing date: 2011-10-12
Publication date: 2012-06-27
Anticipated expiration: 2031-10-12
Also published as: CN102511372B

Abstract

The invention relates to a corn inbred line assistant seed selection method by using an IDP molecular marker, which comprises the following steps: 1) screening 20 pairs of IDP primers possessing polymorphism to the corn inbred line seed selection base material S0 generation group; 3) using 10 pairs of IDP primers in the 20 pairs of IDP primers for screening S1 generation group to obtain an single plant of random n pairs of IDP primers lotus homozygosis in the above 10 pairs of IDP primers and obtain the inbred cluster; 3) using the IDP primers possessing polymorphism to the corn inbred line seed selection base material S0 generation group screened in the step 1) for removing the 20-n pairs of IDP primers outside the IDP lotus homozygosis used in the step 2), screening a S2 generation group to obtain the S2 generation group of the 20-n pairs of IDP primers lotus homozygosis which is the corn inbred line. According to the invention, the corn inbred line seed selection system with high efficiency and rapidity is established, and the inbred line seed selection time can be substantially shortened.

Description

Utilize the method for IDP molecular marking supplementary breeding corn inbred line

Technical field

The invention belongs to the plant breeding technique field, relate to a kind of selection of corn inbred line, particularly relate to a kind of method of utilizing molecular labeling means maize autocopulation strain assistant selection and breeding.

Background technology

Corn ( Zea mays) be the important source material crop of modern food, feed, medicine and chemical industry.Maize Production development in recent years is very fast, becomes the big crop in third place in the world, and sowing area and gross yield are only second to paddy rice and wheat.Along with population increase, livestock breeding and farming industry constantly develop, to the demand of corn also with sustainable growth.China is the second largest producing country of world's corn, and corn occupies crucial status in China's agricultural production, national economy.

Existing corn inbred lines method is isolated by continuous multi-generation selfing and selection by corn variety colony or all kinds of crossbreed normally that isozygoty or isozygoty basically in the beneficial gene site, the self progeny of proterties neat and consistent.Above-mentioned colony can be F ₂For colony, backcross population, also can be the colony that comprises two or more offsprings.Mainly there are two problems in the ordinary maize inbred line breeding: first; Corn inbred lines mainly is according to seed selection person's working experience; On the basis that the breeding material phenotypic character is observed, select, this selection is favourable to single-gene-controlled traits, and is unfavorable to the quantitative character of controlled by multiple genes; And there is work and very easily affected by environment mutually in most of proterties by controlled by multiple genes between the gene.The second, conventional method needs 6～8 self-generations at least to the selection of basic material, thereby breeding cycle is lengthy, need expend resources such as more manpower, financial resources.

In crop breeding, effective choice method is directly to select according to the genotype of plant.Molecular marker assisted selection (MAS) can be carried out accurate, stable selection to target gene in generation morning, thereby quickens breeding process, improves breeding efficiency.In recent years, MAS report is on the increase, and in the breeding practice of chief crops such as paddy rice, wheat, corn, has used and obtained bigger progress, mainly comprises gene transfer, gene pyramiding and quantitative character MAS.Tanksley etc. reach a conclusion through computer simulation analysis: adopt MAS only to need for 3 generations can make crossbreed return to the genotype near recurrent parent.Moreau etc., Berloo etc., Hospital etc. and Wu behave etc. and to deliver many pieces of research papers respectively, have set forth method and the influence factor of MAS, and this provides theoretical foundation for the enforcement of MAS.The various countries scholar has made up paddy rice, wheat, corn, cotton, Soybean and Other Crops colony and has developed the molecular labeling of enormous amount, wherein is no lack of the molecular labeling relevant with economical character, and this lays a good foundation for the application of MAS.The International Rice Research Institute utilizes 4 to contain different water rice bacterial leaf blight resistant gene (xa4, xa5, xa3, xa21) NIL and carry out the polymerization of adversity gene, and the strain of the resistant gene that is produced accumulation has higher resistance level and the anti-spectrum wider to pathogen than the strain that contains single resistant gene.Chen Sheng etc. are donor material with IRBB21, utilize 4 to carry out the auxiliary improvement of molecular labeling with the closely linked molecular labeling of xa21 to ' bright extensive 63 ', have obtained bacterial blight-resisting material ' bright extensive 63 '.Some quantitative trait locus bit transitions that American scholar is relevant with output with corn have arrived in the inbred lines such as B73 and Mo17, improve more than 15% by the crossbreed yield increased output of these inbred line assembly.Utilization such as Zhang Shihuang and the closely linked molecular labeling of opaque-2 gene have carried out the molecular marker assisted selection of high-quality protein maize, have set up the economical and practical high-quality protein maize molecular mark system of a cover.A large amount of theoretical research and facts have proved, MAS is the means that realize crop breeding rapidly and efficiently.In general, MAS is to selecting effect obvious by the selection effect comparison single-gene-controlled traits of controlled by multiple genes proterties, and it has the following advantages: do not receive season, environmental factor variable effect, not whether limited by gene expression; Polymorphism is high, has abundant allelic variation; Quantity is abundant, and polymorphism spreads all over whole genome, shows as " neutrality ", does not influence the expression of objective trait, does not have inevitable chain with bad proterties; Many molecular labeling performance codominances can be differentiated the heterozygous genes type that isozygotys, and complete information are provided, reliable results.

