CN103952402A - SNP (single nucleotide polymorphism) site related to characters of plant root system and application thereof - Google Patents
SNP (single nucleotide polymorphism) site related to characters of plant root system and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP (single nucleotide polymorphism) site relates to characters of a plant root system and application thereof. The SNP site related to the characters of the plant root system is a 55th nucleotide from a tail end 5' in SEQ ID No.4, and the 55th nucleotide is C and/or G; a DNA segment shown in SEQ ID No.4 is at a position 19.3cM on a wheat chromosome 1BS. The invention provides a new root system major gene site QRt.caas-1BS and an auxiliary excellent wheat root system screening SNP site; wheat with excellent root system characters can be screened by utilizing the SNP site, so that the SNP site plays an important role in wheat root system related breeding.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of SPN site and the application thereof relevant to root system of plant proterties.
Background technology
Cultivate high yield, wide suitable and liquid manure heavy duty detergent new variety of wheat, not only need good economical character, and need firm and flourishing root system system and lasting improving activity of root system.Due to the complicacy of growth of wheat roots environment and the limitation of research method and means thereof, make the relatively top tissue hysteresis of genetic research of root traits serious, therefore breeding man still lacks the awareness and understanding to wheat root Heredity, limit kind Root inheritance flow of research, restricted the extensively cultivation of suitable new variety of high yield.Along with investigator deepens the awareness of root system importance, the method for some root system research develops in succession, and wherein nutritive medium is cultivated method and can easy and effectively be measured wheat root proterties (An etc., 2006).So far, root traits and molecular biological combination, make the research of root system correlated inheritance also obtain greater advance, but totally it seems, research is in this respect still weak, as for to the autotelic genetic improvement of wheat root, the clear and definite Root inheritance breeding work of carrying out, still in initiation developmental stage.
Due to the vital role of wheat root, utilize genetic and breeding method mould with overground part grow adapt, there is lasting stability vigor, especially late growth stage also the great-hearted root system of tool be the important goal of wheat root breeding.Molecular Marker Assisted Selection Technology can follow the tracks of transformation or polymerization root system basis because of, for cultivating, there are the effective technology means that good root system and high yield wide adaptation type wheat breed provide.Excavating control root traits gene and genetic marker thereof is the prerequisite of carrying out molecular mark.For this reason, the gene mapping and cloning work of relevant gramineous crop root traits is carried out in succession, and especially in paddy rice and corn, progress is larger.For example, the relevant QTL of the control root system degree of depth carrying on paddy rice the 9th karyomit(e) is not only beneficial to raising plant drought resistance, and can strengthen the absorption of plant to root system nutrition element, the mark corresponding to hereditary section RM242-RM201 of this gene has been applied in marker assisted selection, and obtained and carried this gene and the good strain (Steele etc., 2006) of root traits.The gene L-ABA that controls maize leaf ABA concentration can promote Photosynthetic Rate, and is beneficial to Root morphology growth, and this genetic marker is also developed at present, is used for (Giuliani etc., 2005) in marker assisted selection.In addition, a main effect QTL relevant to corn yield and root system is excavated, and be applied (Landi etc., 2010).Wheat cdna group structure is complicated compared with paddy rice and corn, root traits is subject to controlled by multiple genes as root length, surface-area, volume and dry matter weight etc., conventionally be a bunch existence, mainly be distributed on 1D, 2A, 2D, 3A, 5A, 6A and 7D karyomit(e), the 3.2%-19.6% of the soluble phenotypic variation in single QTL site, there is epistatic effect in part QTL.Several root character gene is associated (Bai etc., 2013) with rich water utilization, economical character and yield traits genes involved, but rarely has the report of close linkage mark.
SNP(Single-nucleotide polymorphism, single nucleotide polymorphism) mark is the genetic polymorphism that the variation of single base causes in DNA sequence dna, and mutation frequency is high, is the highest mark of genome distribution density.Along with detecting the method for SNP, get more and more, and practical more economically, make it become the molecule marker of new generation after SSR.SNP is marked at the aspects such as structure, map based cloning and QTL location, spore, the marker assisted selection breeding of rice genetic collection of illustrative plates be used widely (Pan Cunhong etc., 2007).In wheat cdna group research process, SNP mark is mainly used in genetic map construction and assignment of genes gene mapping aspect research (Cavanagh etc., 2013) at present.
