CN107505303B - A kind of seedling stage Rapid identification hops plant male and female method for distinguishing - Google Patents
A kind of seedling stage Rapid identification hops plant male and female method for distinguishing Download PDFInfo
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- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 1
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- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The present invention relates to cytogenetics fields, more particularly to a kind of seedling stage Rapid identification hops plant male and female method for distinguishing, by preparing chromosome slide sample, CMA/DAPI dyeing, fluorescence detection, image analysis, emphasis solves existing identification hops male and female plant identification evening in period, complicated cumbersome molecular biology method, provides a kind of easy, quick, accurate method.Method provided by the invention can preferably show the structure of hops chromosome, especially the caryosome tissue area morphosis of female plant and staminiferous plant compared with single fluorescent staining is sent out.
Description
Technical field
The present invention relates to cytogenetics fields.Specifically, the present invention relates to a kind of plants of seedling stage Rapid identification hops
Strain male and female method for distinguishing.
Background technique
Hops, scientific name Humulus lupulus L., alias hops, top must be spent, and be Urticales Cannabaceae humulus grass
Belong to, perennial tendril herbaceous plant, stem, branch and the dense villus of petiole and delavay raspberry herb.Hops dioecism, female flower spike are male
Panicle is spent, is female flower used in brewing.Hops can grow in many areas in the world, and most of planting area concentrates on
Between 35 °~55 ° of north and south latitude.Currently, its main place of production is distributed in the U.S., the ground such as Europe, Australia, South America and China
Area.Hops is added in brewing can assign the fragrance of the salubrious bitter taste of beer and fragrance, and have anti-corrosion and clarification wheat
The function of bud juice.Therefore hops is known as " soul of beer ", and since 12nd century, its most important purposes is still so far
Brewing for beer.
For hops as a kind of important medicinal plant, its applicating history is also very long.13rd century, hops
Begin to come as herbal medicine using.In one of Song Dynasty " Kaibao Bencao " that the Compendium of Material Medica of China's Li Shizhen of the Ming Dynasty is drawn
What medicine " top must be colored " referred to is exactly hops.Recent studies indicate that hops not only have calm, tranquilizing the mind, stomach invigorating, sterilization,
The effects of anti-inflammatory and diuresis, also has the multiple efficacies such as anti-oxidant, antiviral and antitumor and phytoestrogen sample effect.
Common chromosome banding technique (chromosome banding technique) based on dyestuff has N band, C band, G
Band, R band etc., can show grown form the feature such as size, primary and secondary contriction of chromosome.These methods can reflect certain
Biological significance has played very big effect in biological study, such as plant evolution biology, the mankind and mammal dyeing
Body research.With the discovery of fluorescent dye, application of the fluorescence banding technology in karyotyping is increasingly extensive.Chromosome fluorescence is aobvious
Band is to carry out dyeing to chromosome using fluorescent dye to divide band, and principle is the specific binding using fluorescent dye and base-pair.
Common nucleotide fluorescent dye mainly has CMA (chromomycin A3), DAPI (4', 6- diamidino -2-phenylindone), PI (iodate third
Pyridine) etc..Wherein CMA is GC Idiotype fluorescent dye, and DAPI is AT Idiotype fluorescent dye.
Chromosome sectioning and double fluorescent staining specific methods are different because of species.Different plants needs different p-dichlorobenzenes
Processing time, temperature and different tip of a root enzymolysis times etc..These factors can all influence final chromosome morphology structure
And dyeing effect.Such as: different p-dichlorobenzene processing time and temperature can make the concentrating degree of chromosome different.If the tip of a root
Enzymolysis time too long, does not then observe chromosome probably under fluorescence microscope.Enzymolysis time is inadequate, then cell wall is still deposited
, Chromosome spread is not opened, be unfavorable for chromosome structure observation.The dye selection and dyeing time of the double fluorescent stainings of chromosome
Sequence, dyeing time are presented the details of final chromosome morphology structure all very crucial.Single fluorescent dye cannot usually be shown
Show the overall structure of chromosome.CMA just can be complementary with both fluorescent dyes of DAPI.But CMA is dyed in actual operation
There are certain difficulty, dyeing time is too short to be unfavorable for chromosome coloring, the too long details knot for being displayed without chromosome of dyeing time
Structure.And it is also critically important with the dyeing order of DAPI, because DAPI coloring is often very fast, it is easy the fluorescence of covering CMA.This patent
Provide the concrete scheme of the film-making of hops root tip chromosomes and double fluorescent stainings.