The genetic marker quantity that IDP (insertion-deletion length polymorphism) molecular labeling is set up is big, can in plant different developmental phases, varying environment condition, different tissues, detect.The IDP molecular marking technique has codominance, can observe the site homozygosity of selecting material from the dna molecular level, can be at inbred line breeding early for finding the higher material of gene loci purity.The IDP molecular labeling does not relate to hybridization, enzyme is cut; On detecting, use Ago-Gel to detect; Detect with SSR polyacrylamide gel electrophoresis at present commonly used and to compare more conveniently, and agarose gel can reuse, and detects more economically than polyacrylamide gel electrophoresis and saves.

Prior art shows that corn inbred lines does not still utilize the IDP labeling technique to separate the technical literature report of the auxiliary corn inbred lines of offspring site homozygote screening at present.

Summary of the invention

To the problems referred to above, the purpose of this invention is to provide a kind of method of the IDP of utilization molecular marking supplementary breeding corn inbred line.

The method of the IDP of utilization molecular marking supplementary breeding corn inbred line provided by the present invention may further comprise the steps:

1) screening is to corn inbred lines basic material S ₀0 pair of the IDP primer 2 that has polymorphism for colony; Said IDP primer sequence is from website www.maizegdb.org.;

Filter out to corn inbred lines basic material S ₀The IDP primer that has polymorphism for colony meets the following conditions: at least 2 pairs of IDP primers on every pair of homologous chromosome, and the genetic distance between two pairs of IDP primer sites on a pair of homologous chromosome is not less than 50cM;

2) utilize 10 couples of primer screening S in 20 pairs of IDP primers that step 1) screens ₁Generation population obtains in above-mentioned 10 pairs of IDP primers at random n to isozygoty individual plant and obtain its selfing fruit ear of IDP primer sites, and said 10 pairs of IDP primers are positioned on 10 pairs of homologous chromosomes;

3) utilize that step 1) screens to corn inbred lines basic material S ₀Have for colony in the IDP primer of polymorphism and remove step 2) in the 20-n of used IDP site outside isozygotying to the IDP primer, screening S ₂Generation population obtains the S that the IDP primer sites is isozygotied at said 20-n ₂The generation population individual plant is candidate's corn inbred line;

To S of the present invention ₀The probability that isozygotys of group size and different loci, the span of n is more suitable in 6≤n≤8 zones.

Wherein, S ₀The seed of expression corn inbred lines basic material and the plant that grows up to; S ₁Expression is by S ₀Seed that the plant selfing produces and the plant that grows up to; S ₂Expression is by S ₁Seed that selfing produces and the plant that grows up to.

Said corn inbred lines basic material can be single cross hybrid, intervarietal cross kind, kind and inbred line intermolecular hybrid kind or synthetic cross variety.

