CN1796572A - Molecule marker of characteristics of cotton yield or quality of fibre - Google Patents

Abstract

This invention relates to a molecule marking method of selecting cotton yields and properties or fiber qualities using SSR technology, which includes: a) cross-fertilization of Sino-G97038 cotton group with the 16th group from China Institute of Cotton (Sino-16), with its F1 generation selfing to produce the F2 generation; b) extraction of the whole DNA in the leaves of F2 (Sino-G97038xSino-16) separate group by means of CTAB; c) establishment of target character extremum bank by means of BSA; d) selection of parent species with SSR primers, further selection of discrepantly behaving primers between parent species with established extremum DNA bank, and amplification of the obtained discrepant primers and all F2 individual plants so as to gain polymorphism markers; e) SSR amplification and detection; f) data analysis. This invention publishes the molecular markers of the cotton fiber qualities, which have a nucleotide sequence from SEQ ID No.1 to SEQ ID No.4. Besides, it also publishes the application of the molecular markers of cotton yield and properties and fiber qualities in seed breeding.

Description

The molecule marker of yield and property of cotton or fibrous quality

Invention field

The present invention relates to the molecule marker of a kind of yield and property of cotton or fibrous quality.Particularly, the present invention relates to the molecular marker method of a kind of SSR of utilization mark and BSA method screening yield and property of cotton or fibrous quality, and the sequence that discloses described molecule marker.In addition, the invention discloses the application of described molecule marker in cotton breeding.

Background technology

Cotton is the important cash crop of China, is bringing into play very important effect in national economy and people's lives.Cotton fibre is the main raw material of textile industry, has characteristics such as insulation, heat insulation, moisture absorption, ventilative, comfortable and easy dyeing, just gains great popularity since ancient times.Advocate natural, pursue that cotton fibre is subjected to people's attention more in comfortable modern society's life, its various advantages also be man-made fiber can't replace.

The quality of cotton fiber quality is determining cotton yarn and the quality of weaving cotton cloth, so quality breeding is cotton breeding man's a major objective always.Economical globalization and the development of textile industry and the raising of living standards of the people are had higher requirement to cotton fiber quality.

The cotton fiber quality common counter is fibre strength, staple length, reguarity, micron value, the ripening degree of cotton.

World's cotton fiber quality improvement increases staple length at first and begins, and up to early 1980s, cotton quality is still main relevant with macrofiber.Late 1980s, the cotton breeding man begins to pay attention to increase the quality-improving research of fibre strength both at home and abroad.

After the 1980s, the fibrous quality of cotton enjoys the concern of breeding man, the breeding emphasis is to the transfer of strengthening interior quality, the important goal of breeding is classified in the improvement of fibrous quality as, obtain remarkable effect, China all will reach the cotton variety of international medium level along with China has successively bred some, be a collection of disease-resistant, high-quality, high yield (the specific tenacity 20.18cNtex of representative with " middle cotton 12 " ^-1) new cotton variety, replaced high yield but " cotton No. 1 of Shandong " (specific tenacity 16.46cNtex susceptible and that the fiber interior quality is relatively poor ^-1) " wait some kinds, make the Cotton in China fibrous quality rise to middle high intensity level by low strength.Most of cotton fibre specific tenacity is 17.8cNtex ^-1(ICC standard), to nineteen ninety-five the fiber specific tenacity more than 70% be 21.7cNtex ^-1(Huang Zikang, 1996).In " 95 " cotton breeding brainstorm project, creating high-quality fiber germplasm and cultivating the high-quality parent material as the important research content, China selects 12 parts of high-intensity fiber germplasms in the first three years, and specific tenacity is at 24.5cNtex ^-1More than.Over nearly 10 years, the fiber specific tenacity is at 22cNtex ^-1Above kind showed increased, micron value is between 3.7-4.2.

The middle and later periods nineties in last century, the Cotton in China quality-improving enters the bottleneck phase, though target is still when keeping other quality parameter, emphasis improves fibre strength, but owing to lack excellent germplasm system and parent material, also lack innovative technology, so the main quality index is taken on a new look and not obvious (Guo Xiangmo, 1999).