In production plantation, people only cultivate the big female hops of economic benefit.Simply, quickly CMA/DAPI is dyed
The application of body Double fluorescence staining method can instruct its production in its gender of hops seedling stage assay, produce chain to hops economy
Development be of great importance.
Summary of the invention
The object of the present invention is to provide a kind of seedling stage Rapid identification hops plant male and female method for distinguishing, and method is simple,
Dyeing effect is good, can fast and accurately judge male female do of hops.
To achieve the above object, the invention discloses following technology contents:
A kind of seedling stage Rapid identification hops plant male and female method for distinguishing, includes the following steps:
Chromosome specimen production:
(1) the hops plant tip of a root is grown to 2-3cm, and top is mellow and full full；
(2) eugonic tip of a root 2.5-3mm is cut, handles 20-24h at 4 DEG C of paracide solution of saturation；
(3) tip of a root is carried out in a conventional manner hypotonic and fixed；
Preferably, the step in step (3) specifically: sop up saturation paracide, add 0.075mol/L KCl solution, room
Hypotonic 30-40min before temperature is lower, the Kano fixer prepared later plus newly (acetic acid: ethyl alcohol=1:3, volume ratio) are fixed at room temperature
24-48h；
(4) fixer is thoroughly cleaned in a conventional manner；
Preferably, the step in step (4) specifically: by the distillation washing 3 times of the tip of a root after fixation, every time on shaking table
5min (shaking speed 15-20 turns/min) is shaken, fixer is sufficiently washed away；
(5) with 1% (mass ratio) pectase Y-23 (Kikkoman Corporation) and 4% (mass ratio) cellulase
Onozuka RS (Yakult Co.LTD) mixed liquor, each tip of a root 15-20 μ l enzyme solution, 37 DEG C of enzymatic hydrolysis 90min；
(6) draw a tip of a root to cleaning on the glass slide that dries, under stereoscope with dissecting needle remove Meristernatic zone with
Outer position and impurity gently breaks the tip of a root into pieces to pulpous state；
(7) 60% glacial acetic acid of 10-15 μ l is added dropwise on the tip of a root, the Kano fixer of 40-50 μ l is added dropwise after drop is transparent
Piece is opened up, slide is placed in 80 DEG C of thermal station dry 5-8min；
(8) microscopy under 20 times of phase contrast microscopes is picked out the more chromosome piece of clean background, split coil method and is done in next step
Fluorescent staining；
The double fluorescent stainings of chromosome:
(1) CMA is added drop-wise to chromosome on piece, 80-100 μ l/ piece, epiphragma is protected from light and is incubated for 2h；
(2) 3-5min is cleaned in phosphate buffer (PBS)；
(3) DAPI is added drop-wise to chromosome on piece, 80-100 μ l/ piece, epiphragma is protected from light and is incubated for 5min；
(4) anti-quencher Citifluor AF1 ((ProSciTech Pty is put into after rapid cleanup in phosphate buffer
Ltd, Australia)) in, it is kept in dark place；
(5) it is observed under fluorescence microscope (Olympus AH3), B excites the chromosome of observation CMA dyeing under filter block, in Huang
Color fluorescence, U excite the chromosome of observation DAPI dyeing under filter block, fluorescence blue.
