CN105238866A - SNP site related to early-maturing traits in upland cotton and application of SNP site - Google Patents
SNP site related to early-maturing traits in upland cotton and application of SNP site Download PDFInfo
- Publication number
- CN105238866A CN105238866A CN201510730586.1A CN201510730586A CN105238866A CN 105238866 A CN105238866 A CN 105238866A CN 201510730586 A CN201510730586 A CN 201510730586A CN 105238866 A CN105238866 A CN 105238866A
- Authority
- CN
- China
- Prior art keywords
- upland cotton
- proterties
- site
- sequence
- g1001asnp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for identification or assisted identification of early-maturing traits in upland cotton. The method for identification or assisted identification of early-maturing traits in upland cotton comprises the following steps: detecting a genotype in a genome DNA of to-be-detected upland cotton on the basis of a G1001ASNP site, and determining the to-be-detected upland cotton as candidate upland cotton having early-maturing traits when the genotype is AA homozygotic type or AG heterozygous type and determining the to-be-detected upland cotton as candidate upland cotton having later-maturing traits when the genotype is GG homozygotic type, wherein the G1001A SNP site is the 1001st nucleotide from 5' terminal of a sequence 1 in a sequence list, on 13562854th site of upland cotton Dt3 chromosome. Experiments prove that the early-maturing traits in upland cotton can be screened by detecting the genotype of the to-be-detected upland cotton based on the G1001A SNP; and the application plays an important role in molecular breeding of normal upland cotton.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a SNP site relevant to upland cotton Early mature apricot and application thereof.
Background technology
Cotton is a kind of important cash crop, in national economy and social development, occupy critical role.But the fundamental realities of the country of China is had a large population and a few land, grain and cotton strive ground contradiction very outstanding.Planting cotton mode due to tradition is one crop per annual, and multiple crop index is low, limits the development of Cotton Production, therefore, in the urgent need to being applicable to precocity, high yield, the high-quality cotton new variety of two ripe or multiple croppings on producing.Cotton prematurity is a complex character, the proterties such as rate are spent before mainly comprising the time of infertility, seedling stage, flower bud phase, flowering and boll-setting period, the first fruit branch position, the first fruit branch position height and frost, these proterties are all quantitative characters, by multiple quantitative character gene locus therefor (Quantitativetraitlocus, QTL) control, Genetic Mechanisms is complicated and be subject to environment because of impact, therefore, traditional breeding way is made slow progress, and efficiency is low.Excavate the excellent allelic variation controlling upland cotton Early mature apricot, and the functional label developing corresponding allelic variation has great importance to following the tracks of the Upland Cotton selecting to have Early mature apricot in breeding process.
Linkage analysis and association analysis are the main method of analysis research Quantitative Trait Genes in Plants type now.Linkage analysis passes through biparent cross, set up mapping population, carry out the drafting of High Density Molecular linkage map, mapping population is carried out to the phenotypic evaluation of various proterties pinpoint accuracy, carry out linkage analysis again, the QTL of corresponding proterties is positioned in specific genetic linkage section.Utilize two F
2colony's linkage analysis located the QTL (Li relevant to upland cotton Early mature apricot such as flower rate before breeding time, seedling stage, flower bud phase, flowering and boll-setting period, the first fruit branch position, the first fruit branch position height and frost, etal.QTLanalysisforearly-maturingtraitsincottonusingtwou plandcotton (GossypiumhirsutumL.) crosses.BreedingScience, 2013,63:154-163).
Association analysis is a kind of based on linkage disequilibrium, the analytical procedure of qualification natural population's objective trait and genetic marker or candidate gene relation.This method is without the need to building mapping population, and application kind sources group can make the more allelotrope of detection, can also investigate association site and the allelic variation thereof of the most of QTL of multiple proterties simultaneously, is the up-and-coming means of the complicated quantitative character of a kind of anatomy.The related analysis technology of genome-wide screening is a kind of effective ways of modern Quantitative Trait Genes location and mapping, is used widely in the excavation etc. of the assignment of genes gene mapping of crop important character and clone, favorable genes resource is studied.The research analyzed about cotton prematurity trait associations is relatively less, 3 SSR sites that correlated character precocious with upland cotton significantly associates are reported at present, wherein CER0098-400 significantly associated with the time of infertility, DPL0375-250 with HAU2414-147 and fruit branch begin to save position and significantly associate (Liang Bing etc., the association analysis of upland cotton economical character and SSR marker, Cotton Science, 26 (5): 387-395).Highdensity single nucleotide polymorphism (Singlenucleotidepolymorphism, SNP) is not utilized at present to mark the main effect QTL of being in depth correlated with to upland cotton Early mature apricot or the site be associated is studied.
