CN110749699A - Method for screening triploid new germplasm from white loquat seedling - Google Patents
Method for screening triploid new germplasm from white loquat seedling Download PDFInfo
- Publication number
- CN110749699A CN110749699A CN201910956514.7A CN201910956514A CN110749699A CN 110749699 A CN110749699 A CN 110749699A CN 201910956514 A CN201910956514 A CN 201910956514A CN 110749699 A CN110749699 A CN 110749699A
- Authority
- CN
- China
- Prior art keywords
- triploid
- loquat
- leaves
- leaf
- sample
- 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.)
- Pending
Links
- 235000009008 Eriobotrya japonica Nutrition 0.000 title claims abstract description 71
- 241001092070 Eriobotrya Species 0.000 title claims abstract description 69
- 208000026487 Triploidy Diseases 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000012216 screening Methods 0.000 title claims abstract description 25
- 239000004576 sand Substances 0.000 claims abstract description 24
- 230000000877 morphologic effect Effects 0.000 claims abstract description 11
- 239000000523 sample Substances 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 28
- 238000000227 grinding Methods 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 14
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 claims description 12
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 10
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 102000006382 Ribonucleases Human genes 0.000 claims description 7
- 108010083644 Ribonucleases Proteins 0.000 claims description 7
- 210000003462 vein Anatomy 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 239000013074 reference sample Substances 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 5
- 239000013504 Triton X-100 Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 229910052564 epsomite Inorganic materials 0.000 claims description 5
- 238000000684 flow cytometry Methods 0.000 claims description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 4
- 230000002745 absorbent Effects 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 229920001778 nylon Polymers 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 210000000349 chromosome Anatomy 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 4
- 244000061508 Eriobotrya japonica Species 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 13
- 238000012360 testing method Methods 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 238000003801 milling Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 241000201976 Polycarpon Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 235000021012 strawberries Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/0098—Plants or trees
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for screening triploid new germplasm from white loquat seedlings. The method comprises (1) primarily screening triploid from seedling group according to morphological characteristics three times of white sand loquat by morphological observation, wherein suspected white sand loquat triploid satisfies the following conditions; (2) and further identifying the ploidy of the suspected eriobotrya japonica triploid white by a flow cytometer. The method has the advantages of simple solution preparation, easy operation steps, high screening success rate and reliable result, and has obvious advantages compared with other methods applied to loquat flow type ploidy screening at present. Compared with the traditional chromosome counting technology, the screening efficiency can be improved by 4-5 times by applying the method. The method can realize large-scale rapid identification of the triploid seedlings of the white sand loquat.
Description
Technical Field
The invention belongs to the field of biological detection, and relates to a method for screening triploid new germplasm from white loquat seedlings.
Background
The white sand loquat is popular with consumers due to the characteristics of fine and smooth meat quality, high sweetness and the like, and Jiangsu Suzhou is one of the main production areas of the white sand loquat. Most of the white sand loquats have many seeds and are large, so that the eating experience of the white sand loquats is influenced. The problem can be effectively solved by cultivating the triploid white loquat, and the method has important significance for promoting the development of the loquat industry. The seedling screening is an important way for breeding new loquat varieties, and in the current main loquat cultivars: white jade (chiffon flood, 2001), Jiefang (Libi lotus, 1999), Dawuxing (Chenguihu et al, 2002) and the like are all obtained by seedling breeding. In several approaches of loquat triploid breeding, seedling screening is an important approach, in recent years, the Beam national Lu professor (2011) of the university in southwest obtains a natural triploid mutant single plant through a 'one-step method', and then 2 plants are preferably selected from 300 plants to obtain a 'Changbai No. 1' Q11, '77-1' K375, a new excellent triploid seedless loquat line. According to the loquat triploid identification, in the past, chromosome counting is mainly carried out under a microscope through chromosome tabletting of root tips, and the method is not only complicated in process, but also time-consuming and labor-consuming and low in screening efficiency. At present, a method capable of screening triploid new germplasm from white sand loquat seedlings in a large scale is urgently needed.
