CN105925722A - Acquisition method of soybean-protein-content-related QTLs (quantitative trait loci) and molecular markers, molecular markers and application thereof - Google Patents

Abstract

The invention provides two soybean-protein-content-related QTLs (quantitative trait loci) and molecular markers, and an acquisition method and application of the molecular markers. The QTLs are respectively positioned on linkage groups A2 and N and are respectively positioned by the molecular markers Satt409 and Satt584. The 5'-3' primer of the molecular marker Satt409 is disclosed as SEQ ID NO.3, and the 3'-5' primer is disclosed as SEQ ID NO.4. The 5'-3' primer of the molecular marker Satt584 is disclosed as SEQ ID NO.5, and the 3'-5' primer is disclosed as SEQ ID NO.6. The acquisition method of the molecular markers comprises the following steps: by respectively using the corresponding soybean individual genome DNA as a template, carrying out PCR (polymerase chain reaction) amplification by using the 5'-3' primers and 3'-5' primers, and separating the PCR amplification products by polyacryloyl ammonia gel electrophoresis to obtain the target molecular markers. The obtained molecular markers can be used for soybean molecular breeding.

Description

The QTL relevant to soy protein content and the preparation method of molecular labeling, molecule mark Note and application

Technical field

The invention belongs to biology field, be specifically related to the QTL relevant to soy protein content and molecular labeling Preparation method, molecular labeling and application.

Background technology

Soybean be China so that world wide in the middle of be all important industrial crops, its reason is that soybean is people One of main source of vegetable protein needed in daily life, soybean is again the important feed work of livestock breeding industry simultaneously Thing.Therefore, the protein content in the middle of Seeds of Soybean becomes one of important indicator weighing soybean varieties.But, current China The main high protein variety soybean produced cannot meet people's daily productive life needs.Therefore, modernization molecular breeding is utilized Technology improves existing soybean varieties protein content and becomes the main task of breeding scientific research personnel in the present age.

Soybean protein content belongs to the Inheritance of Quantitative Characters of controlled by multiple genes, and this proterties is carried out qtl analysis and location, The SSR site of value can be excavated, to cultivate the new varieties of good quality and high output.Both at home and abroad soybean protein QTL was done Big component analysis, but efficient site effectively not being determined also does not has the true effect in clear and definite site, thus cannot be direct It is applied in the middle of actual breeding, and is likely to occur the loss in important site.

Molecular mark has been obtained for certain development in recent years, but in place of but having some shortcomings.First, Soybean protein is by the Inheritance of Quantitative Characters of controlled by multiple genes, and this mode of inheritance is by controlled by multiple genes, because single Site is the least on the impact of proterties, it is necessary to by its manipulation of multiple gene pairs, add the envirment factor impact for phenotype The biggest, so the effect in single site is difficult to be evaluated directly out, cause cannot applying in the middle of actual breeding.For number The Preliminary study of amount character inheritance, is minor-polygene to be regarded as an entirety biometrical method analyze.Go out the most again Show minor-polygene hypothesis, it is believed that quantitative inheritance is to have multiple minor gene co-controlling.Up-to-date achievement in research channel syndrome This hypothesis real is defective.Inheritance of Quantitative Characters is to have a major gene resistance and multiple minor gene jointly to regulate and control, and Gene prediction programs is the biggest on the impact of phenotype.It may be said that the performance of quantitative character be exactly main effect site, minor effect site and Complete under envirment factor joint effect.Therefore from the point of view of from the aspect of gene, it is judged that each site is to protein content Effect value just seems particularly important.

Additionally layered queuing networks is a kind of statistical method verifying as means with Chi-square Test and T, for analyzing one group of impact The effect value that a certain effect of factor pair produces.It it is a kind of statistical method with a certain proterties of Comprehensive Evaluation standard analysis.It is at present Only layered queuing networks application in the middle of biological field is actually rare.At present, lot of domestic and foreign soybean breeder scholar is big in research The quantitative inheritance proterties of beans.The article of relevant MAS and the QTL location delivered at present mostly is the mark of research and the potential of site Using value, is seldom really applied to it in actual breeding, and rarer people effect value genetic to quantity provides conclusion, At Seeds of Soybean protein content, on the one hand this be also a blank.It is known that Seeds of Soybean protein content is quantitative Shape heredity, by controlled by multiple genes.The impact of this proterties of gene pairs necessarily having in these genes is relatively big, and some then affects relatively Little.Before the concrete impact learning each gene pairs this proterties of protein content, it is difficult to these are directly applied to reality In the middle of breeding.Taking layered queuing networks is a relatively effective solution.

Summary of the invention

For the problems referred to above, the present invention is by statistical analysis techniques such as layered queuing networks, it is provided that 5 kinds with soybean protein in soybean The closely-related QTL of matter content, and verify each soy protein content accurately and be correlated with the effect value of QTL and effect side By the calculating of rate of change, formula, finds that 3 crucial QTL can increase protein content, 2 crucial QTL can reduce soybean egg White matter content.Thus directly applied in the middle of actual molecules breeding.

In five QTL that the present invention provides, Satt683 belongs to the main effect QTL of Seeds of Soybean protein content.Satt409、 Satt180, Satt584, Satt181 belong to efficient QTL.

Allele Satt683-3, Satt409-2 and the Satt584-5 wherein comprised for soybean protein content this Trait expression is positive correlation, i.e. the allele of its correspondence effect in Seeds of Soybean is to promote that soy protein content increases Add, and Satt181-2 and Satt180-5 is negative correlation for this trait expression of soybean protein content, be i.e. the equipotential of its correspondence Gene effect in Seeds of Soybean is that suppression larger protein content increases.