Single nucleotide polymorphism refers to the polymorphism of the DNA sequence dna in genomic level caused by single nucleotide diversity.The variation of DNA molecular single core thuja acid has the forms such as base replacement, insertion and disappearance, and SNP does not comprise insertion and the disappearance of base.According to the difference of variation generation area, SNP can be divided into 2 kinds of forms in genome: first is a large amount of single nucleotide variations being arranged in genome sequence; Second is the functional single nucleotide variations being positioned at gene coding region.The latter is owing to being distributed in gene coding region, therefore the cSNP that is otherwise known as (CodingSNP).Along with the development of technology, SNP detection method gets more and more, and testing cost is also more and more economical, makes it become molecule marker of new generation after SSR.
Summary of the invention
Technical problem to be solved by this invention how to identify or assistant identification upland cotton Early mature apricot.
For solving the problem, the invention provides a kind of method of qualification or assistant identification upland cotton mature trait.
A kind of qualification provided by the invention or the method for assistant identification upland cotton mature trait can comprise the steps: to detect the genotype based on G1001ASNP site in the genomic dna of upland cotton to be measured, if be the upland cotton with Early mature apricot that the homozygous or AG heterozygous of AA, upland cotton to be measured are candidate, if for homozygous, the to be measured upland cotton of GG be the upland cotton with late-maturing proterties of candidate.
In aforesaid method, the method of described " detecting the genotype based on G1001ASNP site in the genomic dna of upland cotton to be measured " can be: the genomic dna extracting upland cotton to be measured, pcr amplification contains the target region in described G1001ASNP site, obtains the genotype based on G1001ASNP site; The primer pair that described pcr amplification adopts can be made up of primers F and primer R, and the nucleotide sequence of described primers F can as shown in the sequence 2 of sequence table, and the nucleotide sequence of described primer R can as shown in the sequence 3 of sequence table.
In aforesaid method, the method of described " detecting the genotype based on G1001ASNP site in the genomic dna of upland cotton to be measured " specifically can be: extract upland cotton to be measured genomic dna and as template, carry out pcr amplification with described primers F and described primer R for primer, then utilize PCR-SSCP technology for detection based on the genotype in G1001ASNP site.
Present invention also offers a kind of method that screening has the upland cotton of Early mature apricot.
The method that the method that a kind of screening provided by the present invention has the upland cotton of Early mature apricot can comprise the steps: according to above-mentioned qualification or assistant identification upland cotton mature trait identifies the mature trait of upland cotton to be measured, and screening has the upland cotton of Early mature apricot.
Present invention also offers a kind of method that screening has the upland cotton of late-maturing proterties.
The method that the method that a kind of screening provided by the present invention has the upland cotton of late-maturing proterties can comprise the steps: according to above-mentioned qualification or assistant identification upland cotton mature trait identifies the mature trait of upland cotton to be measured, and screening has the upland cotton of late-maturing proterties.
The primer pair be made up of primers F and primer R also belongs to protection scope of the present invention, and the nucleotide sequence of described primers F can as shown in the sequence 2 of sequence table, and the nucleotide sequence of described primer R can as shown in the sequence 3 of sequence table.
Test kit containing above-mentioned arbitrary described primer pair also belongs to protection scope of the present invention.
Described test kit also can comprise the reagent detected for PCR-SSCP.
Above-mentioned arbitrary described G1001ASNP site be the sequence 1 of sequence table from 5 ' end the 1001st Nucleotide, the 1001st Nucleotide is A or G; This SNP site is at upland cotton D
tthe 13562854th on 3 karyomit(e)s.
Above-mentioned arbitrary described G1001ASNP site also belongs to protection scope of the present invention.
Described G1001ASNP site is relevant to upland cotton mature trait, and be specially the sequence 1 of sequence table from 5 ' end the 1001st Nucleotide, the 1001st Nucleotide is A or G; This SNP site is at upland cotton D
tthe 13562854th on 3 karyomit(e)s.
The DNA fragmentation containing above-mentioned arbitrary described G1001ASNP site relevant to upland cotton mature trait also belongs to protection scope of the present invention.
The nucleotide sequence of described " DNA fragmentation containing above-mentioned arbitrary described G1001ASNP site relevant to upland cotton mature trait " can as shown in sequence in sequence table 1, and this DNA fragmentation can be positioned at upland cotton D
tthe 13561854th on 3 karyomit(e)s the-the 13563854th.