When said corn inbred lines basic material is a single cross hybrid, the male parent of said single cross hybrid and maternal when known can be screened corn inbred lines basic material S as follows ₀The IDP primer that has polymorphism for colony: the genomic DNA with single cross hybrid, female parent and male parent is a template, utilizes candidate IDP primer to carry out pcr amplification, detects the polymorphism of PCR product.Be corn inbred lines basic material S polymorphism being arranged between male parent and the female parent and in single cross hybrid PCR product, comprise the pairing IDP primer of this PCR of father and mother product ₀The IDP primer that has polymorphism for colony.In this case, the individual plant that isozygotys of said IDP primer sites is S ₁Generation population or S ₂In the generation population with single cross hybrid male parent or the consistent individual plant of maternal this IDP primer PCR amplified production banding pattern.

When said corn inbred lines basic material is a single cross hybrid, when the male parent of said single cross hybrid and maternal the unknown, can screen corn inbred lines basic material S according to following method ₀The IDP primer that has polymorphism for colony: with S ₁The genomic DNA of generation population individual plant is a template, utilizes candidate IDP primer to carry out pcr amplification, detects the polymorphism of PCR product.At S ₁The PCR product can produce three kinds of banding patterns in the generation population, and wherein two PCR product banding patterns are a band, and note is made banding pattern

,

The third banding pattern product comprises banding pattern

,

Product, note is made banding pattern

Produce banding pattern

,

The individual plant number meet the ratio of 1 ︰, 2 ︰ 1 basically (three kinds of banding pattern ratios be 1 ︰, 2 ︰ 1 in theory; But when the site has inclined to one side segregation phenomenon, do not meet 1 ︰, 2 ︰, 1 ratio, in this experiment partially segregation phenomenon do not influence the screening of isozygotying of IDP site) the IDP primer be corn inbred lines basic material S ₀The IDP primer that has polymorphism for colony.In this case, the individual plant that isozygotys of said IDP primer sites is S ₁Generation population or S ₂In the generation population with said S ₁The banding pattern of this IDP primer in the generation population

Or

Consistent individual plant.

The invention provides the method for utilizing IDP molecular marking supplementary breeding corn variety Zheng Dan 958 inbred lines, this method may further comprise the steps:

1) be the corn inbred lines basic material with Zheng Dan 958, from the IDP primer that website www.maizegdb.org. provides, filter out following 20 pairs to Zheng Dan 958 corn inbred lines basic material S ₀The IDP primer that has polymorphism for colony: IDP182, IDP126, IDP2485, IDP456, IDP125, IDP484, IDP483, IDP281, IDP4030, IDP89, IDP3887, IDP177, IDP4082, IDP705, IDP535, IDP119, IDP709, IDP549, IDP582 and IDP1471;

2) utilize primer I DP182, IDP2485, IDP125, IDP483, IDP4030, IDP3887, IDP4082, IDP535, IDP709 and IDP582 screening S ₁Generation population obtains wherein at random n to isozygoty individual plant and obtain its selfing fruit ear of (6≤n≤8) IDP primer sites;

3) utilize primer I DP126, IDP456, IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549, IDP1471 and step 2) 10-n that do not isozygoty in the used IDP primer to the IDP primer altogether 20-n to IDP primer screening S ₂Generation population obtains at said 20-n the S that the IDP primer sites is isozygotied ₂The generation population individual plant is candidate's corn inbred line.

Wherein, do not isozygoty said step 2) 10-n to the IDP primer for different S ₁The menu strain has different combinations in generation.

In order to take into account workload and screening effeciency, S according to the invention ₀Form S for colony by 20 individual plants ₁Form S for colony by 2000 individual plants ₂Form by 340 individual plants for colony.

And then the present invention combines S with candidate's corn inbred line that said method obtains ₁, S ₂The economical character of generation population and S ₂Coordinate force/blood relationship is measured the result from generation to generation, comes the selecting and breeding corn inbred line.