Schemes described above, preferred determination method are as follows: the karyotype formulas of diploid males hops is: 14m+4sm+
Xm+Ym；There are two the sites NORs on No. six chromosomes；The karyotype formulas of diploid female hops is: 14m+4sm+2Xm.
Schemes described above, preferred determination method are as follows: whether NORs there are two female or male beer Huadus,
But dyed by CMA, find two NORs of female hops, only one site is active, is in extended state, another site
Silencing is in enrichment stage.However in male hops, two NORs are active, are all in extended state.
Compared with the prior art, the invention has the benefit that
(1) present invention in prior art basis, with saturation 4 DEG C of paracide solution at handle 20-24h the tip of a root,
The hypotonic 30-40min of room temperature in KCl solution is digested after the tip of a root wash with distilled water after being fixed more than for 24 hours using Kano fixer,
Acetic acid on the rocks is transparent, and Kano fixer opens up piece, makes the cohesion of hops metaphase chromosome preferably, and root-tip cells dissociation is abundant, dyeing
Body is uniformly dispersed, is easy observation.
(2) present invention uses the bis- fluorescent staining methods of CMA/DAPI, and step is easy, pollution-free, and can be more clear
Hops metaphase chromosome image.
(3) present invention clearly demonstrates the chromosome of hops, and fluorescence signal is strong, it is determined that is suitble to the dyeing of hops
The method of body fluorescent staining.
(4) present invention is by the CONSTRUCTED SPECIFICATION of observation metaphase in cell division chromosome, such as chromosome arm length, centromere,
The difference in hops male and female plant such as satellite, discrimination hops male and female, analyze hops chromosome structure, draw in seedling stage
The Sustainable use of kind cultivation and resource has great theoretical and practical significance.
(5) colouring method provided by the invention is utilized, is found for the first time, whether there are two female or male beer Huadus
NORs, but dyed by CMA, find two NORs of female hops, only one site is active, is in extended state, separately
One site silencing is in enrichment stage, however in male hops, two NORs are active, are all in extended state, for from now on
The identification of male and female plant of hops provide new reference.
Detailed description of the invention
Fig. 1 is hops tip of a root body cell metaphase chromosome DAPI/CMA Two Colour Fluorescence dye image schematic diagram.
Wherein A, B are male hops metaphase chromosome in Fig. 1, and C, D are female hops chromosome.There is tail arrow signified
For on chromosome be in extended state, the site active nucleolus organizer region (nucleolus organizer region, NOR),
Anury arrow meaning is on chromosome in enrichment stage, inactive NOR bit point.
Fig. 2 is male hops idiogram；Illustrate male hops chromosome relative length and NOR bit point.
Fig. 3 is female hops idiogram；Illustrate female hops chromosome relative length and NOR bit point.
Specific embodiment
The present invention is further described below combined with specific embodiments below.Technical solution of the present invention, such as not especially
Illustrate, belong to conventional scheme, the reagent or material derive from commercial channel if not otherwise specified.
