CN1119928C - Method for determining cytoplasmic male sterility breed of Chinese cabbage - Google Patents
Method for determining cytoplasmic male sterility breed of Chinese cabbage Download PDFInfo
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- CN1119928C CN1119928C CN 99114126 CN99114126A CN1119928C CN 1119928 C CN1119928 C CN 1119928C CN 99114126 CN99114126 CN 99114126 CN 99114126 A CN99114126 A CN 99114126A CN 1119928 C CN1119928 C CN 1119928C
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Abstract
The present invention belongs to a method for identifying novel cytoplasmic male sterility germplasm of a non-heading Chinese cabbage, which comprises: field identification, cell chromosome identification, esterase and peroxide isozyme electrophoresis analysis and molecular biology identification. The identification of floral organ morphology, male sterility and setting percentage, the cell chromosome identification, the isozyme analysis and the molecular biology identification for later generations of inosculated regeneration plants are carried out by the present invention, and results are accurate and reliable. Identified 98H autumnn-45 is the novel germplasm, the sterility rate and the sterility degree of the identified 98H autumnn-45 are 100%, leaves of the identified 98H autumnn-45 can not become yellow at low temperature in a seedling stage, the setting percentage is identical to that of a maintenance line and is obviously higher than that of an original sterility material, and the novel germplasm is a somatic cell hybrid by the verification of PCR amplification cpDNA and the electrophoresis of mtDNA.
Description
One, technical field
The invention belongs to a kind of authentication method of Chinese cabbage cytoplasmic male sterilty new germ plasm, be exclusively used in the evaluation of Chinese cabbage cytoplasmic male sterilty burdo new germ plasm.
Two, technical background
Chinese cabbage (Brassica campestris ssp.chinensis Makino) originates in China, and in recent years, states such as Southeast Asia, Japan and the United States and Europe extensively introduce a fine variety, and become worldwide vegetables gradually.My school Vegetable Research Institute is since the mid-1970s, seed selection and utilize male sterile two-purpose line to carry out the production of hybrid seeds at home and abroad takes the lead in, and priority is bred " short assorted ", " short anti-" serial good hybrid large scale application is in production, but dual-purpose be breeding method pull out can educate strain not only take a lot of work, time-consuming, increase cost, and be difficult for guaranteeing hybrid purity.Utilize Chinese cabbage cytoplasmic male sterile line (CMS) then can overcome these shortcomings.Chinese cabbage (CMS) system that obtains via the transformation of radish cytoplasmic sterility but is difficult to be applied to production because low temperature yellow in seedling stage and floral organ nectary development are bad.Studies show that the yellow in seedling stage is the major obstacle that this CMS system uses, it mainly is the bad reflection that causes because of radish kytoplasm inharmonious between examining with Chinese cabbage.
Three, summary of the invention
Technical problem: the authentication method that the object of the present invention is to provide a kind of Chinese cabbage cytoplasmic male sterilty new germ plasm, to obtaining the scientific verification of burdo regeneration plant, determine Chinese cabbage cytoplasmic male sterilty new germ plasm, for the application of Chinese cabbage cytoplasmic male sterilty new germ plasm lays the foundation.
Technical scheme: the authentication method of Chinese cabbage cytoplasmic male sterilty new germ plasm provided by the present invention:
1) identify in the field: identify that new germ plasm has or not yellow seedling stage, florescence is identified fertility, solid situation under the natural conditions simultaneously compares solid situation under the blade yellowing under the cryogenic conditions, sterile rate and sterile degree, the natural conditions with maintenance line and former sterile material;
2) cell chromosome is identified: for planting experimentally son respectively in the culture dish that is lined with wet filter paper, normal temperature germinates down, after seed shows money or valuables one carries unintentionally, move to and handle 20～30h in the refrigerator, move in 20～28 ℃ of incubators and cultivate 16～20h, 2 roots of clip are put and are handled about 24h in 0 ℃ of mixture of ice and water, fix with the Kano fixer then, take out the tip of a root during film-making, 45ml/100ml acetic acid compressing tablet is observed counting, identifies chromosome number and the maintenance line and the raw-material uniformity of new material;
3) esterase, peroxidase isozyme electrophoretic analysis: for the sowing germination under normal temperature condition simultaneously of examination material, 3 seeds of every part of material are pressed radicle, hypocotyl, cotyledon three parts prepare sample respectively, ice bath grinds in the 1.5ml plastic centrifuge tube, every pipe adds extraction buffer solution 0.1ml, adopt vertical dull and stereotyped discontinuous polyethylene acrylamide gel electrophoresis, each sample pipetting volume amount 30 μ l, 0.01g/100ml bromine powder orchid makes indicator, electrophoresis is finished in 4 ℃ of refrigerators, time 3～4h, adopt fast blue decoration method dyeing esterase isozyme gel, new material radicle and hypocotyl The esterase isozyme result and former sterile material and maintenance line comparing difference, adopt benzidine staining method dyeing peroxidase isozyme gel, the radicle of new material and cotyledon peroxidase isozyme and maintenance line, former sterile material comparing difference.