Association analysis be take linkage disequilibrium as basis, is to identify the relation between proterties and genetic marker in natural population and in conjunction with a kind of method of genetic linkage maps Mapping of QTL, be widely used in the assignment of genes gene mapping of the crops such as paddy rice, corn and wheat.Thornsberry etc. (2001) utilize 92 corn inbred lines to carry out association analysis to the allelic variation of dwarf8 gene first, have proved that dwarf8 gene not only controls plant height but also affect flowering period.Breseghello and Sorrells (2006) utilize 62 SSR marks to carry out association analysis to 95 wheat breeds, find to carry on 2D, 5A and 5B karyomit(e) the major gene loci of control Grain Morphology proterties; And these sites and the result consistent (Breseghello and Sorrells, 2007) that they utilize linkage mapping colony to locate, affirmed the validity of association analysis in wheat cdna location.Crossa etc. (2007) utilize 813 DArT marks and 831 other genetic markers to carry out whole-genome association to the yield traits of 170 portions of CIMMYT spring wheat and stem rust, leaf rust, bar rust and Powdery Mildew, find that most of yield traits is consistent with the result of having reported with disease-resistant gene site, and located many new resistance genes site.Xiao Yonggui etc. (2012) utilize the association analysis of backbone parent week 8425B Derived Populations, identify Stripe Rust Resistance Gene QYr.caas-2BL, and excavate SSR primer Xgwm501 and can be used for this gene-assisted selection.
Capital 411 is the semi-dwarf mutant wheat breeds that are bred as early 1990s, has the advantages such as winter resistance is strong, ability for tillering is strong, the percentage of earbearing tiller is high, disease resistance is good, stable high yield, wide adaptability, is one of the backbone parent in Northern Winter district.At present, utilize capital 411 to be bred as 14 new variety (being), that the middle wheat 175 wherein selecting has is cold-resistant, high yield, wide suitable, many anti-, the advantage such as noodle quality is good, and has realized stable high yield and noodle quality is good, cold-resistant, water saving and precocial good combination.Between 2007-2012, by Beijing, Shanxi Province, national northern water ground, the fertile ground of national the Yellow River and Huai He River drought, Qinghai Province, Gansu Province, authorize respectively, because its plant type is compact, the percentage of earbearing tiller is high, plant height water relatively little with nonirrigated farmland luffing, be applicable to irrigated land, supplement irrigation ground and drought and plant fertilely, become the kind that Northern Winter district and the Yellow River and Huai He River drought have important promotional value fertilely.As seen from Figure 1, compared with capital 411 improvement significantly, backbone parent capital 411 still has good lateral root proterties to the proterties such as the main root of middle wheat 175 length and root dry matter weight.On the 3A karyomit(e) of Ren Yongzhe etc. (2012) in Beijing 411, locate 3 QTL that control the longest root length in seedling stage, root dry matter weight and ground material dry weight, interpret table form variation is all more than 10%, and several root character gene site is participated in regulation and control liquid manure utilising efficiency (An etc., 2006 directly; Xu etc., 2013).Therefore, why capital 411 and middle wheat 175 become the extensively Representative Cultivars of suitable wheat breed of Northern Winter district high yield, the root system basis that may mainly carry with it is because of relevant, locate and excavate its carry new root system basis because of mark, in breeding process from now on, follow the tracks of select to carry good root system basis because of kind there is important practical and be worth.
Summary of the invention
The DNA fragmentation that an object of the present invention is to provide a kind of SNP site relevant to root system of plant proterties and contain this SNP site.
The SNP site relevant to root system of plant proterties provided by the present invention, this SNP site be in SEQ ID No.4 from 5 ' end the 55th Nucleotide, this 55bp position Nucleotide is C and/or G; The 19.3cM position of DNA fragmentation shown in SEQ ID No.4 on chromosome of wheat 1BS.
The DNA fragmentation that contain SNP site relevant to root system of plant proterties provided by the present invention, the nucleotide sequence of this DNA fragmentation is as shown in SEQ ID No.4, and wherein, this sequence 55bp place base is C and/or G; And the 19.3cM position of this DNA fragmentation on chromosome of wheat 1BS.