Domestic and international cotton breeding man is except utilizing the conventional breeding method over nearly 15 years: by backcrossing and hybridization such as mutual friendship mode, obtaining bigger progress aspect the negative correlation of breaking output and fibrous quality proterties, but the traditional breeding method cycle is long, the cost height, foresight poor (Zhang Tianzhen, 2000).The at present domestic molecular breeding research that has utilized molecular biology method to carry out the improvement cotton fibre quality on a large scale.Compare with conventional breeding, molecular breeding is quick, foresight good, the cycle is short, efficiency of selection is high.

Identifying, separate and clone good fibrous quality gene, is one of focus of domestic and international cotton biotechnology.Utilize the molecule means that cotton fiber traits is carried out the gene that genetic manipulation depends on the specific fibrous texture proterties of identification and the growth of separating controlling controlling fiber or remote effect.In the improvement of cotton fiber, specific promoter is primary genetically engineered element, particularly drives foreign gene and expresses in fiber, more needs high specificity, active high promotor, to realize directional operation.The separating clone of therefore strong specificity, highly active promotor and functional gene and identification research are with extremely important (Zhao Guangrong etc., 2002).

Cotton from chasmogamy to the embryo occur to that sprouting can be divided in early days that the embryo forms in early days, mid-term, late period three phases, the mRNA that approximately the 25000-30000 kind is different in embryo's generating process participates in, these mRNA can be divided into six big classes by its time difference of expressing in embryo's generating process, wherein the 5th class mRNA takes place to occur late period the embryo, content is very abundant, and rapidly disappears behind seed germination.This class mRNA is referred to as the embryo mRNA takes place to enrich late period.The gene of these mRNA of encoding is referred to as the Lea gene family.The cDNA library that people such as Galau utilize upland cotton Coker 201 to make up takes the lead in separating 17 cDNA clones of Lea gene family by the method for subtractive hybridization, and these genes are divided into LeaI, Lea2 (2A and 2B) dpd gene family.The LeaI gene family is expressed and regulated by ABA, and the expression of LeaE gene family is regulated by ABA seldom, and the mRNA behavior shows coordinately regulated feature in family.

At present, separate and tens genes that check order by three kinds of approach.Article one, approach is to utilize the clone and the cDNA library clone value direct cross of known array in other crop, filters out and external sequence height homologous cDNA clone.Adopt this method to be separated to gene at present several genes such as P36A, rbcI, trn4, rbcs, Cab-151 are arranged.The second approach is the cDNA library that makes up upland cotton (as Coker201, Coker312), utilize the method for subtractive hybridization to filter out the cDNA clone relevant then with cotton development, and relevant cDNA clone checked order, carry out structural analysis of protein by external translating system, as E6 gene, Lea gene family.The cotton tissue mRNA of extracting different developmental phases carries out reverse transcription, and the DDRT-PCR method of carrying out the analysis of sequence glue then is that present cotton carries out gene clone and isolating up-to-date approach.The method of utilizing this species diversity to show has obtained the cDNA segment of coding resisting verticillium hemiterpene class plant protecting chemical, key gene during fiber is grown.In addition, M.E.John etc. also are separated to the promotor of specifically expressing in cotton flower pesticide, and by engaging with Gus, obtaining transgene tobacco can specifically expressing.The structural gene coding that links to each other with this promotor is similar to PG gene function (left side drives a well, 1998).

Wang Xuede etc. (2000) are contrast with wild-type upland cotton heredity standard system " TM-1 ", the mRNA and the gene thereof of predominant expression in fibrocyte from the fibrocyte RNA of 4 kinds of fiber mutant and genomic dna thereof, have been cloned respectively with the RT-PCR technology, comparison by nucleotide sequence between wild-type and mutant, tangible variation has taken place in the structure and the expression of discovery this gene in fiber mutant gene group DNA, and has carried out preliminary discussion with regard to the relation that may exist between these variations and cotton fiber development.