Disclosure of Invention
The invention aims to provide a method for screening triploid new germplasm from white loquat seedlings, aiming at the defects in the prior art.
The purpose of the invention can be realized by the following technical scheme:
a method for screening triploid new germplasm from white loquat seedlings comprises the following steps:
(1) through morphological observation, triploid is primarily screened from seedling groups according to morphological characteristics three times of white sand loquats, and suspected white sand loquats meet the following conditions: the leaf area of the same-node fully-unfolded leaf of the triploid loquat is more than 1.3 times larger than that of the diploid, the triploid leaf shape index is 2.7 +/-0.25, and the diploid leaf shape index is 3.5 +/-0.3; the included angle between the triploid leaf and the branch is larger than that of the diploid, and the included angle between the triploid leaf and the branch is 70 degrees +/-10 degrees; the included angle between the diploid leaves and the branches is 44 degrees +/-10 degrees; the triploid leaves are darker in color and sunken in veins; the leaf shape index is leaf length/leaf width;
(2) and further identifying the ploidy of the suspected white sand loquat triploid through a flow cytometer.
The step (2) of further identifying the ploidy of the suspected white loquat leaf triploid through a flow cytometer preferably comprises the following steps:
(a) taking clean tender white loquat leaves, shredding the leaves, adding the leaves into a centrifugal tube, and adding clean zirconia beads and 1.5-2 mL of extracting solution A into the centrifugal tube for sample grinding;
(b) after sample grinding is finished, filtering by using a 400-mesh nylon sieve;
(c) centrifuging the filtrate, discarding supernatant, adding extract B into the precipitate, shaking to remove the precipitate, dyeing at low temperature in dark for 5min, and detecting on machine;
(d) detecting the fluorescence intensity of the PI stained sample by a flow cytometer, acquiring data by using random software CellQuestProTM, and counting the CV value of the data by adopting cytExpert of the flow cytometer;
(e) the ploidy calculation method comprises the following steps: the ploidy level of the sample to be detected is multiplied by the ploidy level of the reference sample multiplied by the fluorescence mean value of the peaks G0/G1 of the sample to be detected, namely the fluorescence mean value of the peaks G0/G1 of the reference sample;
wherein the extracting solution A is obtained by adding 1-2% (w/v) polyvinylpyrrolidone PVP into a nuclear separation buffer solution; the extraction solution B is prepared by adding PI powder into a nuclear separation buffer solution to prepare 0.3-0.5 mg/mL-1Adding 0.03-0.05 mg/mL of PI dye solution-1The RNase of (1); the formula of the nuclear separation buffer solution is 8-12 mmol.L-1MgSO4·7H2O，45～50mmol·L-1KCl，5～8mmol·L-10.25-0.3% (v/v) Triton X-100.
The formula of the nuclear separation buffer solution is preferably 10 mmol.L-1MgSO4·7H2O，50mmol·L-1KCl，5mmol·L-10.25% (v/v) Triton X-100.
The extract A is further preferably obtained by adding 1% (w/v) polyvinylpyrrolidone PVP into the core separation buffer; preferably, the extract B is prepared by adding PI powder to the nuclear separation buffer solution to prepare 0.5 mg/mL-10.05 mg/mL of the PI dye solution was added-1The RNase of (1).
Preferably, in the step (1), young and tender loquat leaves are cleaned by using sterilized double distilled water, water is absorbed by absorbent paper, the leaves are shredded and are placed into a centrifugal tube, leaf veins and other positions are avoided, the volume of the leaves approximately accounts for 1/3 of the centrifugal tube, 6-8 clean zirconia beads are added, extracting solution A to 2mL of scale marks are added, a sample grinding machine is adjusted to 50Hz, the sample grinding time is adjusted to 2min, and sample grinding is started.
The centrifugation in the step (3) is preferably performed by a low-temperature centrifuge at 4 ℃ and 3000rpm for 5 min.