The information of 3 QTLs relevant to soybean protein proterties is shown in Table 1, and the allele wherein listed is for proterties just Relevant allele.

The QTL that 13, table is relevant to soybean protein proterties

The information part table 2 of 2 QTLs relevant to soybean protein proterties, the allele wherein listed is for bearing with proterties Relevant allele.

The QTL that 22, table is relevant to soybean protein proterties

Can directly use primer amplification, identify whether kind to be measured contains this allele, for molecular labeling auxiliary choosing Select.

(1), the present invention provides a QTL relevant to soy protein content: be positioned in linkage group N, at public collection of illustrative plates Genomic locations be 27.787cM, molecular labeling Satt683 position；The 5 '-3 of molecular labeling Satt683 ' primer such as SEQ Shown in ID NO.1,3 '-5 ' primer is as shown in SEQ ID NO.2.

This QTL comprises the allele Satt683-3 that effect value is 12%, and this allele is with soy protein content just Relevant.

The present invention also provides for the preparation method of the molecular labeling of the above-mentioned QTL relevant to soy protein content: this molecule 5 '-3 ' primer as shown in SEQ ID NO.1,3 '-5 ' primer of mark is as shown in SEQ ID NO.2；With soybean varieties Beidou 5 For recurrent parent, hybridize with swan egg ZDD02114, backcross the soybean individuality base obtained afterwards through twice hybridization and twice altogether Because group DNA is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, and its PCR amplification system is 10 μ L, including the DNA profiling 0.8 μ L of 1.5ng/ μ L, the 5 '-3 of 0.5 μm ol/L ' primer, 3 '-5 ' each 0.5 μ L, 200mmol/L's Taq enzyme 0.05 μ L, the 10XBuffer1 μ L of dNTP0.6 μ L, 5U/ μ L, cation uses MgCl₂Final concentration 1.5-2.0mmol/L, Remaining is supplied with ultra-pure water；PCR amplification program is denaturation 94 DEG C 5 minutes；Sex change 94 DEG C 40 seconds, annealing 47 DEG C 40 seconds, extend 72 DEG C 40 seconds, circulate 35 times；Extend 72 DEG C 5 minutes；4 DEG C are terminated reaction.The product above-mentioned PCR amplification obtained is through PCR primer Deformation procedure: 94 DEG C 5 minutes, after electrophoretic separation, the size obtained is the amplified fragments of 450bp, 430bp or 410bp, is mesh Mark molecular labeling.

The present invention also provides for the molecular labeling of the above-mentioned QTL relevant to soy protein content: the 5 '-3 of this molecular labeling ' Primer as shown in SEQ ID NO.1,3 '-5 ' primer is as shown in SEQ ID NO.2.

The present invention also provides for the above-mentioned molecular labeling method for soybean molecular breeding: the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.1,3 '-5 ' primer is as shown in SEQ ID NO.2；With its 5 '-3 ' primer and 3 '-5 ' draw to be measured greatly The genomic DNA of beans kind carries out PCR amplification, and its PCR amplification system is 10 μ L, including the DNA profiling 0.8 μ L of 1.5ng/ μ L, The Taq enzyme 0.05 μ L of dNTP0.6 μ L, the 5U/ μ L of the 5 '-3 of 0.5 μm ol/L ' primer, 3 '-5 ' each 0.5 μ L, 200mmol/L, 10XBuffer1 μ L, cation uses MgCl₂Final concentration 1.5-2.0mmol/L, remaining is supplied with ultra-pure water；PCR amplification program For denaturation 94 DEG C 5 minutes；Sex change 94 DEG C 40 seconds, annealing 47 DEG C 40 seconds, extend 72 DEG C 40 seconds, circulate 35 times；Extend 72 DEG C 5 points Clock；4 DEG C are terminated reaction.By above-mentioned amplified production through PCR primer deformation procedure: 94 DEG C 5 minutes, after electrophoretic separation, if amplification There is the amplified fragments that size is 450bp, 430bp or 410bp in product, then this soybean varieties to be measured has and soybean protein The QTLSatt683 that content is relevant, if there is the amplified fragments that size is 410bp in amplified production, then this soybean varieties to be measured is There is the allele Satt683-3 increasing soy protein content in the QTLSatt683 relevant to soy protein content Kind, otherwise, kind to be measured does not have this QTL or this allele.

(2), another QTL relevant to soy protein content that the present invention provides: be positioned in linkage group A2, in public affairs The genomic locations of collection of illustrative plates is 145.565cM altogether, molecular labeling Satt409 position, the 5 '-3 of molecular labeling Satt409 ' draw Thing as shown in SEQ ID NO.3,3 '-5 ' primer is as shown in SEQ ID NO.4.

This QTL comprises the allele Satt409-2 that effect value is 10%, and this allele is with soy protein content just Relevant.

The present invention also provides for the preparation method of the molecular labeling of the above-mentioned QTL relevant to soy protein content: this molecule 5 '-3 ' primer as shown in SEQ ID NO.3,3 '-5 ' primer of mark is as shown in SEQ ID NO.4；With soybean varieties Beidou 5 For recurrent parent, hybridization yellow with swan egg ZDD02114 or middle, backcross the soybean obtained afterwards through twice hybridization and twice altogether Genes of individuals group DNA is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, and its PCR expands System and program are with (one)；The product above-mentioned PCR amplification obtained is after electrophoretic separation, and the size obtained is 360bp, 330bp Or the amplified fragments of 300bp, it is target molecule mark.