Embodiment 1:
A kind of seedling stage Rapid identification hops plant male and female method for distinguishing: material therefor of the present invention:
1. chromosome specimen makes:
(1) routinely vegetable material culture, by the hops of known male and female, (present invention has chosen two female two male, Yi Gongsi
Strain hops) the cuttage plant tip of a root is grown to 2-3cm, and top is mellow and full full；
(2) eugonic tip of a root 3mm is cut, handles 20h at 4 DEG C of paracide solution of saturation；
(3) saturation paracide is sopped up, 0.075mol/L KCl solution is added, at room temperature preceding hypotonic 30-40min, Zhi Houjia
The Kano fixer (acetic acid: ethyl alcohol=1:3, volume ratio) newly prepared is fixed for 24 hours at room temperature；
(4) by after fixation the tip of a root with distillation washing 3 times, shaken on shaking table every time 5min (shaking speed 15-20 turn/
Min), fixer is sufficiently washed away；
(5) with 1% (mass ratio) pectase Y-23 (Kikkoman Corporation) and 4% (mass ratio) cellulase
Onozuka RS (Yakult Co.LTD) mixed liquor, each tip of a root 15-20 μ l enzyme solution, 37 DEG C of enzymatic hydrolysis 90min；
(6) draw a tip of a root to cleaning on the glass slide that dries, under stereoscope with dissecting needle remove Meristernatic zone with
Outer position and impurity gently breaks the tip of a root into pieces to pulpous state；
(7) 60% glacial acetic acid of 10-15 μ l is added dropwise on the tip of a root, the Kano fixer of 40-50 μ l is added dropwise after drop is transparent
Piece is opened up, slide is placed in 80 DEG C of thermal station dry 5-8min；
(8) microscopy under 20 times of phase contrast microscopes is picked out the more chromosome piece of clean background, split coil method and is done in next step
Fluorescent staining；
2. the double fluorescent stainings of chromosome:
(1) CMA (0.5mg/ml) (Chromomycin A3) is added drop-wise to chromosome on piece, 100 μ l/ pieces, epiphragma is protected from light
It is incubated for 2h；
(2) 5min is cleaned in phosphate buffer (PBS)；
(3) DAPI (2ug/ml) (4', 6- diamidino -2-phenylindone dihydrochloride) is added drop-wise to chromosome on piece, 100
μ l/ piece, epiphragma are protected from light and are incubated for 5min；
(4) it is put into Citifluor AF1antifadent after rapid cleanup in phosphate buffer (PBS)
(ProSciTech Pty Ltd, Australia), 4 DEG C are kept in dark place, overnight microscopy；
(5) chromosome fluorescence detection uses fluorescence microscope (Olympus AH3), is equipped with a U excitation block observation
DAPI, a B excitation block observe CMA, a CCD digital camera (Roper Scientific Photometrics Cool
Snap) and RSImage image software is used.In fluorescence microscopy microscopic observation, CMA dyeing is in yellow fluorescence, and DAPI dyeing is in blue
Color fluorescence.In the method, wherein CMA is containing 5mM MgCl2McIlvaine buffer in (PH=7.0), DAPI exists
In McIlvaine buffer (0.1M citric acid and 0.2M disodium hydrogen phosphate, PH=7.0)；
3. results and discussion
(1) karyotype of diploid male and female hops
Analysis counts the male and female hops chromosome data in conjunction with DAPI and CMA respectively, available diploid male and female
The karyotype formula of hops.The karyotype formulas of diploid males hops is: 14m+4sm+Xm+Ym.It is dyed at No. six
There are two the site NORs (Fig. 2) on body；The karyotype formulas of diploid female hops is: 14m+4sm+2Xm (Fig. 3).
(2) male and female hops nucleolus organizer region Site discrepancy
The sex chromosome of diploid female and male hops exists, in addition to diploid female hops have two X dyeing
Body, and male hops only has an X chromosome, there are also a Y chromosome (Karlov et al 2003；Divashuk et
Al 2011) outside, present invention discover that there is also differences for NOR bit point.Although whether there are two the male beer Huadus of female
NORs, but dyed by CMA, find two NORs of female hops, only one site is active, is in extended state (Fig. 1
Have tail arrow meaning position in middle c, d), another site silencing, in enrichment stage (anury arrow meaning position in c in Fig. 1, d).
However in male hops, two NORs are active, in extended state (having tail arrow meaning position in a in Fig. 1, b).
The male and female for identifying hops at present mainly pass through DNA molecular marker method and plant reproduction period Reproductive organ morphology is seen
It examines.Polley A. et al. utilized randomly amplified polymorphic DNA RAPD (random amplified in 1997
Polymorphism DNA) technology successfully identifies the male and female of hops.2006, Danilova and Karlov discovery passed through letter
Simple sequence, which repeats section ISSR (inter-simple sequence repeat), can also identify the gender of hops.The present invention
The double fluorescent staining methods of the hops plant root tip chromosomes of offer identify the gender of plant as a result, with these plants in early days in seedling stage
Strain grows into the male and female plant final result that flowering phase observation male and female reproductive organ morphosis is determined and is consistent.