The present invention utilizes the method for IDP molecular marking supplementary breeding corn inbred line to utilize the IDP molecular marking technique to have codominant characteristics; From the site homozygosity of dna molecular level detection inbred line breeding basic material segregative generation, early in generation, found the material that gene loci isozygotys at inbred line breeding.This method relies on experience on the phenotypic character level, to infer the genotype of the breeding material more science, intuitively of situation of isozygotying than traditional corn breeding; Combine economical character, coordinate force and blood relationship to identify simultaneously, the selection genotype is isozygotied, phenotype is consistent, the higher inbred line of coordinate force.If the pleomorphism site of selecting is the site relevant with economical character, then select purpose stronger.The inventive method has overcome the defective of conventional breeding; Provide a kind of than more convenient, the cost-effective seed selection inbred line of SSR method; Genotype and phenotypic double selection have been realized; Improve homozygotic efficiency of selection greatly, effectively shortened the seed selection cycle of inbred line, accelerated the breeding progress.

The present invention utilizes that the method for IDP molecular marking supplementary breeding corn inbred line has been set up efficiently, corn inbred lines system efficiently.The IDP molecular marking technique has simple to operate, good reproducibility, and characteristics such as experimental cost is low, and the IDP labeled primer is abundant have had more than 5000 being published on the maizegdb.org website at present, and in constantly increasing, upgrading.The inventive method is simple to operate, can significantly shorten the inbred line breeding time, and the breeding cost has obvious reduction than conventional method at aspects such as manpower, financial resources, helps in present commercial breeding, promoting and utilizing.

Description of drawings

Fig. 1 has polymorphism for screening between Zheng Dan 958, maternal Zheng 58, the prosperous 7-2 of male parent part IDP primer amplification electrophoresis pattern.F1 representes single cross hybrid Zheng Dan 958, and ♀ representes maternal Zheng 58, and ♂ representes the prosperous 7-2 of male parent, and M representes DNA Marker.

Fig. 2 utilizes primer I DP1471 to identify the S of Zheng Dan 958 ₁The part individual plant amplification electrophoresis pattern that IDP1471 isozygotys in the site in the generation

population.Swimming lane

2,3,4,5,6,8,9,10,11,14,15,17,18,21,22,23,30,35,38,39,41,42,44,47 isozygotys, and all the other swimming lanes are the fuzzy swimming lane that is difficult to differentiate of IDP1471 site heterozygosis or banding pattern.

Fig. 3 utilizes primer I DP709 to identify the S of Zheng Dan 958 ₂The part individual plant electrophoresis pattern that IDP709 isozygotys in the site in the generation

population.Swimming lane

1,3,10,13,25,37,42 is IDP709 site heterozygosis or the fuzzy swimming lane that is difficult to differentiate of banding pattern, and all the other swimming lanes isozygoty in this site performance.

Embodiment

Present embodiment is a basic material with single cross hybrid Zheng Dan 958, the selecting and breeding corn inbred line.

Experimental technique in the present embodiment if no special instructions, is conventional method.Percentage composition in the present embodiment if no special instructions, is the quality percentage composition.

The various IDP primers that use in the present embodiment are combined and are formed in the living worker in Shanghai; Biological reagents such as Taq archaeal dna polymerase, dNTP are all available from Transgene biotech firm.

One, screening is to Zheng Dan 958 S ₀The IDP primer that has polymorphism for colony.

One) extracts Zheng Dan 958 single cross hybrid, maternal Zheng 58 and the prosperous 7-2 genomic DNA of male parent.

Extract Zheng Dan 958 single cross hybrid, maternal Zheng 58 and the prosperous 7-2 genomic DNA of male parent according to the CTAB method, concrete grammar is following.

1, gets the fresh blade 1g of Zheng Dan 958, Zheng 58, prosperous 7-2 individual plant respectively, put into the 1.5ml centrifuge tube, put into the liquid nitrogen precooling then.

2, the centrifuge tube in the taking-up liquid nitrogen, the blade that grinds fast in the centrifuge tube is changeed in electricity consumption, till the shape of claying into power.

3, will be equipped with to add in the centrifuge tube of blade powder and be preheated to 65 ℃ CTAB buffer solution (1.4M NaCl; 20mM EDTA, pH=8.0; 100mM TrisHCl, pH=8.0; 3%CTAB; 0.2% beta-mercaptoethanol) 400 μ l, the tight pipe lid of lid is put upside down mixing rapidly.