The amount of the extract B to be added in the step (3) is preferably 0.5 mL/tube.
Has the advantages that:
the inventor discovers that the diploid and the triploid of the white sand loquat have great difference in morphological parameters such as the included angle of leaves, the size, the shape of leaves, the length of stem internodes and the like through observation, identification and summarization, so that the suspected white sand loquat triploid is screened through morphological observation, the screening range is greatly reduced, the ploidy of the actual loquat is quickly identified through a flow cytometer, particularly, a nuclear separation buffer solution is optimized, the sampling speed of cells is high, 10000 cells can be collected in 2min in each tube of sample, the requirement of quick screening of the actual loquat is met, the average CV value of a target cell mass is 6.46 percent and is less than 8 percent, and the experimental result is basically reliable. The flow-type screening method applied in the invention has the advantages of simple solution preparation, easy operation steps, low requirements on environmental conditions and methods in the operation process, high screening success rate and reliable results, and has obvious advantages compared with other methods applied to loquat flow-type ploidy screening at present. Compared with the traditional chromosome counting technology, the screening efficiency can be improved by 4-5 times by applying the method. The method can realize large-scale rapid identification of the triploid seedlings of the white sand loquat.
Drawings
FIG. 1 flow fluorescence intensity map of different methods
FIG. 2.48 ploidy identification of plant
A, a fluorescent intensity peak of a diploid loquat control group; b: fluorescence intensity peak of loquat No. 48; c: a fluorescence intensity peak of the actual diploid loquats;
a, chromosome counting of a diploid loquat control group; b: chromosome count of loquat 48
FIG. 3 comparison of stomatal density of diploid and triploid loquats
Note: i: diploid loquat; II: triploid loquats; bar is 50 μm.
Detailed Description
Example 1 screening of cell Nuclear isolates
1.1 test materials and instruments:
in 2018, seedling-grown seeding seedlings of one-year-old and two-year-old Suzhou mountain east mountain white sand loquats in a resource garden of white sand loquats in Jiangsu province (technical popularization center for evergreen fruit trees in Taihu lake in Jiangsu province) are used as test materials in 10 months.
The flow cytometer adopts CytoFLEX of BECKMAN COULTER company to select PE channel for sample loading detection.
1.2 flow cytometry identification:
the method I comprises the following steps:
preparing an extracting solution:
MgSO4 buffer configuration: the buffer solution was prepared to 10 mmol. L-1MgSO4·7H2O，50mmol·L-1KCl，5mmol·L-1The HEPES solution of (1), 0.25% (v/v) Triton X-100, wherein the HEPES solution is prepared in the order of 1 mol. L- 1HEPES solution: 23.8g HEPES was dissolved in 90mL water, the pH was adjusted to 8.0 with NaOH, and 100mL of water was added thereto.
Extracting solution A: MgSO (MgSO)4Adding 1% (w/v) polyvinylpyrrolidone PVP into the buffer solution, and placing the prepared solution into a refrigerator at 4 ℃ for later use.
And (3) extracting solution B: 1g of PI powder was added to MgSO4 buffer solution to prepare 0.5 mg/mL-1The PI dye solution (2) was added in the dark, and then 0.05 mg/mL was added-1The RNase (RNase) of (1) is most preferably used in the present invention. If the PI is inconvenient to prepare, the PI can be placed in a refrigerator at 4 ℃ to be kept away from light for about one month, and RNase is temporarily added when the PI is used.