The E6 gene of cloning from cotton is the specific expression gene of class predominant expression in fibrocyte.Clone this gene and not only help the research of the molecule mechanism of cotton fiber development, and can be used for the genetically engineered of improvement of cotton fiber traits.For example: the fiber specific expression carrier that people such as John utilize fiber specific promoter to make up, two thermoplastic polyester compound-bases that make microorganism (alcaligenes eutrophus) are because of (Pha B and Pha C) specifically expressing in cotton fiber, and the fiber of transgene cotton has better insulative properties.And for example, in Acetobacter xylinum (Acetobaceter xylinum), had now found that and identified the operon of Mierocrystalline cellulose synthetic enzyme, contain synthetic necessary gene: the bscA of 4 Mierocrystalline celluloses, bcsB bcsC bcsD, wherein bcsB or bcsA may be the Mierocrystalline cellulose synthase genes, and bcsD may participate in cellulosic crystallisation procedure does, just might improve cotton fibre quality.If the Mierocrystalline cellulose synthase gene fusion of E6 promotor and Acetobacter xylinum is transformed in the cotton, just might improves cotton fibre quality.

Selection is one of most important link in the breeding, traditional system of selection based on phenotype exists many shortcomings, efficient lower, molecule marker provides possibility for realizing to genotypic direct selection, be molecular marker assisted selection (Molecular marker-assisted selection, MAS).Utilize with the closely linked molecule marker of objective trait and can judge, select, be not subjected to the influence of field natural condition at the fertility commitment.Main Agronomic Characters is identified in early days and can be shortened breeding time greatly, raise the efficiency the quickening breeding process.In 3 generations, just can be chosen desirable material as long as backcross in theory.

MAS is the binding site of modern molecular biology and traditional genetic breeding, can select from dna level breeding material by molecule marker, thereby reach the comprehensively efficient improvement of proterties such as crop yield, quality and resistance.As screen the molecule marker chain with Other Main Agronomic Characters, then, be beneficial to cotton fundamental research and breeding work and carry out by utilizing molecule marker to carry out linkage analysis.Use molecule marker and make up the cotton genetic map, can improve the foresight of breeding work.

Reddy etc. (1996) utilize the F of long wool sea island cotton 3-79 * upland cotton TM-1 ₂Reach parents and carry out aflp analysis, in employed 64 pairs of primers, each all detects 3～16 AFLP marks to primer: at F ₂Found in the colony that 300 marks are relevant with parent's long wool and high-yield character.Researchers such as Zhang Tianzhen are being done a large amount of work aspect the molecular mark of cotton fiber quality, and win initial success.The auxiliary QTL of molecule marker location is the emerging research field that grows up over past ten years, makes much progress.Many economic characters of cotton also are by QTL control (Quantitative Trait Loci), thereby carry out cotton QTL location and have important theory and practice significance.Shen Xinlian etc. (2001) select 22 RAPD marks and SSR marks such as FSR1933, FSR41047 chain with high-intensity fiber that filter out for use, the stability of research main effect QTL and the effect of molecular marker assisted selection.The result proves that these three are marked at different genetic backgrounds, different segregating generation inheritance stability, has, the fibre strength of unmarked individuality reaches utmost point conspicuous level, and main effect QTL carries out molecular marker assisted selection, and to improve cotton fiber strength be effective.

Seek with the closely linked molecule marker of target gene and be unable to do without strong molecular marking technique and special genetic stocks and analysis means.Searching mainly contains two kinds with the method for the closely linked molecule marker objective trait of target gene gene: the near-isogenic line of target gene is analyzed NIL (Young, 1998; Martin G.B., 1991) and target gene F ₂For the segregating population fractional analysis method of segregating population (BulkedSegregant Analysis, BSA).

The auxiliary QTL of molecule marker location is the emerging research field that grows up over past ten years, makes much progress.It is localized more to carry out cotton QTL quality trait abroad, and how relevant with intensity.

Along with development of biology, the Study on Molecular Marker of cotton also more and more receives publicity.The molecular marking technique that is widely used in cotton at present mainly contains RFLP, RAPD, AFLP and SSR etc., the field of research relates to many aspects such as construction of genetic atlas, genetic marker and the assignment of genes gene mapping, sibship analysis, variety evaluation, molecular marker assisted selection, and makes significant progress.

Many Main Agronomic Characters of cotton are by QTL control (Quantitative Trait Loci), thereby carry out cotton QTL location and have important theory and practice significance.Molecular marking technique is widespread use in the cotton genetic breeding, and plays a greater and greater role, and different molecule markers is the associating acting in conjunction mutually, and the more deep development of cotton molecular biology research is gone down.