4, centrifuge tube is placed 65 ℃ of water-baths, temperature is bathed 30min, and jog is 2-3 time therebetween.

5, take out centrifuge tube, every pipe adds 400 μ l Lv Fang ︰ isoamyl alcohol (24 ︰ 1), slight thoroughly mixing, the centrifugal 10min of 12000g under the room temperature.

6, get supernatant, add 800 μ l absolute ethyl alcohols, put upside down mixing, place-20 ℃ of refrigerator 30min with abundant deposit D NA.

7, the centrifugal 10min of 12000g under the room temperature abandons and adds 70% absolute ethyl alcohol, 800 μ l washing 2-3min, the centrifugal 5min of 8000g under the room temperature behind the supernatant.

8, abandon supernatant, air-dry on super-clean bench, add and contain the abundant dissolving DNA of 10 μ g/ml RNaseA aqua sterilisa 50 μ l.Dissolving is placed in-20 ℃ of refrigerators subsequent use.

The DNA that extracts detects purity and concentration through ultraviolet spectrophotometer, and the DNA that dilution is made into 50ng/ μ l can carry out pcr amplification, and amplified production separates acquisition amplification finger-print through agarose gel electrophoresis.

Two) IDP primer amplification.

Present embodiment obtains 20 couples of primer: IDP182, IDP126, IDP2485, IDP456, IDP125, IDP484, IDP483, IDP281, IDP4030, IDP89, IDP3887, IDP177, IDP4082, IDP705, IDP535, IDP119, IDP709, IDP549, IDP582 and IDP1471 between the male parent of Zheng Dan 958 and female parent, having polymorphism from the screening of randomly drawing totally 68 the IDP primers on the maize chromosome of being distributed in; More than 20 pairs of primers satisfy every pair of homologous chromosome and get 2 pairs of IDP primers respectively, the genetic distance condition of 50cM at least between 2 pairs of IDP primers.Said IDP primer sequence is from website www.maizegdb.org.

Concrete experimental technique is following:

1, in advance pcr amplification appearance (GeneAmp PCR System 9700) is heated to 94 ℃ (thermal starting can guarantee to obtain special pcr amplification product);

2, in the PCR pipe, add following component successively:

3, increase according to following program: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 45sec, 58 ℃ of annealing 1min (different I DP annealing temperature is different, the Tm value that provides with reference to maizegdb), 72 ℃ are extended 2min (asynchronism(-nization) of different I DP primer extension is pressed the product size with the conversion of 1KB/min speed), 30 circulations; Extend 7min after 72 ℃ then; 4 ℃ of maintenances.

Three) electrophoresis, gel imaging.

Mixing PCR product and 0.2 times of volume 6 * year appearance buffer solution carry out 1.0% agarose electrophoresis, firm power 120W, and electrophoresis 0.5-1.5h is split up into standard with concrete IDP amplified production polymorphic bands.Select bands of a spectrum clear based on Ago-Gel development collection of illustrative plates, have the effective polymorphism primer of the primer of polymorphism as this test.The result is presented at 20 IDP primer I DP182, IDP126, IDP2485, IDP456, IDP125, IDP484, IDP483, IDP281, IDP4030, IDP89, IDP3887, IDP177, IDP4082, IDP705, IDP535, IDP119, IDP709, IDP549, IDP582 and the IDP1471 between Zheng Dan 958 female parents and male parent, having polymorphism that selection obtains in 68 IDP primers and is 958 S to Zheng Dan ₀The IDP primer that has polymorphism for colony.Wherein, the Ago-Gel collection of illustrative plates of part primer I DP182, IDP2485, IDP125, IDP483, IDP4030, IDP3887, IDP4082 is as shown in Figure 1.

Agarose gel electrophoresis carries out according to following method.

1, cleans plastic cement groove, glue holder and wiped clean with running water, place on the level table.

2, (1 * TAE) in order to fill electrophoresis tank and configuration gel for the electrophoretic buffer of configuration capacity.

3, be added in the triangular flask or vial of the electrophoretic buffer that fills the amount of reserving by the accurate weighing agar of 1.0% (m/v) concentration dry powder.