Above-mentioned arbitrary described mature trait be the time of infertility proterties and/or seedling flower bud phase proterties and/or flowering and boll-setting period proterties and/or the first fruit branch position proterties and/or the first fruit branch position height proterties and/or frost before flower rate proterties.
Above-mentioned arbitrary described Early mature apricot is the combination of one or more in following (a1)-(a6):
(a1) 120 days are less than the time of infertility;
(a2) the seedling flower bud phase is less than 65 days;
(a3) flowering and boll-setting period is less than 50 days;
(a4) the first fruit branch position is less than 6 leaf figure places;
(a5) the first fruit branch position is highly less than 22 centimetres;
(a6) before frost, flower rate is more than 70%;
Above-mentioned arbitrary described late-maturing proterties is following (b1) combination to one or more in (b6):
(b1) more than the 120 days time of infertility;
(b2) more than the 65 days seedling flower bud phase;
(b3) flowering and boll-setting period more than 50 days;
(b4) more than 6, first fruit branch position leaf figure place;
(b5) the first fruit branch position height is more than 22 centimetres;
(b6) spend rate for being less than 70% before frost.
The present invention tests with 185 parts of upland cotton materials, the investigation time of infertility (WGP), the seedling flower bud phase (SSP), flowering and boll-setting period (FBP), first fruit branch position (NFFB), rate (YPBF) 6 proterties are spent before first fruit branch position height (HNFFB) and frost, classify for the Genotype on Cotton material in above-mentioned arbitrary described G1001ASNP site simultaneously, statistics is: (i) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, the time of infertility of 89.77% is all less than 120 days, mean value is 111.68 days, 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the time of infertility of 84.54% is all more than 120 days, and mean value is 124.18 days.(ii) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and the seedling flower bud phase of 84.10% is all less than 65 days, and mean value is 63.35 days; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the seedling flower bud phase of 91.75% is all more than 65 days, and mean value is 69.53 days.(iii) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and the flowering and boll-setting period of 76.14% is all less than 50 days, and mean value is 47.66 days; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the flowering and boll-setting period of 90.72% is all more than 50 days, and mean value is 50.28 days.(iv) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, the first fruit branch position of 82.95% is all less than 6 leaf figure places, and mean value is 5.68 leaf figure places; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the first fruit branch position of 91.75% is all more than 6 leaf figure places, and mean value is 7.23 leaf figure places.(v) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, the first fruit branch position of 80.68% is highly less than 22 centimetres, and mean value is 20.15 centimetres; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the first fruit branch position of 92.78% is highly all more than 22 centimetres, and mean value is 26.05 centimetres.(vi) 66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and before the frost of 90.91%, flower rate is all more than 70%, and mean value is 79.82%; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, before the frost of 82.47%, flower rate is all less than 70%, and mean value is 61.03%.Statistics shows, detect the genotype of upland cotton to be measured based on G1001ASNP site, if upland cotton to be measured based on the genotype in G1001ASNP site be the homozygous or AG heterozygous of AA, upland cotton to be measured is the upland cotton with Early mature apricot of candidate, if upland cotton to be measured is based on the upland cotton with late-maturing proterties of the genotype in G1001ASNP site to be homozygous, the to be measured upland cotton of GG be candidate.
Experiment proves, can screen upland cotton Early mature apricot by detecting upland cotton to be measured based on the genotype of G1001ASNP, method provided by the present invention plays a significant role in common upland cotton molecular breeding.
Accompanying drawing explanation
Fig. 1 is whole-genome association result.
Fig. 2 is the genotypic gene type of PCR-SSCP technology for detection based on G1001ASNP site.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Mature trait in following embodiment be the time of infertility proterties and/or seedling flower bud phase proterties and/or flowering and boll-setting period proterties and/or the first fruit branch position proterties and/or the first fruit branch position height proterties and/or frost before flower rate proterties.
185 kinds of upland cotton materials used in following embodiment are tetraploid cultivated cotton.185 upland cotton materials are all documented in as in Publication about Document: Liang Bing .2014. upland cotton is precocious, the association analysis of output and fiber quality characteristics and SSR molecular marker. and [master thesis]. Yang Ling: Xibei Univ. of Agricultural & Forest Science & Technology.The public can obtain from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute, to repeat this experiment.