The test steps are as follows:
the young and tender loquat leaves are cleaned by sterilized double distilled water, water is absorbed by absorbent paper, the leaves are simply shredded and put into a centrifugal tube, the leaves and other parts are avoided as far as possible, the volume of the leaves approximately accounts for 1/3 of the centrifugal tube, too much leaves are not beneficial to sample grinding, and too little leaves are not beneficial to extracting sufficient complete cells. Adding 6-8 clean zirconia beads, adding the extracting solution A to 2mL of scale marks (not less than 1.5mL), and placing the centrifuge tube on ice if the sample cannot be ground immediately after the adding is finished. And adjusting the sample grinding machine to 50Hz, adjusting the sample grinding time to 2min, and starting to grind the sample. After the completion of the sample grinding, the mixture was filtered through a 400-mesh nylon sieve, and 1mL of the filtered filtrate was taken up and centrifuged in a low-temperature centrifuge (centrifuge parameters: 3000rpm, 5min, 4 ℃). And (4) discarding the supernatant after centrifugation, adding 0.5mL of the extracting solution B, shaking uniformly to eliminate the precipitate, and dyeing at low temperature in a dark place for 5min to obtain the product which can be tested on a machine.
TABLE 1.3 comparison of the effects of the cell nucleus isolates
1.3 statistics of CV values
Each test method data was statistically analyzed for 10 CV values.
1.4 ploidy calculation method: and the ploidy level of the sample to be detected is multiplied by the ploidy level of the reference sample multiplied by the fluorescence mean value of the peaks G0/G1 of the sample to be detected, and the fluorescence mean value of the peaks G0/G1 of the reference sample.
1.5 results
The application effects of three different cell nucleus separation buffers and three test methods on loquat ploidy identification are shown in table 1 and figure 1. The method I is suitable for flow ploidy identification of the white sand loquat material collected in the test, the average CV value of the target cell mass is 6.46 percent and is less than 8 percent, the experimental result is basically reliable, in addition, the cell loading speed is high under the treatment of the test method, 10000 cells can be collected in 2min in each tube of sample, and the method is suitable for the requirement of fast screening of the actual loquat. The method II refers to BECKMAN COULTER flow cytometer technical manual, is an improved method of OTTO buffer solution, the experiment needs to be repeatedly added with liquid by centrifugation for 3 times, the procedure is complicated, the obtained vertical coordinate value of the target peak is high, the success rate is unstable, the CV value is 11.00 percent, and the accuracy and reliability of the result cannot be ensured. In the method III, reference is made to an application method of Chen-Yi (2013) in strawberries, an ideal target cell mass is difficult to obtain, and a large amount of cell fragments are obtained from a scatter diagram obtained by a flow cytometer, so that the method cannot calculate a CV value and is not suitable for rapid identification of loquat ploidy. In combination with the analysis, the method I is adopted in the test as a flow cytometry method for rapidly identifying the ploidy of the seedling loquat, the solution preparation steps are simple, the CV value control is low, clear target cell clusters can be obtained, and the rapid screening requirement of the test on the ploidy of the seedling loquat can be met.
The ploidy of 924 seedlings of white loquat fruits was identified by flow cytometry, in which 6 seedlings numbered 48, 548, 563, 592, 530 and 893, respectively, were found, the cell mass and the fluorescence intensity peak were located at 120 on the abscissa (shown in fig. 2. B) in the flow cytometry identification, while the peak of diploid (2 n-2 x-34) was determined to be 80 (shown in fig. 2.A, a) in the control group that had been previously identified. Therefore, the plants were preliminarily determined to be triploid, and 51 chromosomes (shown in fig. 2. b) were clearly visible from the chromosome counting result of the chromosome pressing of the root tip chromosomes of the plants, thereby confirming that the plants No. 48, 548, 563, 592, 530 and 893 are new seedling triploid (2n ═ 3x ═ 51) white sand loquat lines. Therefore, the reliability of the flow identification method in loquat ploidy identification is further proved. FIG. 2.C is a graph showing the peak of fluorescence intensity of ordinary fructical diploid Eriobotrya japonica identified in the experiment.