4, cover triangular flask or vial gently with a lid, in micro-wave oven, be heated to agarose and melt.

5, from micro-wave oven, take out triangular flask or vial with isolating gloves or clip, gel to be melted is cooled to adding ethidium bromide in back about 55 ℃ slightly, and final concentration is 0.5 μ g/ml.Rotate gently with abundant mixing gel solution.

When 6, Ago-Gel solution is cooling off, form well with a suitable comb.The position of broach should be on tray bottom surface 0.5-1.0 mm.

7, the warm agarose solution of pouring gets in the glue groove.Room temperature held 20-30min adds a small amount of electrophoresis liquid in the gel top, carefully extracts comb, pours out electrophoresis liquid, and gel is placed in the electrophoresis tank.

8, in electrophoresis tank, add electrophoretic buffer, just do not have the about 1mm of gel.

9, hybrid dna sample and 0.2 times of volume 6 * year appearance buffer solution.

10, with micropipettor and disposable tip sample mix liquid is slowly added in the well of submergence gel.The molecular mass standard should add in the hole on sample well left side or right side.

11, shut the electrophoresis tank lid, insert good electrode plug.DNA answers anode (red plug) breathing arm moving, gives the voltage of 1-5V/cm, and wherein distance is as the criterion with the measurement between anode to the negative electrode.

12, when DNA sample or dyestuff in gel, moved enough apart from the time, shut power supply, extract electrode plug and open the electrophoresis tank lid and take out gel and on the gel imaging appearance, take a picture.

Wherein, test prepares according to following method with solution.

1) 50 * TAE storage liquid

242g Tris alkali

57.1ml glacial acetic acid

0.5mol/L?EDTA(pH8.0)　100ml

After adding an amount of distilled water stirring and dissolving, be settled to 1L normal temperature and preserve.

2) 6 * gel loading buffer

0.25% (m/V) bromophenol blue

The blue or green FF of 0.25% (m/V) xylol

40% (m/V) aqueous sucrose solution

After pressing mass volume ratio weighing stirring and dissolving, be made into 200ml with ultra-pure water, funnel filters, 4 ℃ of preservations.

3) 0.5mol/L EDTA, pH8.0 solution

EDTA-Na·2H ₂O　　　　186.1g

With 186.1g two water disodium ethylene diamine tetraacetate (EDTA-Na2H ₂O) add in the 800ml water, vigorous stirring on magnetic stirring apparatus, the pH value to 8.0 (about 20g NaOH particle) with the NaOH regulator solution is settled to 1L, autoclaving after the packing.

Two, S ₁Homozygotic screening in the colony.

Utilize that step 1 screens to Zheng Dan 958 S ₀For 10 pairs of IDP primers that colony has polymorphism, screening S ₁In the generation population in these 10 IDP primer sites the individual plant that isozygotys of 8 IDP primer sites and obtain its selfing fruit ear at random.Concrete grammar is following.

1, Zheng Dan 958 seeds is pressed conventional method sowing, field management.Choose 20 strains normal Zheng Dan 958 F that grow in the loose powder phase ₁For the strict pollination results of plant selfing fruit ear.20 fringe selfing fruit ear seed (S with results ₁Seed) mixes threshing monoseeding, field cultivation, at S ₁Colony plant three leaves are picked at random 2000 strain marks during one heart stage, the fresh blade of clip 1g ,-20 ℃ of freezing preservations.

2, extract the leaf DNA of 2000 strain individual plants respectively according to step 1 CTAB method, the leaf DNA of being extracted detects purity and concentration through ultraviolet spectrophotometer, and dilution is made into the DNA of 50ng/ μ l, and-20 ℃ of refrigerators are preserved.