The present invention also provides for the molecular labeling of the above-mentioned QTL relevant to soy protein content: the 5 '-3 of this molecular labeling ' Primer as shown in SEQ ID NO.3,3 '-5 ' primer is as shown in SEQ ID NO.4.

The present invention also provides for the above-mentioned molecular labeling method for soybean molecular breeding: the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.3,3 '-5 ' primer is as shown in SEQ ID NO.4；With its 5 '-3 ' primer and 3 '-5 ' draw to be measured greatly The genomic DNA of beans kind carries out PCR amplification, and its PCR amplification system and program are with (one)；Above-mentioned amplified production is divided through electrophoresis From rear, if amplified production exists the amplified fragments that size is 360bp, 330bp or 300bp, then this soybean varieties to be measured tool There is the QTLSatt409 relevant to soy protein content, if amplified production exists the amplified fragments that size is 330bp, then should Soybean varieties to be measured be have in the QTLSatt409 relevant to soy protein content increase soy protein content etc. The kind of position gene Satt409-2, otherwise, kind to be measured does not have this QTL or this allele.

(3), another QTL relevant to soy protein content that the present invention provides: be positioned in linkage group N, public The genomic locations of collection of illustrative plates is 29.37cM, molecular labeling Satt584 position, the 5 '-3 of molecular labeling Satt584 ' primer is such as Shown in SEQ ID NO.5,3 '-5 ' primer is as shown in SEQ ID NO.6.

This QTL comprises the allele Satt584-5 that effect value is 10%, and this allele is with soy protein content just Relevant.

The present invention also provides for the preparation method of the molecular labeling of the above-mentioned QTL relevant to soy protein content: this molecule 5 '-3 ' primer as shown in SEQ ID NO.5,3 '-5 ' primer of mark is as shown in SEQ ID NO.6；With soybean varieties Beidou 5 For recurrent parent, hybridize with swan egg ZDD02114, backcross the soybean individuality base obtained afterwards through twice hybridization and twice altogether Because group DNA is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, its PCR amplification system and Program is with (one)；The product above-mentioned PCR amplification obtained is after electrophoretic separation, and the size obtained is 400bp, 350bp or 300bp Amplified fragments, be target molecule mark.

The present invention also provides for the molecular labeling of the above-mentioned QTL relevant to soy protein content: the 5 '-3 of this molecular labeling ' Primer as shown in SEQ ID NO.5,3 '-5 ' primer is as shown in SEQ ID NO.6.

The present invention also provides for the above-mentioned molecular labeling method for soybean molecular breeding: the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.5,3 '-5 ' primer is as shown in SEQ ID NO.6；With its 5 '-3 ' primer and 3 '-5 ' draw to be measured greatly The genomic DNA of beans kind carries out PCR amplification, and its PCR amplification system and program are with (one)；Above-mentioned amplified production is divided through electrophoresis From rear, if amplified production exists the amplified fragments that size is 400bp, 350bp or 300bp, then this soybean varieties to be measured tool There is the QTLSatt584 relevant to soy protein content, if amplified production exists the amplified fragments that size is 300bp, then should Soybean varieties to be measured be have in the QTLSatt584 relevant to soy protein content increase soy protein content etc. The kind of position gene Satt584-5, otherwise, kind to be measured does not have this QTL or this allele.

(4), another QTL relevant to soy protein content that the present invention provides: be positioned in linkage group C1, in public affairs The genomic locations of collection of illustrative plates is 105.092cM altogether, molecular labeling Satt181 position, the 5 '-3 of molecular labeling Satt181 ' draw Thing as shown in SEQ ID NO.7,3 '-5 ' primer is as shown in SEQ ID NO.8.

This QTL comprises the allele Satt181-2 that effect value is 8%, and this allele is born with soy protein content Relevant.

The present invention also provides for the preparation method of the molecular labeling of the above-mentioned QTL relevant to soy protein content: this molecule 5 '-3 ' primer as shown in SEQ ID NO.7,3 '-5 ' primer of mark is as shown in SEQ ID NO.8；With soybean varieties Beidou 5 For recurrent parent, and understand greatly bean or pea or Chinese mugwort card 166 hybridization, individual through twice hybridization and twice soybean obtained afterwards that backcrosses altogether Body genomic DNA is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, and its PCR expands body System and program are with (one)；By the above-mentioned PCR product that obtains of amplification after electrophoretic separation, the size obtained be 300bp, 280bp or The amplified fragments of 180bp, is target molecule mark.

The present invention also provides for the molecular labeling of the above-mentioned QTL relevant to soy protein content: the 5 '-3 of this molecular labeling ' Primer as shown in SEQ ID NO.7,3 '-5 ' primer is as shown in SEQ ID NO.8.

The present invention also provides for the above-mentioned molecular labeling method for soybean molecular breeding: the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.7,3 '-5 ' primer is as shown in SEQ ID NO.8；With its 5 '-3 ' primer and 3 '-5 ' draw to be measured greatly The genomic DNA of beans kind carries out PCR amplification, and its PCR amplification system and program are with (one)；Above-mentioned amplified production is divided through electrophoresis From rear, if amplified production exists the amplified fragments that size is 300bp, 280bp or 180bp, then this soybean varieties to be measured tool There is the QTLSatt181 relevant to soy protein content, if amplified production exists the amplified fragments that size is 280bp, then should Soybean varieties to be measured be have in the QTLSatt181 relevant to soy protein content reduce soy protein content etc. The kind of position gene Satt181-2, otherwise, kind to be measured does not have this QTL or this allele.