Another object of the present invention is to provide a kind of special primer group for detection of SNP site described in claim 1.
Special primer group for detection of SNP site described in claim 1 provided by the present invention comprises two primers, and the nucleotide sequence of article one primer is as shown in SEQ ID No.1, and the nucleotide sequence of another primer is as shown in SEQ ID No.3.
Above-mentioned special primer group also comprises a primer, and the nucleotide sequence of this primer is as shown in SEQ ID No.2.
Another object of the present invention is to provide the PCR test kit of the good root system plant of a kind of assisting sifting.
The PCR test kit of the good root system plant of assisting sifting provided by the present invention, comprises above-mentioned special primer group and pcr amplification damping fluid;
Described pcr amplification damping fluid comprises archaeal dna polymerase, dNTP, MgCl
2, KCl, Tris and water.
The pH value of described pcr amplification damping fluid is 9.0;
In described special primer group, every primer final concentration in described PCR test kit is 0.25uM;
The final concentration of described Tris-HCl in described PCR test kit is 1mmolL
-1;
The final concentration of described KCl in described PCR test kit is 5mmolL
-1;
Described MgCl
2final concentration in described PCR test kit is 2.5mmolL
-1;
The final concentration of described dNTPs in described PCR test kit is 200 μ molL
-1;
The final concentration of described archaeal dna polymerase in described PCR test kit is 1.5U.
The application in the good root system plant of assisting sifting of the DNA fragmentation in above-mentioned SNP site, the above-mentioned SNP of containing site, above-mentioned special primer group or above-mentioned PCR test kit also belongs to protection scope of the present invention.
In above-mentioned application, described plant can be wheat; Described wheat specifically can be in following kind any or appoint several: green ant No. 1, Gaocheng 8901, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, capital are two 16, Jimai 22, Shanxi wheat 45, good star 66, face drought No. 2, good star 99, Shan 354, Mianyang 26, tobacco grower 19, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, raise wheat No. 9, Zhongyou9507, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32, Jimai 20.
Another object of the present invention is to provide a kind of method of differentiating good root system plant.
The method of the good root system plant of discriminating provided by the present invention, comprises the steps: whether the 19.3cM position of detecting on the karyomit(e) 1BS that treats measuring plants contains above-mentioned SNP site, and detects the genotype in this SNP site; Genotype is that the root system of the plant of CC is better than the plant that genotype is CG or GG.
In the method for the good root system plant of above-mentioned discriminating, described plant can be wheat; Described wheat be specially in following kind any or appoint several: green ant No. 1, Gaocheng 8901, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, capital are two 16, Jimai 22, Shanxi wheat 45, good star 66, face drought No. 2, good star 99, Shan 354, Mianyang 26, tobacco grower 19, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, raise wheat No. 9, Zhongyou9507, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32, Jimai 20;
In the method for the good root system plant of above-mentioned discriminating, described " root system of the plant that genotype is CC is better than the plant that genotype is CG or GG " is longer than the plant that genotype is CG or GG for the root system overall length of the plant that genotype is CC.
Another object of the present invention is to provide the method for the good root system plant of a kind of assisting sifting.
The method of the good root system plant of assisting sifting provided by the present invention, comprises the steps: whether the 19.3cM position of detecting on the karyomit(e) 1BS that treats measuring plants contains above-mentioned SNP site, and detects the genotype in this SNP site; If genotype is CC, described in judging, treat the good root system plant that measuring plants is candidate.
In the method for the good root system plant of above-mentioned assisting sifting, the method for described " detection treats whether the 19.3cM position on the karyomit(e) 1BS of measuring plants contains SNP site described in claim 1, and detects the genotype in this SNP site " is following (1) or (2):
(1) utilize above-mentioned arbitrary described special primer group or above-mentioned arbitrary described PCR test kit, treat measuring plants and carry out pcr amplification, pcr amplification product is checked order; If sequencing result is SEQ ID No.4, and from its 5 ' end the 55th be C, the genotype of judging the SNP site for the treatment of measuring plants is CC; If sequencing result is SEQ ID No.4 and SEQ ID No.5, and from SEQ ID No.45 ' end the 55th be C or G, the genotype of judging the SNP site for the treatment of measuring plants is CG or GG;
(2) utilize above-mentioned arbitrary described special primer group or above-mentioned arbitrary described PCR test kit, treat measuring plants and carry out pcr amplification, pcr amplification product is carried out to gel electrophoresis; If a kind of fragment that electrophoresis showed amplified production is 173bp, the genotype of judging the SNP site for the treatment of measuring plants is CC; If electrophoresis showed amplified production is two kinds of fragments of 173bp and 138bp, the genotype of judging the SNP site for the treatment of measuring plants is CG or GG.