Observation shows that the triploid loquat plant is different from the diploid in morphological appearance, the leaf area of the completely unfolded leaf at the same node of the triploid loquat is more than 1.3 times larger than that of the diploid, and the leaf shape index (leaf length/leaf width) is smaller than that of the diploid (the triploid is 2.70 +/-0.25, and the diploid is 3.5 +/-0.3); the included angle between the leaves and the branches is larger than that of a diploid (70 degrees +/-10 degrees; 44 degrees +/-7 degrees respectively), partial leaves are even bent and slightly droop, the leaf color is darker, and the veins are sunken. The statistical results of the leaf shapes and the included angles of the stems and the leaves of the diploid and the triploid plants are shown in a table 2, the statistical results of the stomatal lengths and the densities of the diploid and the triploid plants are shown in a table 3, and the comparison results of the stomatal densities of the diploid and the triploid loquats (No. 48 plants) are shown in a table 3.
TABLE 2 statistics of leaf shape and included angle between stems and leaves of diploid and triploid plants
Note that P < 0.05 indicates significant difference; p < 0.01 indicates that the difference is very significant.
TABLE 3 statistics of stomatal length and density of diploid and triploid plants
Note that P < 0.05 indicates significant difference; p < 0.01 indicates that the difference is very significant.
According to the results, the morphological parameter criteria for distinguishing the diploid and the triploid of the white sand loquat are determined as follows: the leaf shape index of the triploid is smaller than that of the diploid, the mean value is 2.7 +/-0.25, the included angle between the leaf and the stem is larger than that of the diploid, and the mean value is 70 +/-10 degrees; the density of air holes is far less than that of a diploid, and the density of the air holes is 16.0 air holes/mm2The veins are depressed and the color of the leaves is darker.
Example 2
According to the method, the 924 white sand loquat seedlings are subjected to triploid screening. 17 suspected triploid white eriobotrya japonica were preliminarily screened according to the morphological parameter criteria described in example 1.
The suspected triploid white sand loquat young leaves are cleaned by using sterilized double distilled water, water is sucked by using absorbent paper, the leaves are simply torn and placed into a centrifugal tube, the veins and other parts are avoided as much as possible, the volume of the leaves is about 1/3 of the centrifugal tube, too much leaves are not beneficial to sample grinding, and too little leaves are not beneficial to extracting sufficient intact cells. Adding 6-8 clean zirconia beads, adding the extracting solution A to two milliliter scale marks, adding the extracting solution with the amount of not less than 1.5mL, and placing the centrifuge tube on ice if the sample cannot be immediately ground after the adding is finished. The sample mill was adjusted to 50Hz and the sample milling time was adjusted to 2 minutes to begin the sample milling. After the completion of the sample grinding, the mixture was filtered through a 400-mesh nylon sieve, and 1ml of the filtered filtrate was taken up and centrifuged in a low-temperature centrifuge (centrifuge parameter setting: 3000rpm, 5min, 4 ℃). And (4) discarding the supernatant after centrifugation, adding 0.5mL of the extracting solution B, shaking uniformly to eliminate the precipitate, and dyeing at low temperature in a dark place for 5min to obtain the product which can be tested on a machine. The preparation method of the extract solutions A and B was the same as in example 1.
The flow cytometer adopts CytoFLEX of BECKMAN COULTER company, selects PE channel to sample and detect, counts CV value, and calculates ploidy according to the formula described in the embodiment. The result shows that 6 plants are determined to be triploid, the root tip chromosome tabletting verification is carried out on the plants determined to be triploid, and the result shows that the accuracy rate of the triploid plants determined by the flow-type result is 100%.
Claims (7)
1. A method for screening triploid new germplasm from white loquat seedlings is characterized by comprising the following steps:
(1) through morphological observation, triploid is primarily screened from seedling groups according to morphological characteristics three times of white sand loquats, and suspected white sand loquats meet the following conditions: the leaf area of the same-node fully-unfolded leaf of the triploid loquat is more than 1.3 times larger than that of the diploid, the triploid leaf shape index is 2.7 +/-0.25, and the diploid leaf shape index is 3.5 +/-0.3; the included angle between the triploid leaf and the branch is larger than that of the diploid, and the included angle between the triploid leaf and the branch is 70 degrees +/-10 degrees; the included angle between the diploid leaves and the branches is 44 degrees +/-10 degrees; the triploid leaves are darker in color and sunken in veins; the leaf shape index is leaf length/leaf width;
(2) and further identifying the ploidy of the suspected white sand loquat triploid through a flow cytometer.