Claims (4)
1. a kind of seedling stage Rapid identification hops plant male and female method for distinguishing, includes the following steps:
Chromosome specimen production:
(1) the hops plant tip of a root is grown to 2-3cm, and top is mellow and full full；
(2) eugonic tip of a root 2.5-3mm is cut, handles 20-24h at 4 DEG C of paracide solution of saturation；
(3) tip of a root is carried out in a conventional manner hypotonic and fixed；
(4) fixer is thoroughly cleaned in a conventional manner；
(5) with 1% pectase Y-23 and 4% cellulase Onozuka RS mixed liquor, each tip of a root 15-20 μ l enzyme solution, 37 DEG C
Digest 90min；
(6) tip of a root is drawn to cleaning on the glass slide dried, is removed other than Meristernatic zone under stereoscope with dissecting needle
Position and impurity gently break the tip of a root into pieces to pulpous state；
(7) 60% glacial acetic acid of 10-15 μ l is added dropwise on the tip of a root, the Kano fixer exhibition of 40-50 μ l is added dropwise after drop is transparent
Slide is placed in 80 DEG C of thermal station dry 5-8min by piece；
(8) microscopy under 20 times of phase contrast microscopes picks out the more chromosome piece of clean background, split coil method and does next step fluorescence
Dyeing；
The double fluorescent stainings of chromosome:
(1) CMA is added drop-wise to chromosome on piece, 80-100 μ l/ piece, epiphragma is protected from light and is incubated for 2h；
(2) 3-5min is cleaned in phosphate buffer (PBS)；
(3) DAPI is added drop-wise to chromosome on piece, 80-100 μ l/ piece, epiphragma is protected from light and is incubated for 5min；
(4) it is put into anti-quencher Citifluor AF1, is kept in dark place after rapid cleanup in phosphate buffer；
(5) under the microscope, it is in yellow fluorescence that B excites the chromosome of observation CMA dyeing under filter block to fluorescence microscopy, and U is excited under filter block
Observe the chromosome of DAPI dyeing, fluorescence blue.
2. according to the method described in claim 1, it is characterized by: the step in the step of chromosome specimen makes (3) is specific
Are as follows: saturation paracide is sopped up, 0.075mol/L KCl solution is added, preceding hypotonic 30-40min, is prepared later plus newly at room temperature
Kano fixer fixes -48h for 24 hours at room temperature.
3. according to the method described in claim 1, it is characterized by: the step in the step of chromosome specimen makes (4) is specific
Are as follows: by the distillation washing 3 times of the tip of a root after fixation, 5min is shaken on shaking table every time, sufficiently washes away fixer.
4. according to the method described in claim 1, it is characterized by: after dyeing fluorescence microscopy under the microscope, it is whether female
Or there are two NORs for male beer Huadu, but by the double fluorescent stainings of chromosome, find two NORs of female hops, only
There is a site active, is in extended state, another site silencing is in enrichment stage, however in male hops, two
NORs is active, is in extended state.
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| Karyotype and RAPD Analysis of Male and Female Coccinia grandis L. from Bangladesh;Md. Uzzal Hossain et al.;《The Japan Mendel Society》;20161231;第81卷(第3期);第349-355页 |
| Molecular cytogenetics in hop (Humulus lupulus L.) and identification of sex chromosomes by DAPI-banding;G.I. Karlov et al.;《Euphytica》;20031231;第132卷;第185-190页 |
| 沙田柚实生苗染色体的荧光显带分析;苗茵等;《华中农业大学学报》;20140131;第33卷(第1期);第18-23页 |
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