Summary of the invention

Be to improve the cotton unit yield, the improvement cotton fiber quality, the most direct the most basic way is exactly to find the key factor that influence cotton fibre, and its gene is positioned, and then separation and clone genes involved.And then it is imported to main plant in the cotton variety high quality cotton kind that can obtain to efficiently express.By molecular marker screening to cotton high quality fiber gene, filter out and high quality fiber gene or the chain molecule marker of QTL, be used for assisted Selection, improve efficiency of selection, thereby improve the fibrous quality level of fiber species.

Therefore, the purpose of this invention is to provide the molecular marker method of a kind of SSR of utilization mark and BSA method screening cotton fiber strength, and the sequence that discloses described molecule marker.

Another object of the present invention provides the molecule marker with the linkage of characters of cotton fiber yield and quality.

A further object of the present invention provide the application of the molecule marker that screens in cotton breeding.

The inventor is target with the molecular mark,, uses (in 16 * middle G97038) F directly at the gene of controlling fiber proterties ₂Segregating population, by the SSR molecular marking technique, seek molecule marker with the linkage of characters of cotton fiber yield and quality, foundation as the molecular genetic improvement, thereby just identify individual plant in seedling stage with good fiber trait, improve the efficiency of selection of fiber trait, thereby accelerate the breeding process of Cotton in China kind.Wherein the molecular marker method of fibre strength is spent in screening, comprises step:

A). utilize in the cotton G97038 strain system, hybridize with 16 strains system of middle cotton institute (in 16), F1 produces F2 generation for selfing;

B). extract the total DNA of blade of F2 (middle G97038 * in 16) segregating population with the CTAB method;

C). utilize the BSA method to set up objective trait extreme value storehouse;

D). by the SSR primer parent is screened, the discrepant primer of performance is used the extreme value DNA pond screening of being set up again between the parent, and the discrepant primer to extreme value DNA pond is obtained carries out augmentation detection to all F2 individual plants, obtains polymorphism mark;

E). carry out SSR amplification and detection;

F). data statistic analysis.

Be appreciated that by setting up molecular mark available SSR molecule marker,, be beneficial to follow-up study person and carry out relevant fiber each character gene separation and work such as clone in a deep going way for the cotton molecular breeding lays the foundation.This research is the practice of molecular mark on cotton, and research of upland cotton molecular breeding and application are had the theory and practice meaning.

SSR in the screening method (Simple Sequence Repeat) mark, promptly the simple repeated sequence mark is also referred to as little satellite (microsatellite).The beginning of the seventies, people find that just ubiquity simple repeated sequence in the eukaryotic gene group, the two ends of tumor-necrosis factor glycoproteins are tending towards the dna sequence dna guarded often, therefore can be at the special primer of the two ends of tumor-necrosis factor glycoproteins design, through pcr amplification reaction, gel electrophoresis and dyeing, thereby obtain polymorphism, i.e. the SSR mark because of the different amplified fragments that cause of simple repeated sequence units.The characteristics of SSR mark are: general detected is a single multiple alleles site; Show as codominant inheritance, so can differentiate heterozygote and homozygote; Good reproducibility as a result.SSR is widely used in the resource evaluation, linkage map is drawn and the researchs such as molecule marker of objective trait gene.Yet ssr analysis needs more genome sequence column information and synthetic a large amount of Oligonucleolide primers, and this will drop into great amount of manpower and material resources, financial resources undoubtedly, has therefore limited the use of this technology.

(Bulked Segregant Analysis, BSA) method is that Michelmore etc. proposed in 1991 to BSA, for the mark and the location of goal gene provides more simple and effective method.Be about to the objective trait studied in the F2 segregating population, F2 colony is divided into (as disease-resistant, susceptible) according to phenotypic character and is divided into two groups, the DNA balanced mix of the plant of some amount from every group, form two and press the DNA ponds (Pool) that phenotypic character is distinguished, all the other are basic identical except that the dna fragmentation at goal gene place is variant in two ponds.Therefore the polymorphism difference of analyzing DNA in two ponds can obtain the molecule marker chain with goal gene rapidly.Near isogenic line or BSA analyze and combine is the desirable effective ways of searching and the closely linked molecule marker of target gene.And then meticulous genetic map and physical map that can establishing target gene region.