2. The method of claim 1, wherein said step (2) of further identifying the ploidy of the suspected white loquat leaf triploid by flow cytometry comprises the steps of:
(a) taking clean tender white loquat leaves, shredding the leaves, adding the leaves into a centrifugal tube, and adding clean zirconia beads and 1.5-2 mL of extracting solution A into the centrifugal tube for sample grinding;
(b) after sample grinding is finished, filtering by using a 400-mesh nylon sieve;
(c) centrifuging the filtrate, discarding supernatant, adding extract B into the precipitate, shaking to remove the precipitate, dyeing at low temperature in dark for 5min, and detecting on machine;
(d) detecting the fluorescence intensity of the PI stained sample by a flow cytometer, acquiring data by using random software CellQuestProTM, and counting the CV value of the data by adopting cytExpert of the flow cytometer;
(e) the ploidy calculation method comprises the following steps: the ploidy level of the sample to be detected is multiplied by the ploidy level of the reference sample multiplied by the fluorescence mean value of the peaks G0/G1 of the sample to be detected, namely the fluorescence mean value of the peaks G0/G1 of the reference sample;
wherein the extracting solution A is obtained by adding 1-2% (w/v) polyvinylpyrrolidone PVP into a nuclear separation buffer solution; the extraction solution B is prepared by adding PI powder into a nuclear separation buffer solution to prepare 0.3-0.5 mg/mL-1Adding 0.03-0.05 mg/mL of PI dye solution-1The RNase of (1); the formula of the nuclear separation buffer solution is 8-12 mmol.L-1MgSO4·7H2O，45～50mmol·L-1KCl，5～8mmol·L-10.25-0.3% (v/v) Triton X-100.
3. The method of claim 2, wherein the nuclear separation buffer formulation is 10 mmol-L- 1MgSO4·7H2O，50mmol·L-1KCl，5mmol·L-10.25% (v/v) Triton X-100.
4. The method according to claim 3, wherein extract A is obtained by adding 1% (w/v) polyvinylpyrrolidone PVP to the said nuclear separation buffer; the extract B was prepared by adding PI powder to the above nuclear separation buffer solution to give a solution of 0.5 mg/mL-10.05 mg/mL of the PI dye solution was added-1The RNase of (1).
5. The method according to any one of claims 1 to 4, wherein in the step (a), young and tender loquat leaves are taken and washed clean by sterilized double distilled water, water is absorbed by absorbent paper, the leaves are shredded and placed into a centrifuge tube, the positions of veins and the like are avoided, the volume of the leaves is about 1/3 of the centrifuge tube, 6-8 clean zirconia beads are added, the extracting solution A is added to a two-milliliter scale line, the amount of the extracting solution is not less than 1.5mL, a sample grinder is adjusted to 50Hz, the sample grinding time is adjusted to 2 minutes, and sample grinding is started.
6. The method according to any one of claims 1 to 4, wherein the centrifugation in step (c) is performed by a low temperature centrifuge at 4 ℃ and 3000rpm for 5 min.