The discovery of embodiment 1, the SNP site relevant to upland cotton Early mature apricot and association analysis
1, the investigation of upland cotton mature trait
Test with 185 parts of upland cotton materials, completely random district group test design scheme is adopted in field, in triplicate, the step of each repetition is as follows: 50, the seed of often kind of upland cotton material, the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute Anyang is seeded in experimental field respectively in late April, 2013 (SP-2013), in late May, 2013 (SU-2013), in late April, 2014 (SP-2014) or in late May, 2014 (SU-2014), the long 5m of row, line-spacing 0.8m, spacing in the rows 0.2m, carries out normal field management.In upland cotton vegetative period, investigation (WGP in the time of infertility, number of days from being seeded into cotton boll blowing), seedling flower bud phase (SSP, number of days from being seeded into cotton and blooming), flowering and boll-setting period (FBP, number of days from blooming to cotton boll blowing), the first fruit branch position (NFFB, from cotyledonary node to the leaf figure place of the first fruit branch position), the first fruit branch position height (HNFFB, from cotyledonary node to the height of the first fruit branch position) and frost front flower rate (YPBF, October 24, results seed cotton yield accounted for the ratio of ultimate production) 6 proterties.After investigation terminates, the data of same month in time, same upland cotton material are averaged, obtains the mature trait data of this upland cotton material.Utilize these data mining SNP site.
2, SLAF-seq develops SNP marker
Utilize the methods of genotyping of SLAF-seq technology to develop SNP marker by Beijing Biomarker Technologies Co., Ltd., be mainly divided into following steps:
(1) the tender upland cotton blade of children is got according to CTAB method (PatersonAH, BrubakerCL, WendelJF.Arapidmethodforextractionofcotton (Gossypiumspp.) genomieDNAsuitableforRFLPandPCRanalysis.PlantMOlRep, 1993,11:122-127).
(2) with reference to the method (Sun of Sun etc., X., Liu, D., Zhang, X., etal.2013, SLAF-seq:AnEfficientMethodofLarge-ScaleDeNovoSNPDiscover yandGenotypingUsingHigh-ThroughputSequencing.PloSone, 8, e58700), simplification gene order-checking is carried out to the genomic dna that step (1) is extracted: select cotton AD genome to cut prediction for carrying out electronics enzyme with reference to genome, finally determine that using restriction enzyme RsaI and HaeIII to carry out enzyme cuts, endonuclease bamhi length is SLAF label at the sequence definition of 314-344bp, in experiment, the digesting efficiency of restriction enzyme RsaI and HaeIII is 95.91%, obtain 428.93Mreads altogether.
By bioinformatic analysis, obtain 678,397 SLAF labels, wherein the SLAF label of polymorphism has 505,823.Screen according to MAF>0.05 and integrity degree >0.80, obtain 81 altogether, 675 SNP site.
3, whole-genome association (GWAS)
Utilize general linear model (generalizedlinearmodel, and mixed linear model mixture model (mixedlinearmodel GLM), MLM), adopt GAPIT software (Lipka, A.E., Tian, F., Wang, Q., etal.2012, GAPIT:genomeassociationandpredictionintegratedtool.Bioin formatics, 28, 2397-2399) carry out whole-genome association analysis, from step 2 obtains 81, in 675 SNP site, under GLM and MLM two kinds of analytical models, jointly have found one with the SNP site of the time of infertility (WGP) and seedling flower bud phase (SSP) phenotype significant correlation, namely in sequence table, sequence 1 sports A from the 1001st Nucleotide of 5 ' end by G, by its called after G1001ASNP site.Experimental result is shown in Fig. 1. (wherein A is the time of infertility, and B is the seedling flower bud phase).This SNP site is positioned at the D of upland cotton
ton 3 karyomit(e)s.According to upland cotton with reference to genome sequence (Li, F.G., Fan, G.Y., Lu, C.R., etal.2015, GenomesequenceofcultivatedUplandcotton (GossypiumhirsutumTM-1) providesinsightsintogenomeevolution.NatBiotechnol, 33,524-242), G10101ASNP site is at D
ton 3 karyomit(e)s the 13562854th.
4, excellent site
With reference to (Zhang such as zhang, T., Qian, N., Zhu, X., etal.2013, VariationsandTransmissionofQTLAllelesforYieldandFiberQua litiesinUplandCottonCultivarsDevelopedinChina.PloSone, 8, e57220) the calculating allelic variation phenotypic effect method proposed, the phenotypic effect of estimation marker site allelic variation.SNP site allelic variation phenotypic effect method of calculation are:
a
i＝∑x
ij/n
i-∑N
k/n
k
Wherein a
irepresent the phenotypic effect value of i-th allelic variation, x
ijfor carrying the jth material proterties phenotype test value of the i-th allelic variation, n
ifor there is the number of materials of the i-th allelic variation, N
kfor all material phenotype test value, nk is the number of all material.In this research, if a
ivalue is negative, then think that this allelic variation is synergy allelic variation, otherwise for subtracting effect allelic variation.Result shows, in G1001ASNP site, excellent allelic variation A is to the phenotypic effect value a of WGP and SSP
ibe-7.53 days and-3.88 days respectively, show that, in G1001ASNP site, excellent allelic variation base A has active effects to contribute to WGP and SSP.