Utilize SSR mark and BSA method that high-strength and low strong 2 the DNA ponds that make up are screened, obtain the SSR mark of cotton quality intensity, and F2 colony analyzed, thereby obtain mark and the fibrous quality rheobase distance (concrete steps are seen embodiment) between therefore

Material used in the present invention is for being the F2 segregating population of (in 16 * middle G97038), its parent material is upland cotton, wherein maternal " in 16 " be the abbreviation of kind " middle cotton 16 ", by The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute's cultivation, is the short season cotton typical species.Male parent " middle G97038 " is a fine parent material, provides a high fiber strength strains by the seed selection of system of middle cotton institute in professor Li Youcheng of Sichuan agricultural college.In 16 breeding times short, cotton boll is big, ginning outturn is high, the knot bell is strong, yield traits is better.Middle G97038 shows as more late-maturing, but fibre strength is high.The structure of combination selection and this experimental population thereof is finished by middle cotton.Summer 2000 biparent cross, add generation then winter to form the F1 plant in Hainan Island, all individual plant selfing.Sow F2 generation at middle cotton institute sample plot summer calendar year 2001.The long 8m of experiment cell row, line-spacing 80cm, spacing in the rows 25cm～30cm.In 16 specific tenacity be 31.4cN/tex, the specific tenacity of middle G97038 is 40.8cN/tex, the differing greatly of fibre strength between the parent, middle G97038 than in 16 high 9.4cNtex-1, high by 29.94%, relatively be fit to make up the segregating population of molecular marker analysis.Institute's combo is combined into middle cotton 16 * middle G97038.By high-strength and low strong 2 DNA ponds being carried out the SSR screening, obtained the SSR difference banding pattern relevant with fibre strength, through population analysis, the QTL of the maximum effect of fibre strength the LOD value be 4.3 o'clock on the position apart from M111 mark 2cM between the M111-M683, the phenotypic variation that can explain is 48.2%, and additive effect is that 0.2412 dominant effect is 5.587.

The yield traits of field investigation comprises the bell number among the present invention, bell is heavy and ginning outturn.The fibrous quality proterties has staple length, reguarity, specific tenacity, elongation, micron value.Individual plant numbering, branch individual plant carry out economical character investigation and fiber sampling.The detection of fibrous quality is carried out with large vol fiber testing system (HVI900) by cotton quality supervision and inspection center of the Ministry of Agriculture.Test atmosphere temperature and relative humidity are respectively 20 ℃ and 65%.

Adopt SAS software, above-mentioned 8 quantitative characters are done the test of hypothesis of normal distribution.With the W statistic that provides in the Univariate process is foundation, when the probability of Wa＜W less than 0.01 the time, be small probability event, think that it does not meet normal distribution.All data are divided into groups with frequency distribution, make each proterties histogram.

Accompanying drawing and explanation

Fig. 1: SSR111 is marked at the separation in the F2 colony, and wherein 1 is that Marker 2 is P1, and 3 is P2, and all the other are the F2 individual plant;

Fig. 2: SSR 683 is marked at the separation (8% non-denaturing polyacrylamide gel) in the F2 colony, 1 be Marker all the other be the F2 individual plant.

Embodiment

Embodiment 1:

The cotton genomic dna extraction and purification

With reference to (1998) reported method such as Song Guoli, and revise a little, basic step is summarized as follows:

(1) cotton genomic dna extracts:

The first step: add 2% beta-mercaptoethanol in extracting solution, and place 65 ℃ water preheating.

Second step: in very low temperature (70 ℃) refrigerator, take out freezing blade an amount of (about 2g), put into the mortar of precooling immediately, add liquid nitrogen, quick grind into powder, pack into immediately in the centrifuge tube of the 1.5ml that is placed with small amount of activated, add 0.6ml extracting solution mixing, 65 ℃ of water-bath 60min.(the 10min left and right sides, every interval mixing once fully scatters sample).