7. The method according to any one of claims 1 to 4, wherein the amount of the extract B added in step (c) is 0.5 mL/tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910956514.7A CN110749699A (en) | 2019-10-10 | 2019-10-10 | Method for screening triploid new germplasm from white loquat seedling |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910956514.7A CN110749699A (en) | 2019-10-10 | 2019-10-10 | Method for screening triploid new germplasm from white loquat seedling |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110749699A true CN110749699A (en) | 2020-02-04 |
Family
ID=69277893
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910956514.7A Pending CN110749699A (en) | 2019-10-10 | 2019-10-10 | Method for screening triploid new germplasm from white loquat seedling |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110749699A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111562209A (en) * | 2020-05-12 | 2020-08-21 | 广西大学 | Method for rapidly identifying tetraploid eggplant rootstock |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316373A (en) * | 2014-10-22 | 2015-01-28 | 江苏省农业科学院 | Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry |
| CN104878089A (en) * | 2015-04-29 | 2015-09-02 | 广西现代农业科技示范园 | Method for rapidly identifying ploidy of wax gourds by using flow cytometry |
| CN105259097A (en) * | 2015-10-28 | 2016-01-20 | 中国科学院植物研究所 | Method for authenticating ploidy of blueberries through flow cytometry |
| CN105606519A (en) * | 2016-01-08 | 2016-05-25 | 南京林业大学 | Method for rapid identification of ploidy of salicaceae plant |
| CN106226137A (en) * | 2016-08-10 | 2016-12-14 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN106248561A (en) * | 2016-07-21 | 2016-12-21 | 西南大学 | Be suitable to buffer and preparation, the using method utilizing flow cytometer that various plants carries out Ploidy Identification |
| CN107014740A (en) * | 2016-01-28 | 2017-08-04 | 辽宁省农业科学院玉米研究所 | The early stage identification technology of tetraploid silage corn ploidy |
| CN108982192A (en) * | 2017-05-31 | 2018-12-11 | 河北农业大学 | Rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination |
| CN109187315A (en) * | 2018-08-18 | 2019-01-11 | 杭州市农业科学研究院 | Doubling of strawberry chromosome method and ploidy rapid identification method used |
| CN109596502A (en) * | 2018-11-30 | 2019-04-09 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of method and its application of Rapid identification sweet potato Genome Size |
| CN109937876A (en) * | 2019-03-22 | 2019-06-28 | 南京农业大学 | A method of improving Chinese cabbage microspore embryo's inductivity |
| CN110055298A (en) * | 2018-12-19 | 2019-07-26 | 山东省果树研究所 | A kind of detection method of crop field Cherry dwarf rootstock ploidy |
-
2019
- 2019-10-10 CN CN201910956514.7A patent/CN110749699A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316373A (en) * | 2014-10-22 | 2015-01-28 | 江苏省农业科学院 | Extraction method for cell nucleuses of eggplant leaves suitable for flow cytometry |
| CN104878089A (en) * | 2015-04-29 | 2015-09-02 | 广西现代农业科技示范园 | Method for rapidly identifying ploidy of wax gourds by using flow cytometry |
| CN105259097A (en) * | 2015-10-28 | 2016-01-20 | 中国科学院植物研究所 | Method for authenticating ploidy of blueberries through flow cytometry |
| CN105606519A (en) * | 2016-01-08 | 2016-05-25 | 南京林业大学 | Method for rapid identification of ploidy of salicaceae plant |
| CN107014740A (en) * | 2016-01-28 | 2017-08-04 | 辽宁省农业科学院玉米研究所 | The early stage identification technology of tetraploid silage corn ploidy |
| CN106248561A (en) * | 2016-07-21 | 2016-12-21 | 西南大学 | Be suitable to buffer and preparation, the using method utilizing flow cytometer that various plants carries out Ploidy Identification |
| CN106226137A (en) * | 2016-08-10 | 2016-12-14 | 浙江大学 | A kind of method of quick detection Brassica genus hexaploid new germ plasm ploidy |
| CN108982192A (en) * | 2017-05-31 | 2018-12-11 | 河北农业大学 | Rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination |
| CN109187315A (en) * | 2018-08-18 | 2019-01-11 | 杭州市农业科学研究院 | Doubling of strawberry chromosome method and ploidy rapid identification method used |
| CN109596502A (en) * | 2018-11-30 | 2019-04-09 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of method and its application of Rapid identification sweet potato Genome Size |
| CN110055298A (en) * | 2018-12-19 | 2019-07-26 | 山东省果树研究所 | A kind of detection method of crop field Cherry dwarf rootstock ploidy |
| CN109937876A (en) * | 2019-03-22 | 2019-06-28 | 南京农业大学 | A method of improving Chinese cabbage microspore embryo's inductivity |
Non-Patent Citations (5)
| Title |
|---|
| 周先容等: "《分子生物技术与植物保护生物学》", 30 April 2016, 光明日报出版社 * |
| 张志珂等: "借助细胞流式仪进行枇杷基因组学测序材料的倍性鉴定", 《果树学报》 * |
| 浙江农业大学等: "《果树育种学》", 31 March 1980, 上海科学技术出版社 * |
| 陈方永等: "浙江２个地产白沙枇杷种质资源亲缘关系鉴定", 《浙江大学学报（农业与生命科学版）》 * |
| 黎月娟等: "90 份广西桑树种质资源的染色体倍性鉴定", 《中国蚕业》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111562209A (en) * | 2020-05-12 | 2020-08-21 | 广西大学 | Method for rapidly identifying tetraploid eggplant rootstock |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Omidbaigi et al. | Induction of autotetraploidy in dragonhead (Dracocephalum moldavica L.) by colchicine treatment | |
| Cui et al. | In vitro induction of tetraploid Ziziphus jujuba Mill. var. spinosa plants from leaf explants | |
| Broertjes et al. | Mutation breeding of Dahlia variabilis | |
| He et al. | Induction and identification of hexadecaploid of Pinellia ternate | |
| CN110749699A (en) | Method for screening triploid new germplasm from white loquat seedling | |
| Ho et al. | The use of stomatal chloroplast number for rapid determination of ploidy level in maize | |
| CN101015276A (en) | Method for inducing primula forbesii tetraploid and ploidy early stage authentication technique | |
| Dell et al. | Anatomy of Pilostyles hamiltonii CA Gardner (Rafflesiaceae) in stems of Daviesia | |
| Beatson et al. | Flow cytometric identification of sexually derived polyploids in hop (Humulus lupulus L.) and their use in hop breeding | |
| Li et al. | An improved protocol for flow cytometry analysis of dragon fruit (Hylocereus spp.)—Species with a high polysaccharide content | |
| CN108513908B (en) | Method for cultivating tetraploid loose-leaved Chinese cabbage | |
| Olanj et al. | Biosystematics of Tanacetum tenuissimum, a new record from NW of Iran | |
| Kreuger et al. | Genetically stable cell lines of cucumber for the large‐scale production of diploid somatic embryos | |
| CN102498847B (en) | Screening method of advantage-based stock seed of isatis root | |
| CN109041875A (en) | The yield of rape based on economical character and the prediction technique of lodging resistance | |
| Rao et al. | Haploid plants from in vitro anther culture of the leguminous tree, Peltophorum pterocarpum (DC) K. Hayne (Copper pod) | |
| Perera et al. | Regeneration from sweetpotato protoplasts and assessment of growth conditions for flow-sorting of fusion mixtures | |
| Joseph et al. | Investigation of aneuploidy in germplasm of African Yam Bean (Sphenostylis Stenocarpa Hochst. Ex A. Rich) harms using pollen and mitotic data | |
| Nordal et al. | Taxonomic studies of the genus Hypoxis in East Africa | |
| CN112913682A (en) | Screening method for configuration combination of Cenxi soft-branch camellia oleifera cultivars | |
| Maraschin et al. | Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells | |
| CN115326686B (en) | Quick identification method for sex of holly based on genome size difference | |
| CN109997548A (en) | A kind of method of quick screening Queensland nut plantation configuration kind | |
| Fávero et al. | Morphological, cytogenetic, and molecular characterization of Arachis kuhlmannii Krapov. & WC Greg.(Leguminosae) | |
| CN109566545B (en) | Screening method of gallnut aphid winter host moss |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200204 |