The application in embodiment 2, G1001ASNP site
Select 185 parts of upland cotton materials of embodiment 1 as test material.
One, detect the genotype of 185 parts of upland cotton materials based on G1001ASNP site, concrete grammar is as follows:
1, the synthesis of primer pair
According to G1001ASNP site place D
tthe nucleotide sequence of the gene of the position at 13562854bp place on 3 karyomit(e)s, designing and synthesizing (Jin Weizhi bio tech ltd, Suzhou), comprise the primer pair of the target sequence in G1001ASNP site for increasing as follows:
Primers F: 5 '-ACAGGTTATCTCGCATTGTTCA-3 ' (sequence 2 of sequence table);
Primer R:5 '-CCTTCTTTGCCAGAGGGATATG-3 ' (sequence 3 of sequence table).
Special primer to amplification target sequence as shown in the sequence 1 of sequence table.Sequence 1 is positioned at upland cotton D
tthe 13561854th on 3 karyomit(e)s to the 13563854th.
2, the genomic dna of test material is extracted.
3, with the genomic dna of test material for template, with step 1 synthesize primer pair carry out pcr amplification, obtain pcr amplification product.The reaction system (10 μ L) of pcr amplification: ddH
2o4.7 μ L, DNA profiling (30ng μ L
-1) 3 μ L, primers F (10 μm of olL
-1) 0.5 μ L, primer R (10 μm of olL
-1) 0.5 μ L, 10 × Buffer be (containing the Mg of 15mM
2+) the Taq enzyme 0.1 μ L (Beijing Quanshijin Biotechnology Co., Ltd's product) of 1 μ L, 10mMdNTP0.2 μ L and 5U/ μ L.The reaction conditions of pcr amplification is: 95 DEG C of 5min; 95 DEG C of 30s, 62 DEG C of 45s, 72 DEG C of 1min, 30 circulations; 72 DEG C of 5min.
4, utilize PCR-SSCP technology for detection based on the genotype in G1001ASNP site: in 6 μ L sample-loading buffers, add 10 μ LPCR amplified productions, after mixing in PCR instrument 98 DEG C of sex change 10min, take out and put into ice bath immediately, loading after 5 ~ 10min, then use 12% polyacrylamide gel electrophoresis, treat that tetrabromophenol sulfonphthalein moves to bottom gel, stop electrophoresis, glue is immersed in stationary liquid (10% ethanol, 0.5% acetic acid) and fix 10min, rinsed with deionized water 3min, then uses 0.1%AgNO
3dyeing 10 ~ 15min, with rinsed with deionized water 1 time (must not more than 5s), finally uses nitrite ion (2%NaOH, 0.04%Na
2cO
3, 0.4% formaldehyde) and colour developing, band is clear can be stopped.Sample-loading buffer is 98% deionized formamide, 10mmolL-1EDTA (pH8.0), the dimethylbenzene cyanogen of 0.25% and the tetrabromophenol sulfonphthalein of 0.25%.Electrophoretic buffer is 1 × TBE.Deposition condition is 4 DEG C of cryostates, voltage 180V, electrophoresis 5 ~ 8h.
Part of test results is shown in that (in Fig. 1, swimming lane 1,2,3,4,5,15,19,20,21,22,25 and 28 is that AA is homozygous to Fig. 1; Swimming lane 6,8,10,11,12,13,14,16,17,18,23,24,26,27,29,30,31,32,33,35,36,37,38,39,41,42,43,44,45 is that GG is homozygous; Swimming lane 7,9,28,40,46 is AG heterozygous).Result shows, test material is that AA is homozygous, GG is homozygous or AG heterozygous based on G1001ASNP loci gene type.
According to the method described above based on the genotype in G1001ASNP site, somatotype is carried out to 185 parts of upland cotton materials.Experimental result is in table 2, table 3, table 4 and table 5.The upland cotton of 66 kinds is that AA is homozygous based on the genotype in G1001ASNP site, and the upland cotton of 22 kinds is AG heterozygous based on the genotype in G1001ASNP site, and the upland cotton of 97 kinds is that GG is homozygous based on the genotype in G1001ASNP site.