The 3rd step: water-bath finishes, add isopyknic chloroform-primary isoamyl alcohol (24: 1, V/V), behind the cover lid, behind the centrifuge tube 30～50 times of slowly turning upside down, with chloroform-primary isoamyl alcohol (24: 1) equilibrium centrifugation pipe, the centrifugal 15min of 12000g.

The 4th step: get supernatant, extracting is once again to add isopyknic chloroform-primary isoamyl alcohol (24: 1).

The 5th step: get supernatant, add the ice-cold Virahol of 0.6 times of volume, slowly put upside down centrifuge tube, until there being flocks to assemble.

The 6th step: (or put into-20 ℃ of refrigerator 20min～40min), precipitation is chosen the precipitation hook go out, transfer in the 1.5ml centrifuge tube, wash 2～3 times with 70% alcohol, dehydrated alcohol is washed 1 time, and is air-dry to leave standstill 30min.

(2) cotton genomic dna purifying:

The first step exsiccant is slightly carried among the DNA and to be added 500 μ lTE, 20min hydrotropy or spend the night in 65 ℃ of water-baths.

Second step: TE dissolved DNA is according to the RNA enzyme of the no DNA of 10 μ g RNase A/100 μ lDNA ratios adding, and 37 ℃ are incubated 1h.

The 3rd step: add isopyknic chloroform-primary isoamyl alcohol (24: 1), slowly put upside down centrifuge tube 30～50 times, equilibrium centrifugation pipe, the centrifugal 15min of 12000g.

The 4th step: get supernatant, add the NaAc (PH=5.2) of the 3mol/l of 0.1 times of volume, behind the mixing, slowly add the ice-cold dehydrated alcohol of 2 times of volumes, after static 5 minutes, the flocks hook goes out, and forwards in the centrifuge tube of 1.5ml.

The 5th step: the alcohol of adding 70% and 100% is respectively washed once, and is air-dry, is dissolved in the TE of 200 μ l, preserves standby in-20 ℃ refrigerator.(annotate: 100ml extract recipe: Tris 0.01mol; EDTA0.002mol; PvP40 2g; CTAB 2g; NaCl 0.15mol; With preceding adding mercaptoethanol 2ml)

(3) mensuration of DNA concentration

Use the BACKMAN ultraviolet spectrophotometer to measure.

Embodiment 2:

The objective trait utmost point is planted the foundation in storehouse

According to the BSA method that Michelmore (1991) proposes, from colony, choose extreme performance individual plant 5 strains of each proterties of F2 individual plant respectively, get its DNA balanced mix, make up the extreme value gene pool of each proterties, final concentration is 10ng/ μ l.

Embodiment 3:

The screening of mark

Fiber trait performance according to F2 colony, 6 proterties extreme value DNA mixing pits such as fibre strength, length, micron value, reguarity, ginning outturn and elongation have been made up, by 386 pairs of SSR primers at first to parental line selection, the discrepant primer of performance is used the extreme value DNA pond screening of being set up again between the parent, the discrepant primer that extreme value DNA pond is obtained, all F2 individual plants are carried out augmentation detection, obtain 5 polymorphism marks altogether.

Embodiment 4:

The statistics of SSR amplification, detection and banding pattern

(1) SSR amplification method

＜1〉agents useful for same and source thereof: cotton SSR primer is synthetic by Shanghai Sangon company; Taq enzyme, MgCl2, dNTP and 10 * Reaction buffer are all available from Beijing Hua Lvyuan reagent company.

＜2〉SSR reaction system: dna profiling 30ng; Each 0.3 μ mol/L of SSR primer; DNTP 200 μ mol/L; 1 * Reaction buffer; MgCl21.5mmol/L and Taq enzyme 0.5U, cumulative volume are 20 μ l.

＜3〉SSR amplification program:

194 ℃ of 3min of Step; 1 circulation;

294 ℃ of 50sec of Step, 58 ℃ of 45sec, 72 ℃ of 1min; 35 circulations;

Step?372℃10min；

44 ℃ of insulations of Step.

Amplified reaction carries out on eppendorf mastercycler gradient pcr amplification instrument.