4) molecular biology identification: half seedling 3d before sampling of new material and maintenance line carries out the shading yellow to be handled, extract mitochondria NDA and chloroplast NDA, adopt two primers to carry out the pcr amplification of mitochondrial DNA and chloroplast DNA, the sequence of primer is as follows respectively:
Primer?1 5′-ATGGGAAGGGTAGGT-3′
Primer?2 5′-GTCGATATGGCGAGTCC-3′
Primer concentration is 20 μ M, the condition of pcr amplification is: 93 ℃/1min (sex change)--55 ℃/1min (annealing)---72 ℃/2min (polymerization), extend 30 circulations, amplified production is gone up electrophoresis at Ago-Gel (ethidium bromide staining), more former OguCMS and new material and maintenance line cpDNA and mtDNA total amount difference and three parts of mtDNA and cpDNA differences that supply between the examination material.The authentication method of above-mentioned Chinese cabbage cytoplasmic male sterilty new germ plasm, it is characterized in that: used extraction buffer solution is in esterase, the peroxidase isozyme electrophoretic analysis: the 0.05M borate buffer, include 20.0g/100ml sucrose and 1.0g/100ml polyvinylpyrrolidone, pH8.7.
Beneficial effect: the authentication method that adopts Chinese cabbage cytoplasmic male sterilty new germ plasm of the present invention, can obtain Chinese cabbage cytoplasmic male sterilty new germ plasm, not only provide new material, new method, and had important significance for theories seed selection and the heterotic utilization of quickening the Chinese cabbage male sterile line.The phenotype feature helps the evaluation of burdo, and the Identification of somatic hybrids morphological feature is to judge whether the plant that is obtained has the most basic and most important index of the value utilized.Cell chromosome number combining form is learned the hybrid essence that feature can prove fusion product reliably.The electrophoresis banding pattern polymorphism analysis of isodynamic enzyme is to identify kind of the conventional method of interior burdo.The molecular biology identification means that development in recent years is got up make authenticate technology advanced more, reliable.The organelle gene group comprises chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), and cpDNA that extraction is obtained and mtDNA compare with the organelle DNA that known primer increases then to burdo and its fusion parent.Whether result relatively just can identical with fusion parent's a side or both sides in order to chloroplast in the decision fusion product or mitochondria, or new a kind of.Pair cell DNA carries out pcr amplification, is a kind of good method of Identification of somatic hybrids organelle gene group, and it is not only accurate, and only needs a spot of vegetable material.The present invention has carried out morphology of tea flower organ, male sterility and ripening rate evaluation to merging the regeneration plant offspring, and cell chromosome is identified, Isozyme Analysis and molecular biology identification, and authentication method is feasible, and qualification result is accurately and reliably.