In the method for the good root system plant of above-mentioned assisting sifting, described plant can be wheat; Described wheat be specially in following kind any or appoint several: green ant No. 1, Gaocheng 8901, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, capital are two 16, Jimai 22, Shanxi wheat 45, good star 66, face drought No. 2, good star 99, Shan 354, Mianyang 26, tobacco grower 19, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, raise wheat No. 9, Zhongyou9507, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32, Jimai 20;
Described good root system is that root system overall length is long; Described root system overall length length refers to wheat seedlings root in tri-leaf period overall length >=340cm, is specially root system overall length >=349cm.
The present invention has the following advantages: the SNP site that experiment showed, the good root system wheat of assisting sifting that the invention provides a new root system major gene loci QRt.caas-1BS and this site of the present invention.Utilize this SNP site can screen the wheat that root traits is good, in the relevant breeding of wheat root, play a significant role.
Accompanying drawing explanation
Fig. 1 is the Seedling root figure of capital 411 and middle wheat 175
Fig. 2 utilizes SNP site to screen the result of the pcr amplification of good root system wheat
Fig. 3 is the linkage map of 1 SNP mark and QRt.caas-1BS gene
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
All primers are synthetic to be completed by Beijing AudioCodes biotechnology limited liability company.In following examples, all wheat lines of using are all preserved center from country of Chinese Academy of Agricultural Sciences farm crop germplasm.
The acquisition of embodiment 1, one, the SNP mark BS00022505 relevant to root traits
Select winter wheat backbone parent capital 411 and 14 derived varietiess (being) thereof, comprise 6 parts of derivative generation, 8 parts of derivative two generations.
1, root traits investigation
Root test adopts water culture, 50, the seed that every kind is chosen, the processing 20-30 minute with 10%, aseptic water washing 5-6 time.Choose H
2o
2full and the culture dish that is covered with filter paper that is placed in the same size of the seed processed, in the vernalization of incubator darkroom.Each material is selected the consistent seed that germinates and is sent out seedling cultivation, and culture plate is placed in nutritive medium and cultivates at controllable greenhouse, nutrient solution reference literature (Ren etc., 2012).Cultured continuously after 15 days to seedling stage Analysing Root Characters investigate.Investigation proterties comprises that the longest root is long, lateral root is long, main root is long, total root length, lateral root surface-area, main root surface-area, total root surface area, always tip of a root number and weight of root system.
2, primer obtains and labeled analysis
SNP(single nucleotide polymorphism) be marked at Boao Biological Co., Ltd (CapitalBio Corporation, Beijing, China; Http:// bioservices.capitalbio.com) utilize Illumina SNP genotype tests, be mainly divided into following steps: 1) wheat breed genomic dna to be measured is carried out to whole genome amplification; 2) random disconnectedization of endonuclease digestion for amplified production; 3) by DNA fragmentation with DNA fragmentation and chip are hybridized, on the microballon of chip, be connected with 50-mers length specificity capture probe, gDNA enzyme is cut after product and is combined with probe complementary sequence; 4) clean the DNA fragmentation on that removal is not hybridized or mismatch hybridization; 5) the Nucleotide substrate (A/T and C/G) of dinitrophenol (dinitrophenol) and vitamin H (biotin) mark carries out single-basic extension on capture probe, only has the probe that complementary combination occurs with gDNA just can be extended; By dyeing, A/T will distinguish the different fluorescence dye of mark with C/G; 6) chip scanning, and utilize software according to two kinds of fluorescence interpretations and export somatotype result.
Utilize Illumina SNP gene type research platform to carry out 90kSNP chip somatotype to capital 411 and derived varieties (being) DNA thereof, comprise the series of markings such as BS, BobWhite, CAP, D_contig, amount to 81587, wherein in 24080 SNP mark 411 Derived Populations in Beijing, there are differences.