(5), another QTL relevant to soy protein content that the present invention provides: be positioned in linkage group H, public The genomic locations of collection of illustrative plates is 87.597cM, molecular labeling Satt180 position, the 5 '-3 of molecular labeling Satt180 ' primer is such as Shown in SEQ ID NO.9,3 '-5 ' primer is as shown in SEQ ID NO.10.

This QTL comprises the allele Satt180-5 that effect value is 8%, and this allele is born with soy protein content Relevant.

The present invention also provides for the preparation method of the molecular labeling of the above-mentioned QTL relevant to soy protein content: this molecule 5 '-3 ' primer as shown in SEQ ID NO.9,3 '-5 ' primer of mark is as shown in SEQ ID NO.10；With soybean varieties north beans 5 Number being recurrent parent, and Biogedulote or Chinese mugwort card 166 or auspicious rich No. 2 hybridization, altogether after twice hybridization and twice backcross Soybean genes of individuals group DNA obtained is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR expansion to above-mentioned template Increasing, its PCR amplification system and program are with (one)；The product above-mentioned PCR amplification obtained is after electrophoretic separation, and the size obtained is The amplified fragments of 350bp, 340bp or 300bp, is target molecule mark.

The present invention also provides for the molecular labeling of the above-mentioned QTL relevant to soy protein content: the 5 '-3 of this molecular labeling ' Primer as shown in SEQ ID NO.9,3 '-5 ' primer is as shown in SEQ ID NO.10.

The present invention also provides for the above-mentioned molecular labeling method for soybean molecular breeding: the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.9,3 '-5 ' primer is as shown in SEQ ID NO.10；Draw to be measured with its 5 '-3 ' primer and 3 '-5 ' The genomic DNA of soybean varieties carries out PCR amplification, and its PCR amplification system and program are with (one)；By above-mentioned amplified production through electrophoresis After separation, if amplified production exists the amplified fragments that size is 350bp, 340bp or 300bp, then this soybean varieties to be measured There is the QTLSatt180 relevant to soy protein content, if amplified production existing the amplified fragments that size is 300bp, then This soybean varieties to be measured is to have the minimizing soy protein content in the QTLSatt180 relevant to soy protein content The kind of allele Satt180-5, otherwise, kind to be measured does not have this QTL or this allele.

Above-mentioned electrophoretic separation, for polyacrylamide gel electrophoresis or Capillary Electrophoresis.Understand the effect of protein related gene Should be worth, whether we can be just eggs by checking the soybean whether having efficient site to judge this kind in the middle of certain soybean varieties The high quality soybean that Bai Hanliang is high, thus realizes the actual application in the middle of marker assisted selection of this gene.

The present invention determines the QTL important, effect is big in known a large amount of soybean protein qualitative correlation QTL, and determines each The concrete impact of individual gene pairs soybean protein shape: be positive correlation or negative correlation, and utilize these QTL to realize with molecule mark The means of being designated as carry out breeding.

Accompanying drawing explanation

Banding pattern in Fig. 1 soybean Satt683 numbering 1-61 in the resource soybean of 250 shown in table 1 part；

The allelic chromatographic analysis of Fig. 2 chi-square analysis resource；

The allelic chromatographic analysis of resource of Fig. 3 T-test；

Fig. 4 allele is at the ssr analysis of colony；

The allelic chromatographic analysis of Fig. 5 chi-square analysis colony；

The allelic chromatographic analysis of colony of Fig. 6 T-test；

Each allele of Fig. 7 is rate of change in resource and Bei Dou colony；

The primer of Fig. 8 Satt683 to female parent be Beidou 5 male parent be the genomic DNA of the colony of swan egg ZDD02114 Extension product Capillary Electrophoresis figure；

The primer of Fig. 9 Satt409 to female parent be Beidou 5 male parent be the base of the colony of swan egg ZDD02114 or middle Huang Because group DNA extends product Capillary Electrophoresis figure；

The primer of Figure 10 Satt584 to female parent be Beidou 5 male parent be the genome of the colony of swan egg ZDD02114 DNA extends product Capillary Electrophoresis figure；

The primer of Figure 11 Satt181 to female parent be Beidou 5 male parent be to understand greatly bean or pea or the base of colony of Chinese mugwort card 166 Because group DNA extends product Capillary Electrophoresis figure；

The primer of Figure 12 Satt180 to female parent be Beidou 5 male parent be Biogedulote or Chinese mugwort card 166 or auspicious rich No. 2 Colony genomic DNA extension product Capillary Electrophoresis figure.Detailed description of the invention

In following example: soy protein content measures and all uses FOSS Infratec 1241 grain analyzer； Capillary electrophoresis analysis all uses full-automatic HPCE, and Advanced Analytical Technologies is (beautiful AATI company of state), model Fragment Analyzer^TM。

The plantation of embodiment 1 experiment material and protein content detection:

(1) plantation of experiment material and sampling

Resource material: choose is the main cultivation soybean varieties of 250 parts of CHINESE REGION.Table 1 is the variety name of 250 parts of resources And grain protein content of winter wheat.

Plantation place: academy of agricultural sciences of Jilin Province.

Implantation methods: 1m row is long, often 21 seeds of row.

Management method: same to field management.

The method of sampling: vegetative growth phase, takes the youngest tender place, plant top blade for extracting DNA, PCR reaction.Results Time collect Seeds of Soybean for measuring protein content by individual plant.