Four, description of drawings
Fig. 1 is under the cryogenic conditions, new material (in) with the blade yellowing of former OguCMS material (left side), maintenance line (right side) relatively
Fig. 2 be maintenance line (left side), new material (in), the floral organ of former OguCMS material (right side) relatively
Five, embodiment 1 vegetable material: 1.1 new materials: 91H autumn-100 hypocotyl protoplast handles by gamma-radiation and 91H autumn-21 cotyledon protoplast is handled through rhodamine (R-6G), and electricity merges the ZS that the back obtains
6(A) sterile strain obtains F with the maintenance line pollen pollination that normally can educate
1Generation, its offspring ZS
6(A)
3And ZS
6(A)
10(complete sterility, not yellow under the low temperature, and nectary is arranged) continue with the pollination of maintenance line pollen individual plant results seed.With ZS
6(A)
10Offspring (98H autumn-45) tries material as the present invention.1.2 former sterile material: ck
1, 91H autumn-100 (OguCMS, flower pesticide petal-shaped, the yellow of low temperature lower blade, no nectary).1.3 maintenance line: ck
2, 91H autumn-21.Identify in 2 methods, 2.1 fields
Identify that new material (98H autumn-45) has or not yellow seedling stage, the florescence is identified fertility (floral organ detection), and the solid situation under the natural bar condition compares with maintenance line and former sterile material simultaneously.2.2 cell chromosome is identified (tip of a root film-making mitosis metaphase)
Three parts of confessions are planted experimentally son respectively in the culture dish that is lined with wet filter paper, normal temperature germinate down (about 20 ℃).After seed shows money or valuables one carries unintentionally, move in the refrigerator and handle about 24h.Cut root and move in 22 ℃ of incubators noon the previous day and to cultivate, be warming up to about 25 ℃ about at 8 o'clock in morning next day, behind the 2h 2 roots of clip put preserve in 0 ℃ of refrigerator standby.Take out the tip of a root during film-making, 45ml/100ml acetic acid compressing tablet is observed counting under phase contrast microscope.2.3 esterase, peroxidase isozyme electrophoretic analysis 2.3.1 sample preparation
Three parts supply the examination material, and sowing is germinateed under normal temperature condition simultaneously.3 seeds of every part of material prepare sample respectively by radicle, hypocotyl, cotyledon three parts, and ice bath grinds in the 1.5ml plastic centrifuge tube, and every pipe adds extraction buffer solution 0.1ml.Extract buffer solution: the 0.05M borate buffer includes 20.0g/100ml sucrose and 1.0g/100ml polyvinylpyrrolidone, pH8.7.2.3.2 electrophoresis
Adopt vertical dull and stereotyped discontinuous polyethylene acrylamide gel electrophoresis, the method that Hu Nengshu introduces is pressed in the preparation of gel storage liquid, each sample pipetting volume amount 30 μ l, and the 0.01g/100ml bromjophenol blue is made indicator, and electrophoresis is finished in 4 ℃ of refrigerators, time 3～4h.2.3.3 gel-colored method
The fast blue decoration method is adopted in esterase isozyme dyeing.
The benzidine staining method is adopted in peroxidase isozyme dyeing.2.4 the extraction of molecular biology identification 2.4.1 mitochondria NDA and chloroplast DNA
Three parts of seeds for the examination materials, on September 21st, 1998 simultaneously at field sowing.Half seedling 3d before sampling of new material and maintenance line carries out the shading yellow to be handled, and is beneficial to the extraction of mitochondrial DNA.2.4.2 the pcr amplification of mitochondrial DNA and chloroplast DNA
Two primers are adopted in this test, and are synthetic by Shanghai Physiology Inst., Chinese Academy of Sciences.The sequence of the primer is as follows respectively:
Primer?1 5′-ATGGGAAGGGTAGGT-3′
Primer?2 5′-GTCGATATGGCGAGTCC-3′
Primer concentration is 20 μ M.Test adopts the PCR instrument of U.S. genome company to increase.The condition of pcr amplification is: 93 ℃/1min (sex change)-55 ℃/1min (annealing)-72 ℃/2min (polymerization), extend 30 circulations.Amplified production is gone up electrophoresis at Ago-Gel (ethidium bromide staining), and takes a picture with Gel Docamenstation Egstem.Result of implementation is as follows: 1 field evaluation 1) the blade yellowing under the cryogenic conditions
1997～1998 years, new material blade aetiolation (among Fig. 1) do not occur under the temperature condition below 12 ℃, and is consistent with maintenance line material (Fig. 1 right side) performance; And former OguCMS material shows serious yellow (Fig. 1 left side).2) sterile rate and sterile degree
Field investigation 120 strain new materials (30 strain/districts repeat 4 times), its sterile rate, sterile degree are 100%, and be consistent with former OguCMS material.The plant of maintenance line all can educate.The flower pesticide of former OguCMS material is petal-shaped, no nectary (Fig. 2 right side); And the degeneration of the flower pesticide of new material is lower, still is petal-shaped, but has 4 flourishing nectarys (among Fig. 2); The anther development of maintenance line is normal, nectary prosperity (Fig. 2 left side).3) the solid situation under the natural conditions