Two, the statistics of six precocious correlated character of upland cotton
1, according to the method for step 1 in embodiment 1, respectively in late April, 2013 (SP-2013), in late May, 2013 (SU-2013), in late April, 2014 (SP-2014) or in late May, 2014 (SU-2014), plant each test material, normal water and fertilizer management, in upland cotton vegetative period, investigate WGP, SSP, FBP, NFFB, HNFFB and YPBF6 proterties.Classify according to the genotypic results in step one, then upland cotton material added up, the results are shown in Table 1, table 2, table 3, table 4 and table 5.In order to reduce the impact of environmental change on Characters in Upland Cotton, environment phenotypic character measured value all uses SAS computed in software BLUP value (BLUPs, thebestlinearunbiasedpredictions).
Statistics is as follows:
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and the time of infertility of 89.77% is all less than 120 days, and mean value is 111.68 days; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the time of infertility of 84.54% is all more than 120 days, and mean value is 124.18 days.
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and the seedling flower bud phase of 84.10% is all less than 65 days, and mean value is 63.35 days; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the seedling flower bud phase of 91.75% is all more than 65 days, and mean value is 69.53 days.
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and the flowering and boll-setting period of 76.14% is all less than 50 days, and mean value is 47.66 days; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the flowering and boll-setting period of 90.72% is all more than 50 days, and mean value is 50.28 days.
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, the first fruit branch position of 82.95% is all less than 6 leaf figure places, and mean value is 5.68 leaf figure places; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the first fruit branch position of 91.75% is all more than 6 leaf figure places, and mean value is 7.23 leaf figure places.
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, the first fruit branch position of 80.68% is highly less than 22 centimetres, and mean value is 20.15 centimetres; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, the first fruit branch position of 92.78% is highly all more than 22 centimetres, and mean value is 26.05 centimetres.
66 genotype based on G1001ASNP site are the homozygous Upland Cotton of AA and 22 genotype based on G1001ASNP site is in the Upland Cotton of AG heterozygous, and before the frost of 90.91%, flower rate is all more than 70%, and mean value is 79.82%; 97 genotype based on G1001ASNP site are that in the homozygous Upland Cotton of GG, before the frost of 82.47%, flower rate is all less than 70%, and mean value is 61.03%.
Result shows, detect the genotype of upland cotton to be measured based on G1001ASNP site, if upland cotton to be measured based on the genotype in G1001ASNP site be the homozygous or AG heterozygous of AA, upland cotton to be measured is the upland cotton with Early mature apricot of candidate, if upland cotton to be measured is based on the upland cotton with late-maturing proterties of the genotype in G1001ASNP site to be homozygous, the to be measured upland cotton of GG be candidate;
Early mature apricot is for following (a1) is to the combination of one or more in (a6): be less than 120 days (a1) time of infertility; (a2) the seedling flower bud phase is less than 65 days; (a3) flowering and boll-setting period is less than 50 days; (a4) the first fruit branch position is less than 6 leaf figure places; (a5) the first fruit branch position is highly less than 22 centimetres; (a6) before frost, flower rate is more than 70%;
Described late-maturing proterties is following (b1) combination to one or more in (b6): more than 120 days (b1) time of infertility; (b2) more than the 65 days seedling flower bud phase; (b3) flowering and boll-setting period more than 50 days; (b4) more than 6, first fruit branch position leaf figure place; (b5) the first fruit branch position height is more than 22 centimetres; (b6) spend rate for being less than 70% before frost.
Experiment proves, can screen upland cotton Early mature apricot, playing a significant role in common upland cotton molecular breeding by detecting upland cotton to be measured based on the genotype of G1001ASNP.
The phenotypic data statistics of the precocious correlated character of table 1. six upland cotton
Note: SP-2013 (in April, 2013 spring sowing), SU-2013 (in late May, 2013 summer sowing), SP-2014 (in April, 2014 spring sowing) and SU-2014 (in late May, 2014 summer sowing)
The statistics of the part upland cotton material of table 2.2013 year sowing in April
The statistics of the part upland cotton material of table 3.2013 year sowing in May
The statistics of the part upland cotton material of table 4.2014 year sowing in April
The statistics of the part upland cotton material of table 5.2014 year sowing in May
Claims (10)
1. a method for qualification or assistant identification upland cotton mature trait, comprises the steps:
Detect the genotype based on G1001ASNP site in the genomic dna of upland cotton to be measured, if be the upland cotton with Early mature apricot that the homozygous or AG heterozygous of AA, upland cotton to be measured are candidate, if for homozygous, the to be measured upland cotton of GG be the upland cotton with late-maturing proterties of candidate;
Described G1001ASNP site is that the sequence 1 of sequence table is from 5 ' end the 1001st Nucleotide.