Checking material: be that main cultivation soybean varieties Beidou 5 is as recurrent parent, with Thomas's Kato profit using Heilongjiang Province Sa, slope Huang, Harosoy, green 75, middle special No. 1, NOVA, Century, east agriculture 163, Du Najika, Amsoy, middle beans 27,95- 5383, Chinese mugwort card 166, understand bean or pea, Dongshan 69, auspicious rich No. 2, middle Huang, Biogedulote and swan egg ZDD02114 these 19 greatly The soybean of kind hybridizes respectively, altogether backcrosses through twice hybridization and twice and gathers in the crops BC2F2 afterwards.Colony's 330 individualities altogether.

Plantation place: Land Reclamation Research and Breeding Center of Heilongjiang.

Implantation methods: 5m row is long, often 100 seeds of row.

Management method: same to field management.

The method of sampling: vegetative growth phase, takes the youngest tender place, plant top blade for extracting DNA, PCR reaction.Results Time collect Seeds of Soybean for measuring protein content by individual plant.

(2) mensuration of protein content: measure the seed egg of every kind of soybean in above-mentioned resource material and checking material respectively White matter content.

Use step

1) power supply of Infratec 1241 grain analyzer is opened.

2) when instrument carried out self-inspection preheated after, from application model list hurdle, find required mould by upper and lower key Type.Application model title has additional " STM's ".

3) pour Seeds of Soybean into sample cell, after-poppet load docking unit, note there is the side of gear bar on the right. Press support gently downwards, to guarantee gear bar and gear correct engagement.Sample cell support is put into docking unit, at this time little The heart, in order to avoid the little gear within Sun Huaiing.

4) press analysis key to start to analyze in keyboard region.

The data that FOSS Infratec 1241 grain analyzer is measured are " .crd " forms.Need to be first converted into EXCEL Form just can be opened.Derivation data step:

IFT file comprises two catalogues of Disk1, Disk2 and a supporting paper, runs under Disk1 catalogue setup.exe。

Beginning → FOSS → IFT；

Result Data/CRD→TXT-format(columns)。

In Source folder, select the path of 20071030.crd, and before file, choose √.Reselection output road Footpath, such as Desktop, clicks on Convert selected.

Give a name for file, preserve.Exit IFT.Obtain a TXT file.

A newly-built Excel, opens TXT file, selects " list separator " to click on next step, choosing in text import wizard Selecting " tab key " and " comma ", click on next step, click completes.Just FOSS Infratec 1241 grain analyzer can be surveyed The data Excel form of the soy protein content obtained is opened.

1 250 parts of soybean resource titles of table and grain protein content of winter wheat thereof

Embodiment 2 DNA extracts and PCR expands:

(1) (250 parts of resource soybean are all extracted with checking 330 individualities of material, every kind of soybean in the extraction of soybean leaves DNA Extract respectively)

1) the fresh blade being used for extracting DNA, PCR reaction obtained in embodiment 1 is placed in 1.5ml centrifuge tube, will Centrifuge tube is placed on the centrifuge tube shelf being dipped in liquid nitrogen, bores mill with plastics drill bit low temperature, until sample becomes white fine powder end is Only.

2) open water-bath and be preheated to 65 DEG C, CTAB extract (adding mercaptoethanol according to 40ml:10 μ l) is added simultaneously Heat is to 65 DEG C.Temperature will add 0.7mlCTAB extract, fully mixing after reaching in each centrifuge tube after, water-bath 40 points Clock, and interval jog.

3) centrifuge tube after water-bath will add equal-volume chloroform, then centrifugal 20 minutes of 8000 (revs/min).Take supernatant Liquid, adds equal-volume chloroform again.Centrifugal 8000 (rev/min) 20 minutes.

4) take in the 0.7ml isopropanol that supernatant adds to precooling in advance, until DNA separates out.

5) rinse DNA with absolute ethyl alcohol, dry afterwards, dissolve with 80 μ l ultra-pure waters.

6) 1% agarose gel electrophoresis detection.Voltage 100V electric current 100A runs 20 minutes.

Points for attention: 1) tip portion of pipettor yellow rifle head deducts sub-fraction, prevents shearing force from destroying DNA knot Structure)

2) must be slowly soft when of moving liquid and rinse DNA, in order to avoid damaging DNA structure

Table 2 is for extracting the CTAB extract recipe of soybean leaves DNA

(2) PCR reaction

Choose the 29 pairs of primers (being shown in Table 3) being distributed in 13 linkage groups of soy bean DNA, utilize the 29 pairs of SSR primers chosen The genomic DNA of 250 parts of resource soybean is carried out PCR amplification, and passes through electrophoretic analysis: in system shown in table 4, by following journey Sequence expands respectively, and primer is by Shanghai raw work synthesis.

PCR program: denaturation 94 DEG C 5 minutes

Sex change 94 DEG C 40 seconds

Anneal 47 DEG C 40 seconds

Extend 72 DEG C 40 seconds

Circulate 35 times

Extend 72 DEG C 5 minutes

4 DEG C are terminated reaction.

29 pairs of primer locations that table 3 is chosen in 13 linkage groups of soy bean DNA and sequence

Table 4 PCR reacts (10 μ l system)

Selection principle: 1) in 13 linkage groups every, all choose SSR primer as far as possible

2) the several primer genetic distances chosen in a linkage group are the most remote

(3) polyacrylamide gel electrophoresis: the amplified production that above-mentioned steps (2) obtains carries out polyacrylamide gel respectively Electrophoresis detection, obtains 29 pairs of primers of 250 shown in table 1 varieties of resources soybean and checking 330 individual DNA of resource Pcr amplification product banding pattern, part banding pattern is shown in Fig. 1.