Two, the discovery of associated gene location and linked marker BS00022505
Utilize SAS9.2 software (SAS Institute.2000) to carry out basic statistics amount, multiple comparison analyse, and in conjunction with the Glmselect program of SAS, SNP data and root traits are carried out to successive Regression, according to P value (P<0.01), judge associated site.Orient BS00022505 associated with site QRt.caas-1BS (P<0.001).
Three, the allele specific Marker Identification in BS00022505 site
Extract respectively the complete genome DNA of capital 411 and 14 derived varietiess.Take each genomic dna respectively as the allele specific mark KASP of template with SNP mark BS00022505 site
tMgenotype tests, occurs that the fragment of C:C somatotype shows that SNP mark BS00022505 can effectively identify the long gene of wheat breed root.
Separation in table 1BS00022505 mark allelic variation 411 Derived Populations in Beijing
Result shows, when the genotype in BS00022505 site is only C:C, has the long phenotype of long total root conform to wheat breed, illustrates that BS00022505 site can effectively identify the long gene of the total root of wheat breed and genotype thereof.
//www.triticarte.com.au) and the wheat molecular marker collection of illustrative plates announced of Allen etc. (2011) according to Wheat DArT maps Version1.2(http:, mark BS00022505 is incorporated on wheat genetic collection of illustrative plates, result as shown in Figure 3, is determined the 19.3cM position of QRt.caas-1BS on karyomit(e) 1BS.
The application of embodiment 2, SNP
Select offspring's kind of 29 non-411 and the derived varieties in 2 capital 411.Wherein the derived varieties in 2 capital 411 is control group, comprises middle wheat 175 and CA9722; Offspring's kind of 29 non-411 is test group, comprise that green ant No. 1, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, capital are two 16, Jimai 22, Shanxi wheat 45, good star 66, face drought No. 2, good star 99, Shan 354, Mianyang 26, tobacco grower 19, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, raise wheat No. 9, Zhongyou9507, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32 and Jimai 20.
One, detect the Root Traits at Seedling Stage of the wheat of different varieties
Wheat breed Root Traits at Seedling Stage is identified in 2012 Nian Dong Inst. of Genetics and Development Biology, CAS greenhouses and is carried out.Each kind is chosen 50, seed full and of the same size, the H with 10%
2o
2process 20-30 minute, aseptic water washing 5-6 time; Again seed is placed in to the culture dish that is covered with filter paper, darkroom vernalization 18-24h in incubator; Then select 25 consistent, the seed that germinates and be placed in that to send out seedling online, grow and after 6 days, choose wheat seeding 6 strains of the same size and be transferred in culture plate, each culture plate carries out 3 repetitions; Again culture plate being placed in to nutritive medium cultivates at controllable greenhouse.Nutrient solution reference literature (Ren Y, He X, Liu D, Li J, Zhao X, Li B, Tong Y, Zhang A, Li Z.Major quantitative trait loci for seminal root morphology of wheat seedlings.Molecular Breeding, 2012,30:139-148, public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.)。Seedling culture condition is 22 ± 1 ℃ of temperature, and relative humidity is at 50%-60%.Within every 3 days, change one time of nutrition liquid, cultured continuously after 15 days to seedling stage root gather in the crops.Root system with scanner after to each kind results scans, then use that image analysis software Win RHIZO (Canadian Regent Instruments company) analyzes that Analysing Root Characters comprises that the longest root is long, lateral root is long, main root is long, total root length, lateral root surface-area, main root surface-area, total root surface area, always tip of a root number and weight of root system.
The long definition of total root: the length sum that wheat seedling in tri-leaf period is all, i.e. root system overall length.
Two, detect the genotype in the SNP site of different varieties wheat
Extract respectively the genomic dna of each product grow wheat, take genomic dna as template, adopt allele specific primer group F1(SEQ ID No.1), F2(SEQ ID No.2) and R(SEQ ID No.3) carry out allele specific pcr amplification.
12 μ L PCR reaction systems comprise: special primer group F1(SEQ ID No.1), F2(SEQ ID No.2) and R(SEQ ID No.3), and every primer final concentration is 0.25uM, final concentration is 1mmolL
-1tris-HCl(pH9.0), final concentration is 5mmolL
-1kCl, final concentration is 2.5mmolL
-1mgCl
2(Promega company), final concentration is 200 μ molL
-1dNTPs(TaKaRa company), Taq archaeal dna polymerase 1.5U(Tiangen company), template DNA 15ng.
PCR reaction is carried out on PTC-200PCR amplification instrument, and amplification program is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 20s, 50 ℃ of annealing 30s, 72 ℃ are extended 15s, 35 circulations; 72 ℃ are extended 4min; 10 ℃ of preservations.
Pcr amplification product is carried out to agarose gel electrophoresis detection, concrete operations are: in 12 μ L pcr amplification products, add 2 μ L6 times sample-loading buffer (30mM EDTA pH8.0,0.25% tetrabromophenol sulfonphthalein and 0.1% dimethylbenzene are blue or green), every part of amplification sample is got electrophoretic separation 2h in the sepharose that 4 μ L are 3% in concentration, under 300nm ultraviolet lamp, observes electrophoresis result.
Electrophoresis has 2 kinds of results: the one, and amplified production is only a band, size is 173bp; The 2nd, amplified production contains two bands, and size is respectively 138bp and 173bp.If amplified production is 173bp mono-band, judge that the 55th Nucleotide in the SNP site of wheat to be measured is C, genotype is CC; If amplified production is 173bp and 138bp two bands, judge that the 55th Nucleotide in the SNP site of wheat to be measured is C and/or G, genotype is GG or CG.
Above-mentioned PCR product is further carried out to sequence verification.Sequencing result shows, when amplified production is only 173bp mono-band, its nucleotide sequence is as shown in SEQ ID No.4, and the base at 55bp place is C, and the genotype in SNP site is CC; When amplified production comprises 138bp and 173bp two band, the nucleotide sequence of 173bp is as shown in SEQ ID No.4, and all bases at its 55bp place are C or G, and the nucleotide sequence of 138bp is as shown in SEQ ID No.5, and the genotype in SNP site is CG or GG.
If amplified production is only the sequence shown in SEQ ID No.4 for the sequence of 173bp or amplified production, and the base at 55bp place is all C, and the genotype of the wheat QRt.caas-1BS gene SNP site that this product is corresponding is CC.If amplified production had both contained the fragment of 173bp, the fragment that contains again 138bp, or the sequence of amplified production comprises the sequence shown in SEQ ID No.4 and SEQ ID No.5 simultaneously, the genotype of the wheat QRt.caas-1BS gene SNP site that this product is corresponding is CG or GG.
Electrophoresis partial results as shown in Figure 2.The results are shown in Table 2.
The genotype in the SNP site of table 2,29 wheat breeds and the long result of total root
√: in amplified production, represent to contain this band, *: in amplified production, represent not contain this band.
Above result shows: green ant No. 1, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, Jimai 22, Shanxi wheat 45, good star 66, good star 99, Shan 354, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, No. 9, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32 are all increased and are only obtained the band (sequence shown in SEQ ID No.4 of 173bp to raise wheat, and the base in 55bp site is all C), the genotype that shows the SNP site of these wheats is CC.And these wheats all have longer root system overall length.From the above results, also can find out, total root length of the wheat that the genotype in SNP site is CC is greater than the wheat that genotype is CG or GG.
The above results shows, it is long that above-mentioned SNP site can identify whether wheat breed has long total root quickly and accurately.
Claims (10)
1. a SNP site relevant to root system of plant proterties, this SNP site be in SEQ ID No.4 from 5 ' end the 55th Nucleotide, this 55bp position Nucleotide is C and/or G; The 19.3cM position of DNA fragmentation shown in SEQ ID No.4 on chromosome of wheat 1BS.
2. the DNA fragmentation that contain SNP site relevant to root system of plant proterties, is characterized in that: the nucleotide sequence of this DNA fragmentation is as shown in SEQ ID No.4, and wherein, this sequence 55bp place base is C and/or G;
And the 19.3cM position of this DNA fragmentation on chromosome of wheat 1BS.
3. for detection of the special primer group in SNP site described in claim 1, described special primer group comprises two primers, and the nucleotide sequence of article one primer is as shown in SEQ ID No.1, and the nucleotide sequence of another primer is as shown in SEQ ID No.3.
4. special primer group according to claim 3, is characterized in that, also comprises a primer, and the nucleotide sequence of this primer is as shown in SEQ ID No.2.