3, with 10 pairs of IDP polymorphism primers (IDP126, IDP456, IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549, IDP1471) respectively to S ₁2000 strain corn gene group DNAs in the colony carry out pcr amplification, according to agarose gel electrophoretogram screening obtain at random individual plant 7 strains that all isozygoty in IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549,8 IDP sites of IDP1471 in these 10 IDP primer sites (in to 10 IDP primer sites other at random 44 of all isozygotying in 8 IDP sites be combined into the row filter statistics and obtain the individual plant that isozygotys in 8 IDP sites of 316 strains; Obtain 8 individual plant 323 strains of all isozygotying in the IDP site at random in above-mentioned 10 IDP primer sites altogether).Fig. 2 identifies S for utilizing primer I DP1471 ₁The part individual plant amplification electrophoresis pattern that IDP1471 isozygotys in the site in the colony.The individual plant that said IDP primer sites is isozygotied is S ₁In the generation population with the male parent of Zheng Dan 958 or the maternal consistent individual plant of this IDP primer extension product banding pattern.IDP primer amplification, electrophoresis, gel imaging are operated according to the method for step 1.

The 7 strain individual plants that will in IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549, IDP1471 primer sites, all isozygoty (with select the corresponding S of plant in 316 strains isozygotied in other 8 sites ₁Plant) through menu strain pollination self in the evaluation of field, further to accept or reject again after the species test of results fruit ear, the fruit ear of choosing is preserved.

Lay respectively on 10 pairs of homologous chromosomes (IDP126, IDP456, IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549 and IDP1471 are respectively on 1,2,3,4,5,6,7,8,9, No. 10 chromosome) in order to 10 IDP primers of Analysis and Identification, thus between primer sites at S ₁On behalf of separating at random, do not receive the influence of gene linkage etc.According to law of segregation and the permutation and combination computing formula of n heterozygous genes self progeny homozygous gene (1/2) n of independent inheritance can draw at selfing S mutually in the mendelian inheritance ₁In generation 2000 parts of materials, in 10 sites at random the theoretical value of 8 site homozygous individuals be 351, this test S ₁For obtaining 323 strains of homozygote plant in the colony, actual result and gross data error are 8.0%, and this possibly be to exist cause inclined to one side separation the in various degree because colony is less with part IDP primer.

Three, S ₂Homozygotic screening in the colony

Utilize 12 IDP primers that do not isozygoty, screen corresponding S ₂Generation population obtains the S that isozygotys 12 IDP primer sites ₂The generation population individual plant is candidate's corn inbred line.Said 12 the IDP primer sites of not isozygotying comprise IDP126, the IDP456IDP site of not isozygotying in used 10 IDP sites in IDP182, IDP2485, IDP125, IDP483, IDP4030, IDP3887, IDP4082, IDP535, IDP709, IDP582 and the step 1.(2 IDP sites of not isozygotying in 44 combinations that in other 10 IDP primer sites all isozygoty in 8 IDP sites are for different S ₁In select 2 IDP sites of plant indication different).Concrete grammar is following.

1, plantation last year results and through the field economical character select IDP484, IDP281, IDP89, IDP177, IDP705, IDP119, IIDP549 and, 83 fruit ear S that the IDP primer sites is all isozygotied such as IDP1471 ₂Seed (in 7 strains select field agronomic shape 3 preferably), every fringe kind is a single file, totally 51 individual plants (77 fruit ears plantations single file, totally 1309 individual plants are chosen in 44 combinations that in other 10 IDP primer sites all isozygoty in 8 IDP sites).

2, treat that seedling grows to three leaves during one heart stage, all S of clip ₂The new blade 1g of individual plant of generation extracts leaf DNA according to the CTAB method of step 1, and the DNA of extraction detects through ultraviolet spectrophotometer, and dilution is made into the DNA of 50ng/ μ l, and-20 ℃ of refrigerators are preserved.

3, S ₂In the colony plant, according to field plant table shape qualification result, select 2 comparatively neat head progeny rows of characters with plant, totally 34 individual plants are distinguished the genomic DNA of its plant leaf one by one to S with described 12 the IDP polymorphism primers of step 3 ₂34 strain maize leaf DNA in the colony carry out pcr amplification, screen the individual plant that all isozygotys 12 IDP primer sites according to the agarose gel electrophoresis bands of a spectrum.The individual plant that said IDP primer sites is isozygotied is S ₂In the generation population with the consistent individual plant of this IDP primer extension product banding pattern of Zheng Dan 958 female parents or male parent.The same step 2 of concrete grammar.The result is at S ₂In generation, obtain 4 individual plants that 20 pairs of primer sites are isozygotied fully in 34 individual plants altogether, with these 4 individual plant selfing fruit ears as candidate's inbred line.Wherein, electrophoresis pattern (other 44 combination S as shown in Figure 3 of the part individual plant that in the S2 generation population that screens of IDP709 isozygoty in the IDP709 site ₂Obtain 49 individual plants that 20 pairs of primer sites are isozygotied fully altogether, all S in 306 individual plants of generation 18 head progeny rows ₂53 individual plants of Dai Zhongxuan are distributed in 18 head progeny rows).