(2) electrophoresis of SSR amplified production

Pcr amplification product non-denaturing polyacrylamide gel electrophoresis in 1 * tbe buffer liquid, 280v voltage stabilizing 3h.Perhaps use 6% denaturing polyacrylamide gel electrophoresis in 1 * tbe buffer liquid, electrophoresis is about 2 hours under 1800 constant voltages.Sex change and native polyacrylamide gel electrophoresis method division are as follows:

A, denaturing polyacrylamide gel electrophoresis

＜1〉preparation of PAGE glue

With the careful brushing long and short two of liquid detergent piece sheet glass, sheet glass is placed the stink cupboard drying.

Sheet glass is handled: 0.5ml silication agent is embrocated equably on long sheet glass, embrocate by a direction with 1ml ethanol behind the 2min and set aside.Anti-silication agent 1ml (the anti-silication agent of 2 μ l adds in 1ml 95% ethanol, 0.5% glacial acetic acid) is applied on the short sheet glass equably, embrocates with ethanol equally behind the 2min.

Sealing: edge strip is placed short glass plate edge, add a cover long sheet glass behind the consistency from top to bottom.Clamp with clip, two sheet glass are fixed, the beginning sealing.To pay special attention to glass plate footing during sealing, generally seal two-layerly, guarantee not leak glue.After sealing finishes, offset plate has been filled up at a certain angle, in order to encapsulating.

Encapsulating: once suction PAGE solution (adding 380 μ l concentration are 10% Ammonium Persulfate 98.5 and 38 μ lTEME in the 70mlPAGE solution, and wherein the former is a peptizer, and TEMEP is an accelerator) with the 100ml syringe, at the uniform velocity pour into towards hanging down one.If the glass plate cleans up, then the solution cabling is more straight.If bubble occurs in the encapsulating process, offset plate back can have been inclined, transfer at a slow speed again, so operation can be removed bubble.

Insert comb: after comb is rinsed well and dried, insert along one side between glass plate holder seam, the flicking comb makes it to be close to following glass plate, in case bubble produces.(the PAGE glue of recording generally was solidifiable after 1 hour)

＜2〉PCR product denaturing treatment

In pcr amplification product, add behind the 4 μ l sample-loading buffers in 94 ℃ of sex change 5min, take out and place frozen water immediately, in case the DNA renaturation.

＜3〉prerunning

To solidify completely that PAGE glue places on the Bio-rad 3000DNA sequenator, add 1 * TBE electrophoretic buffer, the permanent power prerunning of 80W 30min, this moment glass sheet temperatures between 45 ℃-55 ℃ for well.

＜4〉electrophoresis

After prerunning finishes, use the irrigation with syringe gel interface, carefully insert comb, last sample 5 μ l, electrophoresis about 2 hours under 1800 constant voltages then.

B, native polyacrylamide gel electrophoresis

＜1〉non-denaturing polyacrylamide gel preparation: the non-denaturing acrylamide gel of 8% (w/v) (acrylamide 76g, N '-methylene diacrylamide 4g add 10 * TBE 100ml distilled water and be settled to 1000ml)

＜2〉point sample: in amplified production, add sample-loading buffer (not containing methane amide) back point sample and get final product.DYY-11128D type electrophoresis chamber gel size: 180 * 120 * 1cm

＜3〉electrophoresis: electrophoretic buffer 1 * TBE constant voltage (electrophoresis DYY-8B type voltage stabilization and current stabilization electrophoresis apparatus) 325V.2-3 hour (degree distinguished according to SSR molecular weight size and difference banding pattern is regulated electrophoresis time)

(3) silver dyes

After electrophoresis finishes, there is tap water to wash sheet glass a little, makes it temperature and drop to room temperature, carefully open sheet glass then.Silver dyes step:

Step 1, fixing offset plate is put into 10% acetic acid stationary liquid, fixing 30min on the shaking table;

Step 2, the rinsing of rinsing distilled water 3 times, each 5min～10min;

Step 3, silver change offset plate over to silver-colored dye liquor (500ml water adds 0.5g Silver Nitrate, 37% formaldehyde, 750 μ l), 80rpm shaking table, dyeing 30min after dying rinsing;

Step 4, rinsing dyeing back rinsing in distilled water are no more than 8sec～10sec, take out rapidly;

After step 5, the development rinsing offset plate is changed over to the yellow soda ash of colour developing liquid 3%, (500ml water adds 15g yellow soda ash, is cooled to 4 ℃.Use and added 37% formaldehyde, 750 μ l, 10mg/ml Sulfothiorine 100 μ l in preceding 5 minutes).Slowly shaking, occur until band, is contrast with marker, grasps developing time.