Spring in 1998, three parts for all pollinations under field conditions (factors) of examination material, each material is unit with the individual plant, the solid situation of random searching 10 strains shows (table 1), the average individual plant of new material bears pods and counts, average individual plant is set seeds number, average single-strain seed heavily is respectively 446.3,6232.6 and 13.40g, all do not have significant difference with three indexs of maintenance line, but all reach utmost point significant difference between three indexs and former OguCMS.Three indexs of new material are respectively 3.6,4.7,4.5 times of three indexs of former OguCMS.2 indoor identification 1) root-tip cells chromosome is identified
Qualification result shows that the chromosome number of new material and maintenance line and former OguCMS material are in full accord, are 2n=20, and the chromosome of a pair of band satellite is all arranged.2) isozyme electrophoresis analyzes 2.1) the esterase isozyme analysis
Three parts of materials are all sowed on October 25th, 1998, October 28 by stages, and November 2 was carried out electrophoretic analysis simultaneously, and it is basically identical as a result.The esterase isozyme of the new material radicle of October 28 sowing has 8 bands of a spectrum, than maintenance line Duo a little less than 1 (the Rf value is bigger) be with; Though the same with the broadband number of former sterile material, the Rf value of bands of a spectrum has a great difference.The hypocotylar EST isodynamic enzyme of new material also has 8 bands of a spectrum, has lacked strong bands of a spectrum than former sterile material; Although consistent with the maintenance line broadband number, the Rf value of bands of a spectrum differs bigger.The EST isodynamic enzyme of new material has 10 bands of a spectrum, Duos 3 bands of a spectrum than maintenance line, lack one than former sterile material a little less than band, have than big-difference between the three.2.2) the peroxidase isozyme analysis
The radicle of new material and cotyledon POD isodynamic enzyme and maintenance line, there were significant differences for former sterile material.The POD isodynamic enzyme of new material radicle has 6 bands of a spectrum, Duos a weak band that the Rf value is bigger than maintenance line; Equate with the bands of a spectrum of former sterile material, but the 5th band is more eager to excel in whatever one does than sterile material.The hypocotylar POD isodynamic enzyme of new material has 4 bands of a spectrum, no matter from the band number, or on Rf value and the power, all with parents' indifference.The POD isodynamic enzyme of new material cotyledon has 5 bands of a spectrum, not only Duos band a little less than than parents, and the bigger band of Rf value is all stronger than parents.3) molecular biology identification 3.1) cpDNA and the comparison of mtDNA total amount
Three parts supply the examination material, and the pure blade weight average of the sample of each material is 400g, extracts the cpDNA and the mtDNA that obtain, adopt agarose gel electrophoresis to measure each sample size, and the point sample amount of each sample is 30 μ l.The result shows, the cpDNA content of maintenance line is the highest in three parts of materials, is respectively new material and former OguCMS 10 times, 30 times; The cpDNA of new material is between parents, and its content is about 10 times of former OguCMS; From mtDNA content, former OguCMS material is the highest, is respectively new material and maintenance line 6 times, 8 times; The mtDNA content of new material is also between parents, a little more than maintenance line.No matter new material and maintenance line are cpDNA, or mtDNA all contains RNA, and rna content is higher among the cpDNA; And all not containing RNA among the cpDNA of former OguCMS material and the mtDNA, this is a very big difference of former OguCMS and new material and maintenance line.3.2) pcr amplification of cpDNA and mtDNA
Three parts for mtDNA and cpDNA significant difference between the examination material.With regard to mtDNA, new material not only has the bands of a spectrum identical with former OguCMS, and new bands of a spectrum occurred; And no matter the bands of a spectrum of the mtDNA of new material and maintenance line mtDNA are broadband numbers, and still strong and weak degree and Rf value are all different.The cpDNA of new material is different with maintenance line, also with former OguCMS very big difference is arranged, and the cpDNA of this explanation new material reconfigures.
The field qualification result confirms ZS
6(A)
10The offspring is the cytoplasmic male sterilty new material that portion is hopeful utilization on producing.People such as Wei Baoqin utilize backcross transformation and select the method for maintenance line, on Chinese cabbage the OguCMS characteristic are improved, and yellow has also had alleviating to a certain degree, but because of the nectary problem does not solve, still have difficulties so utilize on producing.Earle etc. utilize cell fusion method to solve wild cabbage sterile material low temperature yellow problem, but the nectary of sterile material is still very little, can not be normally solid.The not only sterile rate of cytoplasmic sterility new material, sterile degree that we obtain are 100%; In the not yellow of cryogenic conditions lower blade, and 4 flourishing nectarys are arranged; The ripening rate height does not have significant difference with maintenance line, and this result is similar to results of study on Chinese cabbage such as MarinaSigareva.