2. the method for claim 1, is characterized in that:
Described Early mature apricot is following (a1) combination to one or more in (a6):
(a1) 120 days are less than the time of infertility;
(a2) the seedling flower bud phase is less than 65 days;
(a3) flowering and boll-setting period is less than 50 days;
(a4) the first fruit branch position is less than 6 leaf figure places;
(a5) the first fruit branch position is highly less than 22 centimetres;
(a6) before frost, flower rate is more than 70%;
Described late-maturing proterties is following (b1) combination to one or more in (b6):
(b1) more than the 120 days time of infertility;
(b2) more than the 65 days seedling flower bud phase;
(b3) flowering and boll-setting period more than 50 days;
(b4) more than 6, first fruit branch position leaf figure place;
(b5) the first fruit branch position height is more than 22 centimetres;
(b6) spend rate for being less than 70% before frost.
3. method as claimed in claim 1 or 2, it is characterized in that: in described method, the genotypic method detected based on G1001ASNP site in the genomic dna of upland cotton to be measured is: the genomic dna extracting upland cotton to be measured, pcr amplification contains the target region in described G1001ASNP site, obtains the genotype based on G1001ASNP site.
4. method as claimed in claim 3, is characterized in that: the primer pair that described pcr amplification adopts is made up of primers F and primer R; The nucleotide sequence of described primers F is as shown in the sequence 2 of sequence table, and the nucleotide sequence of described primer R is as shown in the sequence 3 of sequence table.
5. screening has a method for the upland cotton of Early mature apricot, and comprise the steps: the mature trait identifying upland cotton to be measured according to described method arbitrary in Claims 1-4, screening has the upland cotton of Early mature apricot.
6. screening has a method for the upland cotton of late-maturing proterties, and comprise the steps: the mature trait identifying upland cotton to be measured according to described method arbitrary in Claims 1-4, screening has the upland cotton of late-maturing proterties.
7. the primer pair be made up of primers F and primer R; The nucleotide sequence of described primers F is as shown in the sequence 2 of sequence table, and the nucleotide sequence of described primer R is as shown in the sequence 3 of sequence table.
8. the test kit containing primer pair described in claim 7.
9. a SNP site relevant to upland cotton mature trait, for the sequence 1 of sequence table is from 5 ' end the 1001st Nucleotide, the 1001st Nucleotide is A or G;
Described mature trait be the time of infertility proterties and/or seedling flower bud phase proterties and/or flowering and boll-setting period proterties and/or the first fruit branch position proterties and/or the first fruit branch position height proterties and/or frost before flower rate proterties.
10. a DNA fragmentation containing claim 1 described in SNP site relevant to upland cotton mature trait, is characterized in that: the nucleotide sequence of this DNA fragmentation is as shown in sequence in sequence table 1;
Described mature trait be the time of infertility proterties and/or seedling flower bud phase proterties and/or flowering and boll-setting period proterties and/or the first fruit branch position proterties and/or the first fruit branch position height proterties and/or frost before flower rate proterties.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510730586.1A CN105238866B (en) | 2015-11-02 | 2015-11-02 | One SNP site related to upland cotton Early mature apricot and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510730586.1A CN105238866B (en) | 2015-11-02 | 2015-11-02 | One SNP site related to upland cotton Early mature apricot and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105238866A true CN105238866A (en) | 2016-01-13 |
| CN105238866B CN105238866B (en) | 2018-04-06 |
Family
ID=55036684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510730586.1A Expired - Fee Related CN105238866B (en) | 2015-11-02 | 2015-11-02 | One SNP site related to upland cotton Early mature apricot and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105238866B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099588A (en) * | 2017-04-28 | 2017-08-29 | 中国农业科学院棉花研究所 | Exploitation and its application for identifying the precocial SSR marker of upland cotton |
| CN108396031A (en) * | 2018-02-11 | 2018-08-14 | 中国农业科学院棉花研究所 | Regulate and control gene and its application of upland cotton plant height |
| CN114032235A (en) * | 2017-12-13 | 2022-02-11 | 山东棉花研究中心 | SSR marker, primer pair and application thereof, and screening method of SSR marker sites related to upland cotton precocity molecular breeding |
| CN115418409A (en) * | 2022-08-15 | 2022-12-02 | 甘肃农业大学 | SNP site related to upland cotton boll opening character and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757030A (en) * | 2014-01-14 | 2014-04-30 | 浙江农林大学 | Alleles for regulating and controlling two types of main stem table fur types of upland cotton, and single stranded conformational polymorphism (SSCP) molecular marker for identification thereof |