After step 6, the fixing fully colour developing, change in color development stopping liquid 10% acetic acid, offset plate can be taken out behind the 10min, rinsing is also dried, tape reading.

Embodiment 5:

Data statistic analysis

＜1〉SSR banding pattern statistical method

Codominant marker: the SSR overwhelming majority is the codominant marker, adopts the ABH writing-method.Parent's the banding pattern that isozygotys is designated as A, and parent two the banding pattern that isozygotys is designated as B, and the heterozygosis banding pattern is designated as H."-" expression data disappearance.

Dominant marker: if dominant marker's banding pattern statistics is complicated, represent that with " A " this site is not from parent's homozygote (having band), represent that with " B " this site from parent's homozygote (do not have band), represents that with " C " this site is from homozygote or heterozygote (band is arranged); Represent that with " D " this site is from parent's homozygote or heterozygote (band is arranged); "-" expression data disappearance.

＜2〉data analysis

Data input computer with collecting uses Mapmaker/Exp3.0 software to carry out data analysis.

The result of SSR label screening

Fiber trait performance according to F2 colony has made up fibre strength proterties extreme value DNA mixing pit.

By of the screening of 386 pairs of SSR primers, obtain 2 polymorphism marks such as M111 and M683 to parent and extreme value DNA pond.Utilize of the analysis of these 2 marks to F2 colony, the position of the QTL of its maximum effect is just on the position apart from M111 mark 2cM between the M111-M683, the LOD value is 4.3, and the phenotypic variation that can explain is 48.2%, and additive effect is that 0.2412 dominant effect is 5.587.

Sequence table

＜110〉The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute

＜120〉molecule marker of yield and property of cotton or fibrous quality

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<170>PatentIn?version?3.1

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Claims (6)

1. screening and the chain molecular marker method of cotton fiber strength comprise step:

A). utilize in the cotton G97038 strain system, hybridize with 16 strains system of middle cotton institute (in 16), F1 produces F2 generation for selfing;

B). extract the total DNA of blade of F2 (middle G97038 * in 16) segregating population with the CTAB method;

C). utilize the BSA method to set up objective trait extreme value storehouse;

D). by the SSR primer parent is screened, the discrepant primer of performance is used the extreme value DNA pond screening of being set up again between the parent, and the discrepant primer with extreme value DNA pond is obtained carries out augmentation detection to all F2 individual plants, obtains polymorphism mark;

E). carry out SSR amplification and detection;

F). data statistic analysis.

2. the discrepant primer that obtains according to the process of claim 1 wherein has the nucleotide sequence of SEQ ID No.1 and SEQ ID No.2 or SEQ ID No.3 and SEQ ID No.4.

3. molecule marker chain with cotton fiber strength, it prepares by following step:

A). utilize in the cotton G97038 strain system, hybridize with 16 strains system of middle cotton institute (in 16), F1 produces F2 generation for selfing;

B). extract the total DNA of blade of F2 (middle G97038 * in 16) segregating population with the CTAB method;

C). utilize the BSA method to set up objective trait extreme value storehouse;

D). by the SSR primer parent is screened, the discrepant primer of performance is used the extreme value DNA pond screening of being set up again between the parent, and the discrepant primer with extreme value DNA pond is obtained carries out augmentation detection to all F2 individual plants, obtains polymorphism mark;

E). carry out SSR amplification and detection;

F). data statistic analysis.

4. according to the molecule marker chain with cotton fiber strength of claim 3, the discrepant primer that is wherein obtained has the nucleotide sequence of SEQ ID No.1 and SEQ ID No.2 or SEQ ID No.3 and SEQ ID No.4.

5. the application of the arbitrary described molecule marker of claim 3-4 in cotton breeding.

6. utilize the chain primer of SSR technology screening and cotton fiber strength right, it is characterized in that having the nucleotide sequence of SEQID No.1 and SEQ ID No.2 or SEQ ID No.3 and SEQ ID No.4.。