Table 5 polyacrylamide gel electrophoresis medicine

Polyacrylamide gel electrophoresis step

Big plate wipes of alcohol is clean, the more affine silane of uniform application (alcohol: affine silane: glacial acetic acid=5ml:50 μ l: 50μl)

Otic placode is put into air extracting cabinet, clean by wipes of alcohol, then uniform application stripping silane.

Clean edge strip is placed on big plate both sides, then covers otic placode.Two boards smears the one side of silane will be inwardly.Use clip Fixing.

200 μ lAPS and 50 μ lTEMED are added in the 6%PA glue that will have configured.Uniformly pour between big plate and otic placode, note There is not bubble in meaning.

Smooth for stripping fork one side is inserted about 5mm between two plates inwardly.Level-off.Stand and can use for about one hour

Upper plate, clamping, buffer solution TBE (upper groove is 1 times of TBE, and lower groove is 1/3 times of TBE) will be poured in electrophoresis tank into.

A small amount of coloring agent, 1800V voltage prerunning about 10 minutes is instilled in point sample groove.Period adds in PCR primer Coloring agent also makees degenerative treatments.

Stripping fork inserts point sample groove (tooth is down), point sample, and 1800V voltage runs 90 minutes.Lower plate.Take off otic placode and limit Bar.

Silver staining: add 1500 μ l silver nitrate solutiones in 1500ml distilled water.Big plate is immersed in silver staining solution 20～30 Minute.

Distilled water flushing half a minute.

10) development: 1500ml distilled water, adds 1500 μ l formaldehyde, 3%NaOH and 6.25gNa2CO3.Big plate is immersed in In silver staining solution 20～30 minutes.

11) statistics banding pattern, records data, scanning.

The clip size that part primer amplification in table 6 layering goes out

Embodiment 3 soy protein content associated alleles layered queuing networks:

(1) soy protein content associated alleles layered queuing networks

The data of the protein content of each kind and reality in 250 varieties of resources shown in the table 1 that embodiment 1 is obtained Executing its banding pattern data one_to_one corresponding obtained in example 2 (3), before choosing protein content, 40% and rear 40% utilizes MATLAB Allelic effect value is analyzed in R2009a Chi-square Test.Using alpha=0.01 and 0.1 as the threshold value of layering, result is divided It is 3 levels.Wherein when checking P value less than 0.01, it is believed that this site is main effect site, and when P value is more than 0.1, recognize For being minor effect site, with reference to Fig. 2, P value be less than 0.01 level (the most notable) allele have Satt683-3, Satt578-4, Satt409-2 and Satt079-1.The level of signifiance has Satt181-2, Satt180-5, Satt126-between 0.1 and 0.01 1、Satt578-2、Satt584-5、Satt409-10、Sat_311-2、Sat_242-2、Satt578-6、Satt584-6、 Satt713-1, Satt012-5, Sat_216-2, Sat_216-3, Satt584-2, Satt700-2 and Satt369-2, remaining Its level of signifiance of allele is all higher than 0.1.

With reference to Fig. 3 after Minitab14.12T checks, the level of signifiance has ten allele less than 0.01, With Chi-square Test before to 4 extremely notable allele contrasts under it appeared that have three allele in the inspection of card side Test with under T inspection, all show as extremely notable, be Satt683-3, Satt578-4 and Satt409-2 respectively.Except this three The allele of the individual pole level of signifiance, remaining allele otherwise show as general significantly or notable, or be exactly The level of signifiance in two kinds of methods of inspection is inconsistent.Can tentatively distinguish major gene resistance and minor gene.

(2) crucial allele checking in Beidou 5 colony

According to resource hierarchy data, select whole primers of the pole level of signifiance and the primer that other level of signifiance is higher. Carry out Chi-square Test and T inspection.Simultaneously in order to verify the establishment of layered effect, one extremely low drawing of significance of reselection Thing.Carry out two kinds of analyses equally.

Choose allele Satt683-3, Satt578-4 and Satt409-2 of the pole level of signifiance, choose Chi-square statistic one As notable and T detection extremely significantly allele Satt180-5, Satt181-2 and Satt584-5, choose Chi-square statistic and T inspection Survey the most inapparent allele Sat_174-2 totally 7 allelic primers (table 3), the group with Beidou 5 as recurrent parent 330 individual genomic DNAs of body are template, in system shown in table 4, carry out PCR amplification by following procedure respectively, and lead to Cross electrophoretic analysis.Primer is by Shanghai raw work synthesis.Part electrophoretogram is shown in Fig. 4.Statistics banding pattern.

PCR program: denaturation 94 DEG C 5 minutes；

Sex change 94 DEG C 40 seconds,

Anneal 47 DEG C 40 seconds,

Extend 72 DEG C 40 seconds,

Circulate 35 times；

Extend 72 DEG C 5 minutes,

4 DEG C are terminated reaction.

Identical to the banding pattern data processing method of resource with the present embodiment (1), to 7 pairs of primers in Beidou 5 colony Banding pattern data carry out Chi-square Test and T checks:

By data and the banding pattern number of the protein content of Beidou 5 introgressive line colony individual (checking 330 individualities of resource) According to one_to_one corresponding

Entirely sort with protein content for keyword.Select front 40% and rear 40%

MATLAB R2009a is utilized to carry out Chi-square Test

Open Minitab14.12 and carry out T inspection.Checking Chi-square Test result.

Result such as Fig. 5 and Fig. 6.

From above two figures it can be seen that Satt683-3 is extremely notable two kinds of detections, this equipotential base is described Because really belonging to the major gene resistance of Seeds of Soybean protein content.Satt409-2 is still extremely notable in T checks, although card side Assay has declined, but stills remain in higher level.Satt180-5 and Satt584-5 but shows at two kinds Inspection is all extremely notable, adds the Satt181-2 consistent with the performance of last round of layered queuing networks, it can be determined that go out these four etc. Position gene should belong to high efficiency gene, simply its action effect not as Satt683-3 obvious.Sat_174-2 is then with last round of Result completely the same, be all notable, illustrate that this gene is really to increase protein content this respect effect value the lowest.Unexpected Be that each allele of the primer Satt578 that the level of signifiance is high level of signifiance in Bei Dou colony is extremely low, very in resource To not finding allele Satt578-4.This pole some phenomena producing cause is probably 20 of Beidou 5 introgressive line colony Not this gene in the middle of the genome of parent, the systematic error that the reasons such as additionally measuring instrument is not accurate enough produce is likely to cause This pole some phenomena occurs.

Therefore, although the layered queuing networks in Liang Ge colony is slightly different, but difference little, level generally still than More apparent, that is, in resource, the level of signifiance is higher, the highest in Beidou 5 colony, the equipotential base of different primers Because the effect in Liang Ge colony is consistent.Thus it is believed that for the allele layered queuing networks of these 29 primers Its conclusion is accurately.

Embodiment 4 utilizes character mutation rate to verify and analyzes allele additive effect:

The present invention indicates whether containing this allelic change by rate of change, this rate of change be based on resource or Phenotypic number difference after the individual in population maturity period, if certain allele has the additivity of positive direction to Seeds of Soybean protein content Effect, then containing the individual phenotypic data of this allelic resource or colony than do not contain this allelic resource or Individual wants height, but is not excluded for the interaction between mark and the impact of the interaction between allele.The calculating of above-mentioned rate of change Method is:

$Rateofchange=\frac{ValueA-ValueB}{ValueB}\×100\%.$

Wherein Rate of change is rate of change, and Value A represents containing the allelic individuality of A or the albumen of resource The mean value of matter content phenotypic number, Value B represents containing B allele or does not contains the allelic individuality of A or resource The mean value of protein content phenotypic number.

Character mutation rate is utilized to verify crucial allele additive effect further

Theoretically, there is certain its protein content of allelic soybean varieties if above there is not this equipotential base The soybean varieties of cause, then then illustrate that this equipotential gene is just showing as positive regulation for this quantitative character of protein content Related gene.Otherwise, this allele is then the gene of negative correlation.The present invention have selected and obtains through twice layered queuing networks screening 5 extremely notable allele, select containing these 5 allelic Beidou 5 microcommunities (table 7).By important primer etc. The rate of change that protein content is produced in resource and Beidou 5 colony by position gene is verified.Result such as Fig. 7.Screening Obtain several primer afterwards and allele verifies colony accordingly.The female parent of colony is the Big Dipper 5, and male parent is shown in Table 7.

Table 7 screening obtains colony for crucial allele

Can clearly find out that from table these 5 allele are on the impact of Seeds of Soybean protein content in resource and colony Effect is consistent.This three allele rate of change in Liang Ge colony of Satt683-3, Satt409-2 and Satt584-5 Being all positive, illustrate that they are positive correlation for this trait expression of soybean protein content, i.e. this allele is big bean or pea Effect in Li is to promote that soy protein content increases.And the rate of change that Satt181-2 and Satt180-5 is in Liang Ge colony It is all negative, it was demonstrated that the two allele is negative correlation for this trait expression of soybean protein content, i.e. this allele Effect in Seeds of Soybean is that suppression larger protein content increases.

The application in molecular marker breeding of the embodiment 5 allele site

The primer (SEQ ID NO.1, SEQ ID NO.2) of QTL site Satt683, the primer of QTL site Satt409 The primer (SEQ ID NO.5, SEQ ID NO.6) of (SEQ ID NO.3, SEQ ID NO.4), QTL site Satt584, QTL position The point primer (SEQ ID NO.7, SEQ ID NO.8) of Satt181, primer (SEQ ID NO.9, SEQ of QTL site Satt180 ID NO.10), 330 individual genomic DNAs of the colony with Beidou 5 as recurrent parent are as template, in system shown in table 4 In, PCR amplification is carried out respectively by following procedure, and by capillary electrophoresis analysis (step and parameter illustrate with reference to instrument).Draw Thing is by Shanghai raw work synthesis.

PCR program: denaturation 94 DEG C 5 minutes；

Sex change 94 DEG C 40 seconds,

Anneal 47 DEG C 40 seconds,

Extend 72 DEG C 40 seconds,

Circulate 35 times；

Extend 72 DEG C 5 minutes；

4 DEG C are terminated reaction.

Capillary electrophoresis analysis result shows, the primer of Satt683 to female parent be Beidou 5 male parent be swan egg The genomic DNA of the colony of ZDD02114 has expanded molecular labeling fragment (Fig. 8) that size is 410bp, and this colony's base is described Because group DNA contains the Satt683-3 allele being improved soybean protein content；

The primer of Satt409 to female parent be Beidou 5 male parent be the genome of the colony of swan egg ZDD02114 or middle Huang DNA has expanded molecular labeling fragment (Fig. 9) that size is 330bp, illustrates in this Meta-genomic DNA containing being improved soybean egg The Satt409-2 allele of Bai Hanliang；