-
2015
- 2015-11-02 CN CN201510730586.1A patent/CN105238866B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757030A (en) * | 2014-01-14 | 2014-04-30 | 浙江农林大学 | Alleles for regulating and controlling two types of main stem table fur types of upland cotton, and single stranded conformational polymorphism (SSCP) molecular marker for identification thereof |
Non-Patent Citations (2)
| Title |
|---|
| JUNJI SU ET AL: "Identification of favorable SNP alleles and candidate genes for traits related to early maturity via GWAS in upland cotton", 《BMC GENOMICS》 * |
| LI F ET AL: "Genome sequence of cultivated Upland cotton（Gossypium hirsutum TM-1） provides insights into genome evolution", 《NATURE BIOTECHNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099588A (en) * | 2017-04-28 | 2017-08-29 | 中国农业科学院棉花研究所 | Exploitation and its application for identifying the precocial SSR marker of upland cotton |
| CN114032235A (en) * | 2017-12-13 | 2022-02-11 | 山东棉花研究中心 | SSR marker, primer pair and application thereof, and screening method of SSR marker sites related to upland cotton precocity molecular breeding |
| CN114032235B (en) * | 2017-12-13 | 2023-08-11 | 山东棉花研究中心 | SSR marker, primer pair, application of primer pair and screening method of SSR marker locus related to upland cotton early-maturing molecular breeding |
| CN108396031A (en) * | 2018-02-11 | 2018-08-14 | 中国农业科学院棉花研究所 | Regulate and control gene and its application of upland cotton plant height |
| CN108396031B (en) * | 2018-02-11 | 2021-05-25 | 甘肃农业大学 | Gene for regulating and controlling height of cotton plant on land and application thereof |
| CN115418409A (en) * | 2022-08-15 | 2022-12-02 | 甘肃农业大学 | SNP site related to upland cotton boll opening character and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105238866B (en) | 2018-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ren et al. | Utilization of a Wheat55K SNP array for mapping of major QTL for temporal expression of the tiller number | |
| CN107278877B (en) | A kind of full-length genome selection and use method of corn seed-producing rate | |
| CN103555717B (en) | Functional molecular markers of related genes of sweetness and sourness characters of muskmelon and application of markers | |
| Ma et al. | QTLs analysis and validation for fiber quality traits using maternal backcross population in upland cotton | |
| CN105238866A (en) | SNP site related to early-maturing traits in upland cotton and application of SNP site | |
| CN107937556A (en) | One and the relevant SNP site of pannage conversion ratio and its application | |
| Ademe et al. | Association mapping analysis of fiber yield and quality traits in Upland cotton (Gossypium hirsutum L.) | |
| CN107090494A (en) | The molecular labeling related to millet code grain number character and its detection primer and application | |
| CN107090495A (en) | The molecular labeling related to the long character of millet neck and its detection primer and application | |
| Zhang et al. | Identification of loci and candidate genes responsible for fiber length in upland cotton (Gossypium hirsutum L.) via association mapping and linkage analyses | |
| CN103993011A (en) | Molecular marker of sesame dominant genic male sterility gene and preparation method and application thereof | |
| CN104988142A (en) | Novel cucumber SNP molecular marker | |
| Ma et al. | Cumulative and different genetic effects contributed to yield heterosis using maternal and paternal backcross populations in Upland cotton | |
| CN107099588B (en) | Development and application of SSR (simple sequence repeat) marker for identifying earliness of upland cotton | |
| Zhang et al. | Efficacy of pyramiding elite alleles for dynamic development of plant height in common wheat | |
| CN104672315B (en) | Control gene and to Cucumber Roll palpus character related SNP marker of the cucumber without tendril character | |
| Liao et al. | Aus rice root architecture variation contributing to grain yield under drought suggests a key role of nodal root diameter class | |
| CN103160584B (en) | Method and special primer for screening or auxiliary screening of wheat with high pre-harvest sprouting resistance | |
| Zhu et al. | Old-growth Platycladus orientalis as a resource for reproductive capacity and genetic diversity | |
| Zhang et al. | Genomic prediction of drought tolerance during seedling stage in maize using low-cost molecular markers | |
| CN115927718B (en) | KASP molecular marker for identifying grain weight and grain width of wheat and application | |
| CN104293774A (en) | Functional SSR labels obviously related with wood quality characters in populus CesAs gene, and applications and kit thereof | |
| Abhijith et al. | Genome-wide association study reveals novel genomic regions governing agronomic and grain quality traits and superior allelic combinations for Basmati rice improvement | |
| CN104818326A (en) | SNP marker related to erhualian sow littering traits | |
| CN111088390B (en) | SNP molecular marker related to upland cotton leaf villi and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180406 Termination date: 20211102 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |