CN110592263B - SSR primer group and kit for identifying blood margin of saccharum arundinaceum in sugarcane and application of SSR primer group and kit - Google Patents
SSR primer group and kit for identifying blood margin of saccharum arundinaceum in sugarcane and application of SSR primer group and kit Download PDFInfo
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Abstract
The invention discloses an SSR primer group and a kit for identifying the blood margin of saccharum arundinaceum in sugarcane and application thereof. The primer group comprises 10 pairs of SSR marker primers SSR 1-SSR 10. The detection kit comprises an SSR primer group and a PCR reagent. The SSR 1-SSR 10 primer pairs are a set of autonomously developed SSR labeled primers with the specificity of the saccharum arundinaceum, and the SSR labels are characterized in that the SSR labels are averagely distributed on a set of chromosome groups of the saccharum arundinaceum, one or more pairs of primers of the labels are used for amplification, and the existence of the blood margin of the saccharum arundinaceum can be judged as long as a saccharum arundinaceum specificity band appears. The identification by using the set of markers can avoid the problem that the blood margin of the festuca arundinacea cannot be detected due to marker failure after the chromosome of the offspring of the festuca arundinacea is lost, so the set of markers is particularly suitable for identifying the high-generation hybrid material of the sugarcane and the festuca arundinacea.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an SSR primer group and a kit for identifying the blood margin of saccharum arundinaceum in sugarcane and application thereof.
Background
The sugarcane is an important crop for both sugar and energy in China, more than 85% of sugar in China is derived from the sugarcane, and the sugarcane is used for cultivating high-yield, high-sugar and strong-stress resistance sugarcane varieties and plays an important role in the sugarcane industry. The breeding of the sugarcane is also called high-priced breeding, and the adaptability, the stress resistance, the perennial root property and other properties of the sugarcane variety are improved through the hybridization of wild resources and tropical seeds and the multi-generation backcross. However, the current available parent materials are few, so that the variety faces the problems of narrow genetic background and reduced performances of the variety such as adaptability, stress resistance, perennial root property and the like. The festuca arundinacea has the excellent characteristics of strong stress resistance, strong ratoon, more tillering, barren resistance and the like, and is an excellent material for improving the variety of the sugarcane. At present, wild species of the saccharum arundinaceum has been successfully introduced into the background of the saccharum arundinaceum through a distant hybridization method, a large number of filial generations of the saccharum arundinaceum and the saccharum arundinaceum are obtained, and part of excellent lines have been applied to the crossbreeding of the saccharum arundinaceum. However, the genetic rule of filial generations of the sugarcane and the festuca arundinacea is complex, chromosomes are reduced along with the increase of the number of backcross generations, and researches show that 1/2 of the festuca arundinacea chromosome is lost almost every generation in the high-generation material of the sugarcane and the festuca arundinacea, so that the high-level loss directly influences the detection of the blood margin of the festuca arundinacea.
At present, methods for identifying whether hybrid progeny of sugarcane and festuca arundinacea contains the blood margin of the festuca arundinacea mainly include Genome In Situ Hybridization (GISH) and molecular marking technology. In the two methods, the period and the technical difficulty for identifying the filial generation by using the GISH technology are high, and the method usually needs a professional to operate and cannot carry out early identification. The identification is carried out by utilizing a molecular marker technology, the technical difficulty is small, the identification can be completed in a seedling stage, and the investment of land, manpower and the like for actual seedling culture is reduced. There are molecular marker technologies for identifying the blood margin of the saccharum arundinaceum in the sugarcane by using molecular markers, such as 5S rDNA, ITS marker, AELP technology and the like, but because of the small number of markers, the markers exist on individual chromosomes, and if the chromosomes are lost, the markers are invalid, and the filial generation of the hybrids losing the chromosomes cannot be effectively detected.
CN 1566362A discloses a DNA identification method for hybrid of Saccharum officinarum and Festuca arundinacea, which comprises genomic DNA extraction of Saccharum officinarum and Festuca arundinacea, reagent preparation, amplification and electrophoresis detection. The identification method can obtain the most direct hybrid evidence from the DNA molecular level, has the characteristics of accuracy, intuition, convenience, high identification speed, small environmental influence factors and high identification accuracy, successfully solves the difficulty of identifying the hybrid of the sugarcane and the festuca arundinacea for a long time, ensures the authenticity of the identified hybrid and improves the effectiveness of breeding and utilization. However, because only two primers are selected for marking, the marking range is limited, and the problem that the blood margin of the saccharum arundinaceum cannot be detected due to the marking failure after the chromosome of the offspring of the saccharum arundinaceum is lost after the sugarcane is hybridized with the saccharum arundinaceum cannot be avoided.
Therefore, the development of more markers specific to the Erythrococcus arundinacea, especially markers uniformly distributed on a whole set of chromosomes, is the key point of the identification process of the blood margin of the Erythrococcus arundinacea in high-generation materials.
Disclosure of Invention
The application mainly aims to provide an SSR primer group for identifying the blood margin of the saccharum arundinaceum in the sugarcane.
Another object of the present invention is to provide a kit for identifying the blood margin of Erythrochloe arundinacea in sugarcane.
The invention further aims to provide the SSR primer group for identifying the blood relationship of the saccharum arundinaceum in the sugarcane and application of the kit.
The above object of the present invention is achieved by the following technical solutions:
an SSR primer group for identifying the blood margin of the saccharum arundinaceum in the sugarcane comprises 10 primer pairs, and the specific sequence is as follows:
(1) primer pair SSR1
F1：5’-GCGTAGAATCTGTCGGCACT-3’(Tm＝60.43℃)；
R1：5’-CAACGCGTAATTTCCATGTG-3’(Tm＝59.99℃)；
(2) Primer pair SSR2
F2：5’-GGGATGGCAATGGCATATAA-3’(Tm＝60.51℃)；
R2：5’-TCTGCGCTCTGGTCAACTTA-3’(Tm＝59.74℃)；
(3) Primer pair SSR3
F3：5’-CCAATCATCCTTGCTCAGGT-3’(Tm＝60.07℃)；
R3：5’-GCGAGGCAGACTGGTTATTC-3’(Tm＝59.84℃)；
(4) Primer pair SSR4
F4：5’-TCGAGTAGTACTGCAGCTGATGA-3’(Tm＝60.23℃)；
R4：5’-CATGGTGTGTCTCATGAACCT-3’(Tm＝58.42℃)；
(5) Primer pair SSR5
F5：5’-ATCTCCTCCACATTGGCTTG-3’(Tm＝60.07℃)；
R5：5’-TTTTCAATGAAGTGGAGCCC-3’(Tm＝60.05℃)；
(6) Primer pair SSR6
F6：5’-CCCCAGTGCTTCGCTACTAC-3’(Tm＝59.90℃)；
R6：5’-TTTTCCTGATTGGAAAACCG-3’(Tm＝59.91℃)；
(7) Primer pair SSR7
F7：5’-TTTCCTGAACACGCAGGAG-3’(Tm＝59.97℃)；
R7：5’-CTGCTCATAGCAAGGGGTGT-3’(Tm＝60.28℃)；
(8) Primer pair SSR8
F8：5’-ACCGACATGAGAGCTGGACT-3’(Tm＝59.87℃)；
R8：5’-GTTTCATGCTTTTCGATTGC-3’(Tm＝58.36℃)；
(9) Primer pair SSR9
F9：5’-GGCTCGTAGGAGCATTCAAC-3’(Tm＝59.84℃)；
R9：5’-TGAGAACAGCATGGAGACCT-3’(Tm＝58.38℃)；
(10) Primer pair SSR10
F10：5’-AGCCTGCAGGTCTCTCTGAC-3’(Tm＝59.74℃)；
R10：5’-ATGCAATGCAACACGACAAT-3’(Tm＝60.00℃)。
The SSR marker has the advantages of high polymorphism, codominance and good stability, and is widely applied to genomes. The invention is based on the research of the genome of the saccharum arundinaceum developed by the unit, takes the sorghum genome as reference, autonomously develops a group of SSR markers which are uniformly distributed on a set of chromosome sets and have the specificity of the saccharum arundinaceum, and has important significance for improving the identification of the blood margin of the saccharum arundinaceum in the distant hybridization progeny of the sugarcane and the saccharum arundinaceum, in particular for the identification of the blood margin of the saccharum arundinaceum in high-generation materials.
The SSR primer group for identifying the blood margin of the saccharum officinarum in the sugarcane is applied to identifying the material containing the blood margin of the saccharum officinarum in the filial generation of the saccharum officinarum and the saccharum officinarum or preparing a kit for identifying the blood margin of the saccharum officinarum in the sugarcane.
A kit for identifying the blood margin of the saccharum arundinaceum in the sugarcane comprises the SSR primer group for identifying the blood margin of the saccharum arundinaceum in the sugarcane.
The kit for identifying the blood margin of the festuca arundinacea in the sugarcane also comprises a reagent for PCR.
The reagent for PCR comprises at least one of a premix for 2 XPCR amplification and water for PCR.
The pre-mixed solution for PCR amplification comprises Ex Taq polymerase with the concentration of 0.05U/. mu.L and PCR buffer solution.
The buffer solution for PCR contains 4mM Mg2+And 0.4mM dNTP buffer.
The kit for identifying the blood margin of the festuca arundinacea in the sugarcane is applied to identifying the material containing the blood margin of the festuca arundinacea in the filial generation of the sugarcane and the festuca arundinacea.
A method for identifying the bloody margin of the saccharum officinarum and the festuca arundinacea in filial generations of the saccharum officinarum and the festuca arundinacea comprises the following steps:
(1) extracting the genomic DNA of a filial generation sample of the sugarcane and the stipa arundinacea to be identified;
(2) taking the genomic DNA of the sample to be identified extracted in the step (1) as a template, and performing PCR amplification by using the primer group or the kit to obtain a PCR amplification product;
(3) carrying out gel electrophoresis detection on the PCR amplification product in the step (2), and counting gel electrophoresis results after treatment;
(4) and (4) analyzing the gel electrophoresis result in the step (3) and identifying whether the filial generation of the sugarcane and the festuca arundinacea contains the festuca arundinacea blood margin.
The genome DNA in the step (1) can be obtained by extracting through conventional technologies, such as a nucleic acid precipitation method, a magnetic bead method, an adsorption column method and the like; preferably prepared by the following steps: grinding plant leaves to be identified by liquid nitrogen, and adding a preheated lysis buffer solution; heating in water bath, mixing, adding chloroform/isoamylol, centrifuging, mixing the supernatant with the precooled DNA precipitation solution, performing ice bath, continuously centrifuging, washing the precipitate with ethanol, drying, dissolving with TE, and storing for later use; wherein the lysis buffer solution is composed of NaCl solution, Tris-HCl buffer solution and Na2-EDTA solution, CTAB solution, said lysis buffer requiring room temperature storage; the DNA precipitation solution is PEG8000 and NaCl solution; more preferably prepared by the following steps: taking plant leaves, grinding by liquid nitrogen, taking about 0.1g of ground sample, adding the ground sample into a 2mL centrifuge tube, adding 700 mu L of lysis buffer solution preheated at 65 ℃, turning and mixing uniformly during water bath at 65 ℃ for 30min, adding chloroform/isoamylol mixed solution with the same volume (the volume ratio of chloroform to isoamylol is 24:1), mixing uniformly, centrifuging at 15 ℃ and 11000rpm for 10min, taking supernatant, adding DNA precipitation solution precooled with the same volume, mixing uniformly, carrying out ice bath for 30min, centrifuging, discarding supernatant, adding 75% ethanol into the precipitate for washing, centrifuging, pouring out supernatant, drying, adding 100 mu L of TE buffer solution for dissolving, and storing at-20 ℃ for later use; wherein the lysis buffer solution comprises 1.4M NaCl solution, 0.1M Tris-HCl buffer solution and 20mM Na2-EDTA solution, 2% CTAB solution; the DNA precipitation solution is 20% (M/v) PEG8000, which contains 2M NaCl solution.
The PCR system in step (2) is preferably: each primer pair is prepared into a reaction system, each 20 mu L of reaction system contains 0.5 mu L of DNA template, 10 mu L of premixed solution for 2 XPCR amplification and 10 mu mol.L-1The amount of the upstream primer and the downstream primer of each primer pair of (1) is 0.5. mu.L, and the balance is water.
The PCR conditions in step (2) are preferably: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 33 cycles; total extension at 72 ℃ for 10 min.
The electrophoresis in the step (3) is non-denaturing polyacrylamide gel electrophoresis.
The electrophoresis condition is 250V constant voltage electrophoresis for 5 hours.
The concentration of the native polyacrylamide gel electrophoresis is 7% (v/v).
The electrophoresis buffer is 0.5 xTBE.
The treatment comprises the steps of rinsing, silver staining, rinsing, color development, gel rinsing and the like.
The identification method in the step (4) comprises the following steps: if the specific stripe of the festuca arundinacea appears on the gel, the sample to be identified can be judged to contain the festuca arundinacea blood margin; on the contrary, the blood margin of the tall fescue is not contained. The specific strip positions of the amplified product of each primer pair are as follows: SSR1 occurs around 240 bp; the SSR2 main band appears at about 185bp, and 1-2 sub-bands appear between 185-195; about 265bp appears in the SSR3 main band, and 1-2 sub-bands appear between 265-289; SSR4 appears at about 250bp and 275bp, and two bands appear simultaneously or only one band appears; the SSR5 band appears at about 185 bp; the SSR6 band appears about 250 bp; the SSS7 band occurs at about 180 bp; SSR8 bands appear about 178bp and 165bp, and the two bands appear at the same time; the SSR9 band appears about 280 bp; the SSR10 band appears around 245 bp.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the SSR 1-SSR 10 primer pairs are a set of self-developed SSR markers with the specificity of the saccharum arundinaceum, and the SSR markers are characterized by being evenly distributed on a set of chromosome groups (10) of the saccharum arundinaceum, and one or more pairs of primers of the markers are used for amplification, so that the existence of the blood margin of the saccharum arundinaceum can be judged as long as a saccharum arundinaceum specificity band appears. The SSR primer provided by the invention is obtained by optimization screening, and has strong specificity. The chromosome set can be used for avoiding the problem that the blood margin of the festuca arundinacea cannot be detected due to the failure of the marker after the chromosome of the offspring of the festuca arundinacea is lost, so that the marker set is particularly suitable for identifying the high-generation hybrid material of the sugarcane and the festuca arundinacea.
(2) The plant material is not limited in position and development period, and can be identified by taking the leaves as the material in the seedling stage of the sugarcane, so that the material without the blood margin of the festuca arundinacea is eliminated in the early stage, and the planting cost is reduced.
(3) The method has the advantages of simple and easy test operation steps and high repeatability, can finish the identification of a large amount of test materials in a short time, and can be widely used for germplasm innovation of the hybridization of the sugarcane and the saccharum arundinaceum and the screening of the saccharum arundinaceum strain containing the saccharum arundinaceum in the future.
Drawings
FIG. 1 is a diagram of the amplification results of SSR1 on different sources of stipa arundinacea, sugarcane parent materials and filial generation materials of the sugarcane and the stipa arundinacea; wherein, Lane M is DL500 DNA maker, Lane 1 is Jiangxi 83-4, Lane 2 is Sichuan 79-I-9, Lane 3 is Hainan 92-77, Lane 4 is Guizhou 78-II-14, Lane 5 is Yunnan 82-85, Lane 6 is Badila, Lane 7 is New Tabane No. 22, Lane 8 is Guangdong sugar 94-128, Lane 9 is HoCP07-613, Lane 10 is YCE07-71, Lane 11 is YCE01-92, Lane 12 is YCE01-105, Lane 13 is YCE06-140, Lane 14 is BC 2-32; the arrows indicate the target bands.
FIG. 2 is a diagram of the amplification results of SSR2 on different sources of the saccharum arundinaceum, sugarcane parent material and filial generation material of the saccharum officinarum and the saccharum arundinaceum; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 3 is a diagram showing the amplification results of SSR3 on different sources of stipa arundinacea, sugarcane parent material and filial generation material of sugarcane and stipa arundinacea; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 4 is a diagram of the amplification results of SSR4 on different sources of stipa arundinacea, sugarcane parent material and filial generation material of sugarcane and stipa arundinacea; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 5 is a diagram of the amplification results of SSR5 on different sources of the saccharum arundinaceum, sugarcane parent material and filial generation material of the saccharum officinarum and the saccharum arundinaceum; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 6 is a diagram of the amplification results of SSR6 on different sources of the saccharum arundinaceum, sugarcane parent material and filial generation material of the saccharum officinarum and the saccharum arundinaceum; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 7 is a graph showing the amplification results of SSR7 on different sources of stipa arundinacea, sugarcane parent material and filial generation material of sugarcane and stipa arundinacea; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 8 is a graph of the amplification results of SSR8 on different sources of stipa arundinacea, sugarcane parent material and filial generation material of sugarcane and stipa arundinacea; the samples corresponding to the lanes are the same as in FIG. 1.
FIG. 9 is a diagram of the amplification results of SSR9 on different sources of stipa arundinacea, sugarcane parent material and filial generation material of sugarcane and stipa arundinacea; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 10 is a diagram of the amplification results of SSR10 on different sources of stipa arundinacea, sugarcane parent material and sugarcane and stipa arundinacea filial generation material; the samples corresponding to each lane are the same as in FIG. 1.
FIG. 11 is a diagram of the amplification result of the primers SSR2, SSR4, SSR10 and ITS markers in identifying the blood margin of the festuca arundinacea in the high-generation backcross generation; wherein: lane M is DL500 marker, lane 1 is YCE07-71, lane 2 is neotame No. 22, lane 3 is Y22-1, lane 4 is Y22-2, lane 5 is Y22-3, lane 6 is Y22-4, lane 7 is Y22-5, lane 8 is Y22-6, lane 9 is Y22-7, lane 10 is Y22-8, lane 11 is Y22-9, and lane 12 is Y22-10.
FIG. 12 is a chart comparing the results of amplification of SSR2 with non-specific marker E02-77186 located on the same chromosome; wherein A is an SSR2 amplification effect graph; b is a non-specific label amplification effect graph; the samples corresponding to each lane are the same as in FIG. 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1: blood margin identification of saccharum arundinaceum, saccharum officinarum and saccharum officinarum hybridized different backcross materials
In the embodiment, a set of SSR marks comprising SSR 1-SSR 10 are used for completely showing the effect of identifying the blood margin of the saccharum arundinaceum in the materials of different backcross generations of the hybridization of the saccharum officinarum and the saccharum arundinaceum by using the set of SSR marks, and simultaneously respectively showing the conditions of specific bands of the saccharum arundinaceum when different pairs of primers are amplified, so that reference is provided for identifying the blood margin of the saccharum arundinaceum by using the primers.
The specific operation steps are as follows:
in the embodiment, 5 original varieties of the saccharum arundinaceum (Jiangxi 83-4, Sichuan 79-I-9, Hainan 92-77, Guizhou 78-II-14 and Yunnan 82-85), 4 parent materials of the saccharum officinarum (Badila, New Tabane No. 22, Guangdong sugar 94-128 and HoCP07-613) and 5 filial generation materials of the saccharum arundinaceum (YCE07-71, YCE01-92, YCE01-105, YCE06-140 and BC2-32) in different areas are selected, and all the materials can be obtained from a saccharum germplasm resource bank in Guangdong province.
(1) Extracting the genomic DNA of the filial generation sample of the sugarcane and the stipa arundinacea to be identified
Firstly, extracting the genome DNA of the 14 parts of materials as a template for standby; the extraction of the genome DNA adopts a CTAB extraction method, and the specific steps are as follows:
1) the 14 young leaves of the material are ground by liquid nitrogen, and about 100mg of the tissue sample is added into a 2mL centrifuge tube.
2) Adding 700 mu L of lysis buffer solution preheated at 65 ℃, carrying out water bath at 65 ℃ for 30min, and uniformly turning over for 2-3 times.
3) Adding 700 μ L chloroform/isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1) mixed solution, shaking vigorously for 10min, centrifuging at 11000rpm at 15 ℃ for 10min, and transferring the supernatant into a 1.5mL centrifuge tube.
4) Adding precooled DNA precipitation solution with the same volume, mixing uniformly, standing for 30min in ice bath, centrifuging for 10min at 11000rpm, and discarding the supernatant.
5) The precipitate was washed 2 times with 1mL of 75% ethanol, the supernatant was decanted, and the solution was dried under vacuum for 10 min.
6) Add 30. mu.L of TE buffer for DNA solubilization and store at-20 ℃ until use.
The lysis buffer solution is prepared by mixing 1.4M NaCl, 0.1M Tris-HCl, 20mM Na-EDT and 2% CTAB and is stored at room temperature; the DNA precipitation solution was 20% (M/v) PEG8000 containing 2M NaCl.
(2) And (3) taking the extracted genome DNA of the sample to be identified as a template, and respectively carrying out PCR amplification by using primer pairs of SSR 1-SSR 10 and PCR reagents to obtain a PCR amplification product.
Wherein, the PCR reaction system is as follows: DNA template 0.5. mu.L, 2 XPCR amplification premix 10. mu.L, 10. mu. mol. L-1The above primers are each 0.5. mu.L, sterile ultrapure water is added to the final volume20 μ L.
The PCR amplification procedure was: denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 30s, 33 cycles, total extension at 72 deg.C for 10min, and storage at 4 deg.C.
(3) Performing gel electrophoresis detection on the PCR reaction product in the step (2), and counting the gel electrophoresis result
Performing gel electrophoresis detection by adopting non-denaturing polyacrylamide gel electrophoresis, wherein the gel concentration is 7% (V/V), the electrophoresis buffer solution is 0.5 xTBE, the 250V constant voltage electrophoresis is performed for 4-5 hours, and after the electrophoresis is finished, rinsing, silver staining, rinsing and developing are performed, and the reading data is recorded after the gel is rinsed.
PCR products were detected by 7% (v/v) native PAGE, as follows:
1) cleaning the glass plate, drying, assembling, and sealing the opening at the bottom end of the glass plate with 1% (m/v) agar gel.
2) The electrophoresis gel (table 1) is prepared in proportion, glue is injected immediately after shaking up lightly (no bubbles need to be generated), a comb is inserted, and the gel is solidified after being laid flat and standing for 1 h.
3) Fixing the glass plate with the solidified glue on an electrophoresis tank, adding a proper amount of 0.5 xTBE buffer solution, covering the liquid surface with comb holes, and pulling out a comb.
4) 2.5. mu.L of the sample loading indicator was added to each PCR product, and after shaking and mixing, 3. mu.L of the sample to be identified described in this example was taken by a micropipette and added to the well.
5) The electrophoresis is stopped when xylene is slowly moved to 3/4 of the gel at a constant pressure of 250V in each electrophoresis tank, and about 5 hours is required.
6) After electrophoresis is finished, the glass plate is pried open, agar gel on the periphery is removed by a blade, the gel is carefully taken out and put into a disc filled with distilled water for slight oscillation and washing twice.
7) The solution was dissolved in 400mL with 20mL of 0.1% silver nitrate, and then poured into a dish for dyeing for 10min, during which time the dish was shaken gently on a shaker, then the silver nitrate was poured out of the dish and rinsed gently twice with distilled water.
8) Then 20mL of mother liquor prepared with color developing solution is dissolved in 400mL of secondary water, 1.0mL of formaldehyde is added, color development is carried out for about 10min, and photographing is carried out to be stored in a JPEG format.
TABLE 17% (v/v) amounts of the components of the non-denatured glue
The preparation method of each reagent formula in the steps comprises the following steps:
1. 10 × TBE electrophoresis buffer: 108g Tris, 55g boric acid, 7.4448g EDTA-Na2(pH8.0) was dissolved in deionized water to a constant volume of 1L.
2. 10% (m/v) ammonium persulfate solution: 30g of ammonium persulfate is weighed and deionized water is added to 300mL, and the mixture can be stored for a plurality of weeks at 4 ℃.
3. Loading an indicator: weighing 500mg of bromophenol blue, adding 20mL of distilled water, standing overnight at room temperature, weighing 500mg of xylene blue, dissolving in 20mL of deionized water, adding 80g of sucrose, adding deionized water for dissolving, mixing the three solutions, adding deionized water to a constant volume of 200mL, and storing at 4 ℃ for later use.
4. 12% PAGE formulation (1000 mL): 114g of acrylamide, 6g of methylene acrylamide, 240g of Urea Urea, and 200mL of 10 XTBE, and adding deionized water to 1000mL (the solution is stored at 4 ℃ for standby).
5、20×0.1％AgNO3Dyeing liquid: weighing 10g AgNO3And adding deionized water to dissolve to a constant volume of 500 mL.
6. 20 × color developing solution: 50g of NaOH and 1.9g of sodium tetraborate are weighed, and deionized water is added for dissolving and metering to 500 mL.
(4) Analyzing the specific band condition of the festuca arundinacea by analyzing the amplified band
Analyzing the electrophoresis result: if the specific stripe of the festuca arundinacea appears, the sample to be identified can be judged to contain the festuca arundinacea blood margin; on the contrary, the blood margin of the tall fescue is not contained.
The identification results are shown in FIGS. 1 to 10, and the results show that: the SSR 1-SSR 10 is used for amplifying the stock seeds of the festuca arundinacea in different areas, the sugarcane and the filial generation of the sugarcane and the festuca arundinacea, and 10 pairs of primers are used for amplifying bands with the specificity of the festuca arundinacea, wherein the SSR2, the SSR3, the SSR4, the SSR5, the SSR8, the SSR9 and the SSR10 mark that polymorphism exists among different festuca arundinacea materials; from the amplification results of the progeny of the hybrids of sugarcane and festuca arundinacea of different generations, YCE01-92 (lane 11) all have markers and specific bands are detected; YCE01-105 (Lane 12) all detected a band specific to Erecta arundinacea except SSR 9; BC2-32 (lane 14) No specific band was detected except SSR 6; the high generation YCE07-71 (lane 10) detected specific bands using primers SSR2, SSR4 and SSR10, and YCE06-140 (lane 13) detected specific bands using primers SSR3, SSR4 and SSR 5. Therefore, the SSR markers can be used for maximally detecting the blood margin of the festuca arundinacea in the filial generation of the sugarcane and the festuca arundinacea, and the situation that the blood margin of the festuca arundinacea cannot be detected by a single marker due to chromosome loss is avoided.
Comparative example 1 comparison of SSR marker and ITS marker of the invention for detecting the blood margin of saccharum officinarum and saccharum arundinaceum in filial generation of saccharum officinarum and saccharum arundinaceum
To further verify the superiority of the marker of the invention in identifying the blood margin of the high-generation material, this example compares the difference between the conventional marker ITS for identifying the blood margin of the festuca arundinacea and the marker of the invention in identifying the blood margin of the festuca arundinacea in the high-generation backcross generation.
In the embodiment, SSR2, SSR4 and SSR10 are taken as representatives, materials are YCE07-71 (including the blood margin of the festuca arundinacea, which is obtained by hybridizing sugarcane with the festuca arundinacea and then backcrossing for four generations, and belongs to high-generation materials), new Tabane No. 22 (without the blood margin of the festuca arundinacea), 10 filial generations of YCE07-71 and new Tabane No. 22: y22-1, Y22-2, Y22-3, Y22-4, Y22-5, Y22-6, Y22-7, Y22-8, Y22-9 and Y22-10, wherein the 10 materials are randomly selected from a cross combination which takes YCE07-71 as a female parent and takes neotame No. 22 as a male parent, and have obvious differences in plant height, stem diameter, stem color, bud type and the like; the SSR markers described herein are compared to ITS labeling methods.
(1) Extracting the genomic DNA of the filial generation sample of the sugarcane and the stipa arundinacea to be identified
Firstly, extracting the genome DNA of the 14 parts of materials as a template for standby; the extraction of the genome DNA adopts a CTAB extraction method, and comprises the following specific steps:
1) the 14 young leaves of the material are ground by liquid nitrogen, and about 100mg of the tissue sample is added into a 2mL centrifuge tube.
2) Add 700. mu.L of lysis buffer pre-heated at 65 ℃ and water bath at 65 ℃ for 30min, during which the mixture was tumbled evenly 3 times.
3) Adding 700 μ L chloroform/isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1) mixed solution, shaking vigorously for 10min, centrifuging at 11000rpm at 15 ℃ for 10min, and transferring the supernatant into a 1.5mL centrifuge tube.
4) Adding precooled DNA precipitation solution with the same volume, uniformly mixing, standing for 30min in an ice bath, centrifuging for 10min at 11000rpm, and discarding the supernatant.
5) The precipitate was washed 2 times with 1mL of 75% ethanol, the supernatant was decanted, and the solution was dried under vacuum for 10 min.
6) Add 30. mu.L of TE buffer for DNA solubilization and store at-20 ℃ until use.
The lysis buffer solution is prepared by mixing 1.4M NaCl, 0.1M Tris-HCl, 20mM Na-EDT and 2% CTAB and is stored at room temperature; the DNA precipitation solution was 20% (M/v) PEG8000 containing 2M NaCl.
(2) Using the extracted genomic DNA of the sample to be identified as a template, and respectively using primers SSR4 and ITS to carry out PCR amplification to obtain PCR amplification products
Wherein, the PCR reaction system is as follows: DNA template 0.5. mu.L, 2 XPCR amplification premix 10. mu.L, 10. mu. mol. L-1The above primers of (4) were each 0.5. mu.L, and sterile ultrapure water was added to a final volume of 20. mu.L.
PCR amplification was performed with markers SSR2, SSR4, and SSR10, respectively, by the following procedure: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and 33 cycles; total extension at 72 deg.C for 10min, and storage at 4 deg.C.
The PCR amplification program of the ITS marker is as follows: denaturation at 95 deg.C for 5min, denaturation at 93 deg.C for 50s, annealing at 52 deg.C for 20s, extension at 72 deg.C for 30s, 30 cycles, total extension at 72 deg.C for 5min, and storage at 4 deg.C.
ITS 7:5’-GGAAGAAAGAAAACAAGGGT-3’；
ITS 8:5’-GGGACGGMCMAAACAAAATT-3’。
(3) Performing gel electrophoresis detection on the PCR reaction product in the step (2), and counting the gel electrophoresis result
Performing gel electrophoresis detection by adopting non-denatured polyacrylamide gel electrophoresis, wherein the gel concentration is 7% (V/V), the electrophoresis buffer solution is 0.5 xTBE, the 250V constant voltage electrophoresis is performed for 4-5 hours, and after the electrophoresis is finished, rinsing, silver staining, rinsing and developing are performed, and the read data is recorded after the gel is rinsed.
(4) Identification results
The ITS primers are used for identification (as shown in FIG. 11), it can be seen that two clear bands (lane 1) of the parent YCE07-71 are directly seen in 300-400 bp, no band (lane 2) is found in New Tab 22, but in the filial generation (lanes 3-12), only the obvious amplified bands are seen in the 4 th, 5 th, 7 th, 8 th and 9 th lanes of five materials, and no band is found in the 3 th, 6 th, 10 th, 11 th and 12 th lanes, which indicates that most of the high-generation backcross of the festuca arundinacea has lost the chromosome containing the marker, so that the ITS marker cannot identify whether the blood margin of the festuca arundinacea is contained. However, the SSR primer group is used for identifying the blood margin of the festuca arundinacea, and three markers with specific bands in YCE07-71 are identified; wherein: the SSR2 marker clearly shows the spotted cogongrass specific bands in lanes 3, 5 and 9; SSR4 marker, the hybridization progeny of the used sugarcane and the zebra grass can see the zebra grass specific band, and only the difference exists in the quantity of the amplification product; SSR10 marker, appearing as a stigmata specific band in lanes 4, 5, 6, 8, 10, and 11. The three pairs of primers are used for amplification, so long as a specific band appears on one pair of primers, the result shows that the blood margin of the festuca arundinacea is contained, and therefore, 10 offspring identified by using the three pairs of primers contain the blood margin of the festuca arundinacea. The invention utilizes a plurality of pairs of primers to carry out combined identification, and avoids the situation that the blood margin of the festuca arundinacea cannot be identified due to chromosome loss to the maximum extent, so that the SSR marker of the invention is obviously superior to the ITS marker in identifying the blood margin of the festuca arundinacea.
Comparative example 2 comparison of amplification of specific SSR marker and non-specific SSR marker of the present invention
In order to prove that the SSR marker disclosed by the invention has a better specific marker effect than other non-specific markers in the identification of the blood margin of the festuca arundinacea, the SSR2 marker is selected in the embodiment to be compared with other markers on the same chromosome.
The specific operation steps are as follows:
the material selected in this example was the same as that described in example 1, namely 5 different sources of Erysia arundinacea (Jiangxi 83-4, Sichuan 79-I-9, Hainan 92-77, Guizhou 78-II-14 and Yunnan 82-85), 4 sugarcane parent materials (Badila, New Tabano No. 22, Guangdong sugar 94-128 and HoCP07-613), and 5 filial generation materials of Erysia arundinacea (YCE07-71, YCE01-92, YCE01-105, YCE06-140 and BC2-32)
(1) Extracting the genomic DNA of the filial generation sample of the sugarcane and the stipa arundinacea to be identified
Firstly, extracting the genome DNA of the 14 parts of materials as a template for standby; the extraction of the genome DNA adopts a CTAB extraction method, and the specific steps are as follows:
1) the 14 young leaves of the material are ground by liquid nitrogen, and about 100mg of the tissue sample is added into a 2mL centrifuge tube.
2) Adding 700 mu L of lysis buffer solution preheated at 65 ℃, carrying out water bath at 65 ℃ for 30min, and uniformly turning over for 2-3 times.
3) Adding 700 μ L chloroform/isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1) mixed solution, shaking vigorously for 10min, centrifuging at 11000rpm at 15 ℃ for 10min, and transferring the supernatant into a 1.5mL centrifuge tube.
4) Adding precooled DNA precipitation solution with the same volume, mixing uniformly, standing for 30min in ice bath, centrifuging for 10min at 11000rpm, and discarding the supernatant.
5) The precipitate was washed 2 times with 1mL of 75% ethanol, the supernatant was decanted, and the solution was dried under vacuum for 10 min.
6) Add 30. mu.L of TE buffer for DNA solubilization and store at-20 ℃ until use.
The lysis buffer solution is prepared by mixing 1.4M NaCl, 0.1M Tris-HCl, 20mM Na-EDT and 2% CTAB and is stored at room temperature; the DNA precipitation solution was 20% (M/v) PEG8000 containing 2M NaCl.
(2) Taking the DNA extracted in the step (1) as a template, and respectively carrying out PCR amplification by using a specific primer SSR2 and a non-specific marker E02-77186 to obtain a PCR reaction product
The primer pair for the non-specific marker E02-77186 is:
forward primer sequence: 5'-GCGCTAATCAAATGCTCCTC-3' (Tm 59.95 ℃)
Reverse primer sequence: 5'-CGTACCGAAACTAAACCCCA-3' (Tm 59.86 ℃)
The PCR reaction system is as follows: DNA template 0.5. mu.L, 2 XPCR amplification premix 10. mu.L, 10. mu. mol. L-1mu.L of each of the above primers was added with sterile ultrapure water to a final volume of 20. mu.L.
The PCR amplification procedure was: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 33 cycles; total extension at 72 deg.C for 10min, and storage at 4 deg.C.
(3) Performing gel electrophoresis detection on the PCR reaction product in the step (2), and counting the gel electrophoresis result
Performing gel electrophoresis detection by adopting non-denatured polyacrylamide gel electrophoresis, wherein the gel concentration is 7% (V/V), the electrophoresis buffer solution is 0.5 xTBE, the 250V constant voltage electrophoresis is performed for 4-5 hours, and after the electrophoresis is finished, rinsing, silver staining, rinsing, developing, rinsing and recording reading data after the gel.
(4) Identification results
The SSR2 is used for identifying that the specific bands of the saccharum arundinaceum are obviously distinguished from other bands (figure 12A), no interference band exists, the non-specific marker E02-77186 is used for identifying, similar amplification bands appear in sugarcane (4-9 lanes in figure 12B) and the saccharum arundinaceum (1-5 lanes in figure 12B), and in filial generations of the sugarcane and the saccharum arundinaceum (10-14 lanes in figure 12), which bands come from the saccharum arundinaceum and cannot be used for identifying the blood margin of the saccharum arundinaceum. Therefore, the SSR markers are beneficial to complete the identification of a large number of test materials in a short time, and can be widely used for germplasm innovation of sugarcane and festuca arundinacea hybridization and screening of the variety of the sugarcane containing the festuca arundinacea.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangdong province bioengineering research institute (Guangzhou sugar industry institute)
<120> SSR primer group and kit for identifying stipa arundinacea blood margin in sugarcane and application thereof
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR1-F
<400> 1
gcgtagaatc tgtcggcact 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR1-R
<400> 2
caacgcgtaa tttccatgtg 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR2-F
<400> 3
gggatggcaa tggcatataa 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR2-R
<400> 4
tctgcgctct ggtcaactta 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR3-F
<400> 5
ccaatcatcc ttgctcaggt 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR3-R
<400> 6
gcgaggcaga ctggttattc 20
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR4-F
<400> 7
tcgagtagta ctgcagctga tga 23
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR4-R
<400> 8
catggtgtgt ctcatgaacc t 21
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR5-F
<400> 9
atctcctcca cattggcttg 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR5-R
<400> 10
ttttcaatga agtggagccc 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR6-F
<400> 11
ccccagtgct tcgctactac 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR6-R
<400> 12
ttttcctgat tggaaaaccg 20
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR7-F
<400> 13
tttcctgaac acgcaggag 19
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR7-R
<400> 14
ctgctcatag caaggggtgt 20
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR8-F
<400> 15
accgacatga gagctggact 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR8-R
<400> 16
gtttcatgct tttcgattgc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR9-F
<400> 17
ggctcgtagg agcattcaac 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR9-R
<400> 18
tgagaacagc atggagacct 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR10-F
<400> 19
agcctgcagg tctctctgac 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SSR10-R
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atgcaatgca acacgacaat 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ITS 7
<400> 21
ggaagaaaga aaacaagggt 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ITS 8
<400> 22
gggacggmcm aaacaaaatt 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> E02-77186 Forward primer sequence
<400> 23
gcgctaatca aatgctcctc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> E02-77186 reverse primer sequence
<400> 24
cgtaccgaaa ctaaacccca 20
Claims (9)
1. An SSR primer group for identifying the blood margin of the saccharum officinarum, which is characterized in that: comprises 10 groups of primer pairs SSR 1-SSR 10, and the sequence is shown as SEQ ID NO. 1-20.
2. The use of the SSR primer set for identifying the blood margin of the saccharum officinarum Dunn in the claim 1 for identifying the material containing the blood margin of the saccharum officinarum Dunn in the filial generation of the saccharum officinarum Dunn and the saccharum officinarum Dunn or preparing a kit for identifying the blood margin of the saccharum officinarum Dunn in the saccharum officinarum Dunn.
3. A kit for identifying the bloody border of festuca arundinacea in sugarcane, which is characterized in that: the SSR primer group for identifying the blood margin of the saccharum officinarum as claimed in claim 1.
4. The kit of claim 3, wherein: reagents for PCR are also included.
5. The use of the kit for identifying the bloodborders of the saccharum arundinaceum in the claim 3 or 4 for identifying the material containing the bloodborders of the saccharum arundinaceum in the filial generation of the saccharum officinanaceum and the saccharum arundinaceum.
6. A method for identifying the blood margin of the saccharum arundinaceum in filial generations of the saccharum officinarum and the saccharum arundinaceum is characterized by comprising the following steps:
(1) extracting the genomic DNA of a filial generation sample of the sugarcane and the stipa arundinacea to be identified;
(2) performing PCR amplification by using the genomic DNA of the sample to be identified extracted in the step (1) as a template and using the primer group of claim 1 or the kit of claim 3 or 4 to obtain a PCR amplification product;
(3) carrying out gel electrophoresis detection on the PCR amplification product in the step (2), and counting gel electrophoresis results after treatment;
(4) analyzing the gel electrophoresis result in the step (3) and identifying whether the filial generation of the sugarcane and the festuca arundinacea contains the festuca arundinacea blood margin or not;
the identification method in the step (4) comprises the following steps: if the specific stripe of the festuca arundinacea appears on the gel, the sample to be identified can be judged to contain the festuca arundinacea blood margin; otherwise, the blood margin of the festuca arundinacea is not contained; the specific strip positions of the amplified product of each primer pair, namely the festuca arundinacea, are as follows: SSR1 band at 240bp position; the SSR2 has a main band at 185bp position and has 1-2 auxiliary bands between 185-195 bp position; the SSR3 has a main band at the position of 265bp and 1-2 auxiliary bands between 265-289 bp; in 250bp and 275bp positions of SSR4, two bands appear simultaneously or only one band appears; SSR5 band at 185bp position; SSR6 band at 250bp position; SSS7 bands appeared at 180bp positions; in the positions of 178bp and 165bp, two bands appear simultaneously in the SSR 8; SSR9 bands appear at 280bp positions; SSR10 appeared as a band at the 245bp position.
7. The method of claim 6, wherein: the PCR system in the step (2) is as follows: preparing a reaction system for each primer pair, wherein the pre-mixed solution containing 0.5 muL and 2 XPCR amplification of the DNA template in each 20 muL reaction system is 10 muL and 10 mumol-1The upstream and downstream primers of each primer pair are 0.5 mu L each, and the balance is water.
8. The method of claim 6, wherein: the PCR conditions in step (2) are as follows: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and 33 cycles; total extension at 72 ℃ for 10 min.
9. The method of claim 6, wherein: the electrophoresis in the step (3) is non-denaturing polyacrylamide gel electrophoresis.
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