The primer of Satt584 to female parent be Beidou 5 male parent be the colony of swan egg ZDD02114 genomic DNA extension Go out molecular labeling fragment (Figure 10) that size is 300bp, illustrated in this Meta-genomic DNA containing being improved soybean protein content Satt584-5 allele；

The primer of Satt181 to female parent be Beidou 5 male parent be to understand greatly bean or pea or the genome of colony of Chinese mugwort card 166 DNA has expanded molecular labeling fragment (Figure 11) that size is 280bp, illustrates in this Meta-genomic DNA containing reducing soybean The Satt181-2 allele of protein content；

The primer of Satt180 to female parent be Beidou 5 male parent be Biogedulote or Chinese mugwort the card 166 or auspicious colony of rich No. 2 Genomic DNA expanded molecular labeling fragment (Figure 12) that size is 300bp, illustrate this Meta-genomic DNA contains Reduce the Satt180-5 allele of soybean protein content.

Claims (10)

1. a QTL relevant to soy protein content, it is characterised in that: it is positioned in linkage group A2, at the base of public collection of illustrative plates Because group position is 145.565cM, molecular labeling Satt409 position, the 5 '-3 of molecular labeling Satt409 ' primer such as SEQ ID Shown in NO.3,3 '-5 ' primer is as shown in SEQ ID NO.4.

QTL the most according to claim 1, it is characterised in that: this QTL comprises the allele that effect value is 10% Satt409-2, this allele and soy protein content positive correlation.

3. a preparation method for the molecular labeling of QTL relevant to soy protein content described in claim 1, its feature exists In the 5 '-3 of this molecular labeling, ' primer as shown in SEQ ID NO.3,3 '-5 ' primer is as shown in the SEQ ID NO.4；With soybean product Planting Beidou 5 is recurrent parent, hybridization yellow with swan egg ZDD02114 or middle, backcrosses through twice hybridization and twice altogether and obtains afterwards Soybean genes of individuals group DNA obtained is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, By the PCR product that obtains of amplification after electrophoretic separation, size 360bp that obtains, the amplified fragments of 330bp or 300bp, it is mesh Mark molecular labeling.

4. the molecular labeling of QTL relevant to soy protein content described in a claim 1, it is characterised in that: this molecule 5 '-3 ' primer as shown in SEQ ID NO.3,3 '-5 ' primer of mark is as shown in SEQ ID NO.4.

5. the molecular labeling described in claim 4 is for a method for soybean molecular breeding, the 5 '-3 of this molecular labeling ' primer As shown in SEQ ID NO.3,3 '-5 ' primer is as shown in SEQ ID NO.4；Draw soybean to be measured with its 5 '-3 ' primer and 3 '-5 ' The genomic DNA of kind carries out PCR amplification, by amplified production after electrophoretic separation, if there is size in amplified production is The amplified fragments of 360bp, 330bp or 300bp, then this soybean varieties to be measured has relevant to soy protein content QTLSatt409, if there is the amplified fragments that size is 330bp in amplified production, then this soybean varieties to be measured is for having and soybean The kind of the allele QTLSatt409-2 increasing soy protein content in the QTLSatt409 that protein content is relevant, Otherwise, kind to be measured does not have this QTL or this allele.

6. a QTL relevant to soy protein content, it is characterised in that: it is positioned in linkage group N, at the gene of public collection of illustrative plates Group position is 29.37cM, molecular labeling Satt584 position, the 5 '-3 of molecular labeling Satt584 ' primer such as SEQ ID NO.5 Shown in, 3 '-5 ' primer is as shown in SEQ ID NO.6.

QTL the most according to claim 6, it is characterised in that: this QTL comprises the allele that effect value is 10% Satt584-5, this allele and soy protein content positive correlation.

8. a preparation method for the molecular labeling of QTL relevant to soy protein content described in claim 6, its feature exists In the 5 '-3 of this molecular labeling, ' primer as shown in SEQ ID NO.5,3 '-5 ' primer is as shown in the SEQ ID NO.6；With soybean product Kind of Beidou 5 is recurrent parent, hybridizes with swan egg ZDD02114, altogether through twice hybridization and twice backcross obtain afterwards big Beans genes of individuals group DNA is template；With above-mentioned 5 '-3, ' primer and 3 '-5 ' primer carries out PCR amplification to above-mentioned template, is expanded by PCR Increase the product that obtains after electrophoretic separation, size 400bp that obtains, the amplified fragments of 350bp or 300bp, it is target molecule Mark.

9. the molecular labeling of QTL relevant to soy protein content described in a claim 6, it is characterised in that: this molecule 5 '-3 ' primer as shown in SEQ ID NO.5,3 '-5 ' primer of mark is as shown in SEQ ID NO.6.

10. the molecular labeling described in claim 9 is for a method for soybean molecular breeding, the 5 '-3 of this molecular labeling ' draw Thing as shown in SEQ ID NO.5,3 '-5 ' primer is as shown in SEQ ID NO.6；With its 5 '-3 ' primer and 3 '-5 ' draw to be measured greatly The genomic DNA of beans kind carries out PCR amplification, by amplified production after electrophoretic separation, if there is size in amplified production is The amplified fragments of 400bp, 350bp or 300bp, then this soybean varieties to be measured has relevant to soy protein content QTLSatt584, if there is the amplified fragments that size is 300bp in amplified production, then this soybean varieties to be measured is for having and soybean The kind of the allele Satt584-5 increasing soy protein content in the QTLSatt584 that protein content is relevant, no Then, kind to be measured does not have this QTL or this allele.