S ₂Consistent for colony's typhon mouth phase plant growing way, show the S that screening obtains through the site homozygote ₂For complete stool type proterties neat and consistent comparatively between plant in colony's head progeny row.

S ₀In colony, the heterozygosis site of 20 independent inheritances does not add any selection, separates the offspring at random at S ₂Homozygotic probability is merely 0.3% (homozygote ratio computing formula is { 1-1/2r} in the colony in generation ⁿ, r representes selfing algebraically, n representes the independent inheritance gene pairs).This test utilizes 20 pairs of IDP polymorphism primers at S ₁, S ₂Identify homozygous individual in the two generation colonies, the result is at S ₂Obtain 53 individual plants that 20 pairs of primer sites are isozygotied fully altogether in 340 individual plants, the homozygote probability reaches 15.6%.Utilize the IDP molecular marker assisted selection to improve efficiency of selection and reach 52 times.Explanation combines the field plant type to identify the effect highly significant that homozygote is selected with the IDP molecular labeling.

Four, coordinate force/blood relationship is identified

At S ₂In colony; Comparatively neat 10 head progeny rows of plant type shape and China corn advantage crowd-Reid advantage crowd in corresponding 18 head progeny rows of 53 individual plants that 20 pairs of primer sites selecting step 3 to obtain are isozygotied fully, Lancaster advantage crowd, Luda red bone advantage crowd, the advantage crowd of Siping City, the pool, PB advantage crowd tuck in 478,5 surveys such as Mo17, B73, yellow morning four, P138 join inbred line and hybridize assembly; Design with incomplete diallel cross NC II; Promptly with tuck in 478, Mo17, B73, yellow early four, P138 is male parent, respectively with selected 10 head progeny rows in a plant be that female parent is hybridized.50 hybrid combinations of assembly altogether, each hybrid combination 34 strain is carried out coordinate force/blood relationship and is identified.8 head progeny rows that obtain output general combining ability higher (greater than 0.10) according to the coordinate force qualification result are as inbred line, and its selfing fruit ear (3 generation seed) is preserved in order to next step corn breeding and used.

Claims

1. utilize the method for IDP molecular marking supplementary breeding corn inbred line, may further comprise the steps:

3) utilize that step 1) screens to corn inbred lines basic material S ₀Have for colony in the IDP primer of polymorphism and remove step 2) in the 20-n of used IDP site outside isozygotying to the IDP primer, screening S ₂Generation population obtains the S that the IDP primer sites is isozygotied at said 20-n ₂The generation population individual plant is candidate's corn inbred line.

2. the method for utilizing IDP molecular marking supplementary breeding corn inbred line according to claim 1, the span that it is characterized in that said n is in 6≤n≤8 zones.

3. the method for utilizing IDP molecular marking supplementary breeding corn inbred line according to claim 1 is characterized in that said corn inbred lines basic material is single cross hybrid, intervarietal cross kind, kind and inbred line intermolecular hybrid kind or synthetic cross variety.

4. utilize the method for IDP molecular marking supplementary breeding corn variety Zheng Dan 958 inbred lines, this method may further comprise the steps:

5. the method for utilizing IDP molecular marking supplementary breeding corn variety Zheng Dan 958 inbred lines according to claim 4, the span that it is characterized in that said n is in 6≤n≤8 zones.

6. the candidate's corn inbred line that utilizes the method for the IDP molecular marking supplementary breeding corn inbred line of claim 1 to obtain combines S ₁, S ₂The economical character of generation population and S ₂Coordinate force/blood relationship is measured selecting and breeding corn inbred line as a result from generation to generation.