5. a PCR test kit for the good root system plant of assisting sifting, is characterized in that:
Described PCR test kit comprises special primer group and pcr amplification damping fluid described in claim 2 or 3;
Described pcr amplification damping fluid comprises archaeal dna polymerase, dNTP, MgCl
2, KCl, Tris and water.
The pH value of described pcr amplification damping fluid is 9.0;
In described special primer group, every primer final concentration in described PCR test kit is 0.25uM;
The final concentration of described Tris-HCl in described PCR test kit is 1mmolL
-1;
The final concentration of described KCl in described PCR test kit is 5mmolL
-1;
Described MgCl
2final concentration in described PCR test kit is 2.5mmolL
-1;
The final concentration of described dNTPs in described PCR test kit is 200 μ molL
-1;
The final concentration of described archaeal dna polymerase in described PCR test kit is 1.5U.
6. the application of PCR test kit in the good root system plant of assisting sifting described in the special primer group described in DNA fragmentation, claim 3 or 4 or claim 4 described in SNP site, claim 2 described in claim 1.
7. differentiate a method for good root system plant, comprise the steps: to detect 19.3cM position on the karyomit(e) 1BS that treats measuring plants and whether contain SNP site described in claim 1, and detect the genotype in this SNP site; Genotype is that the root system of the plant of CC is better than the plant that genotype is CG or GG.
8. a method for the good root system plant of assisting sifting, comprises the steps: to detect 19.3cM position on the karyomit(e) 1BS that treats measuring plants and whether contains SNP site described in claim 1, and detects the genotype in this SNP site; If genotype is CC, described in judging, treat the good root system plant that measuring plants is candidate.
9. according to the method described in claim 7 or 8, it is characterized in that: the method for described " detection treats whether the 19.3cM position on the karyomit(e) 1BS of measuring plants contains SNP site described in claim 1, and detects the genotype in this SNP site " is following (1) or (2):
(1) utilize PCR test kit described in special primer group described in claim 3 or 4 or claim 4, treat measuring plants and carry out pcr amplification, pcr amplification product is checked order; If sequencing result is SEQ ID No.4, and from its 5 ' end the 55th be C, the genotype of judging the SNP site for the treatment of measuring plants is CC; If sequencing result is SEQ ID No.4 and SEQ ID No.5, and from SEQ ID No.45 ' end the 55th be C or G, the genotype of judging the SNP site for the treatment of measuring plants is CG or GG;
(2) utilize PCR test kit described in special primer group described in claim 3 or 4 or claim 4, treat measuring plants and carry out pcr amplification, pcr amplification product is carried out to gel electrophoresis; If a kind of fragment that electrophoresis showed amplified production is 173bp, the genotype of judging the SNP site for the treatment of measuring plants is CC; If electrophoresis showed amplified production is two kinds of fragments of 173bp and 138bp, the genotype of judging the SNP site for the treatment of measuring plants is CG or GG.
10. application according to claim 6, the arbitrary described method of claim 7-9, is characterized in that:
Described plant is wheat;
Described wheat be specially in following kind any or appoint several: green ant No. 1, Gaocheng 8901, Handan 6172, Huaihe River wheat 18, Huaihe River wheat 20, Jimai 19, Jimai 21, capital are two 16, Jimai 22, Shanxi wheat 45, good star 66, face drought No. 2, good star 99, Shan 354, Mianyang 26, tobacco grower 19, Anhui wheat 38, river in Shangdong Province agriculture 14, western agriculture 979, littlely lay down 54, raise wheat No. 9, Zhongyou9507, Henan wheat 63, middle wheat 895, all wheats 16, all wheats 18, all wheats 22, all wheats 31, all wheats 32, Jimai 20;
Described good root system is that root system overall length is long;
Described " root system of the plant that genotype is CC is better than the plant that genotype is CG or GG " is longer than the plant that genotype is CG or GG for the root system overall length of the plant that genotype is CC;
Described root system overall length is long for wheat seedlings root in tri-leaf period overall length >=340cm, is specially root system overall length >=349cm.
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| CN105132428A (en) * | 2015-09-22 | 2015-12-09 | 中国农业大学 | ZmLRT gene related to plant root system characters as well as relevant biological material and application thereof |
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