Indoor identification proof ZS
6(A)
10Be burdo, its offspring 98H autumn-45 are the cytoplasmic male sterilty new germ plasm.Isodynamic enzyme is the multiple molecular form of the identical enzyme of function, is special gene outcome.Merging the regeneration plant offspring compares with parents in EST, POD isodynamic enzyme and new bands of a spectrum occur or lack some bands of a spectrum, be since OguCMS hypocotyl protoplast and maintenance line cotyledon protoplast in the somatic cell fusion process, new combination appears in dna molecular, produces some new genetic fragment.This is similar with the result of study of people on wild cabbage such as Quiros in the research on the paddy rice with Hayashi etc., Terada etc.The Isozyme Analysis of this experiment shows, merges plant offspring ZS
6(A)
10Has the burdo characteristic.
The specificity of pcr amplification determined by primer, the specific DNA difference that different primers shows, and the special cpDNA that different materials shows is different with mtDNA band line, thereby identifies for burdo molecular Evidence is provided.The PCR primer of this experiment is consistent, and the difference that the chloroplast DNA of new material, mitochondrial DNA and parents' bands of a spectrum occur shows that then the gene order of new material and fragment length are different with parents, thereby confirmed ZS from molecular level
6(A)
10Be burdo, its offspring 98H autumn-45 are the cytoplasmic male sterilty new germ plasm.
The different solid situations of individual plant under table 1 natural conditions for the examination material
*
The average individual plant of the material average individual plant of number (individual) average single-strain seed of number (grain) of setting seeds that bears pods weighs (g)
Former OguCMS 123.5
B1333.4
B2.97
B
New material 446.3
A6232.6
A13.40
A
Maintenance line 456.1
A6494.3
A13.12
A
*Adopt the Deng Kenshi multiple range test, K=3, n=10.Expression difference reaches utmost point significance level between the capitalization.
Claims (2)
1. the authentication method of a Chinese cabbage cytoplasmic male sterilty new germ plasm comprises:
1.1) field identifies: identify that new germ plasm has or not yellow seedling stage, florescence is identified fertility, solid situation under the natural conditions, simultaneously compare 12 ℃ of blade yellowing, sterile rate and sterile degree, solid situations under the natural conditions under the following condition with maintenance line and former sterile material, the blade aetiolation does not appear in new material, sterile rate, sterile degree are 100%, average individual plant the set seeds number, average single-strain seed of number, average individual plant that bear pods heavily is respectively 446.3,6232.6 and 13.40g, but and all reach utmost point significant difference between former sterile material;
1.2) the cell chromosome evaluation: for planting experimentally son respectively in the culture dish that is lined with wet filter paper, normal temperature germinates down, after seed shows money or valuables one carries unintentionally, move to and handle 20～30h in the refrigerator, move in 20～28 ℃ of incubators and cultivate 16～20h, 2 roots of clip are put and are handled about 24h in 0 ℃ of mixture of ice and water, fix with the Kano fixer then, take out the tip of a root during film-making, 45ml/100ml acetic acid compressing tablet is observed counting, identifies chromosome number and the maintenance line and the raw-material uniformity of new material, and the chromosome number of new material and maintenance line and former sterile material are in full accord, be 2n=20, and the chromosome of a pair of band satellite is all arranged;
1.3) esterase, peroxidase isozyme electrophoretic analysis: for the sowing germination under normal temperature condition simultaneously of examination material, 3 seeds of every part of material are pressed radicle, hypocotyl, cotyledon three parts prepare sample respectively, ice bath grinds in the 1.5ml plastic centrifuge tube, every pipe adds extraction buffer solution 0.1ml, adopt vertical dull and stereotyped discontinuous polyethylene acrylamide gel electrophoresis, each sample pipetting volume amount 30 μ l, 0.01g/100ml bromjophenol blue is made indicator, electrophoresis is finished in 4 ℃ of refrigerators, time 3～4h, adopt fast blue decoration method dyeing esterase isozyme gel, new material radicle and hypocotyl The esterase isozyme result and former sterile material and maintenance line comparing difference, adopt benzidine staining method dyeing peroxidase isozyme gel, the radicle of new material and cotyledon peroxidase isozyme and maintenance line, former sterile material comparing difference, the esterase isozyme of new material has 10 bands of a spectrum, Duo 3 bands of a spectrum than maintenance line, band a little less than lacking one than former sterile material, the peroxidase isozyme of new material cotyledon has 5 bands of a spectrum, not only Duo band a little less than, and the bigger band of Rf value is all stronger than parents than parents;
1.4) molecular biology identification: half seedling 3d before sampling of new material and maintenance line carries out the shading yellow to be handled, extract mitochondria NDA and chloroplast DNA, adopt two primers to carry out the pcr amplification of mitochondrial DNA and chloroplast DNA, the sequence of primer is as follows respectively:
Primer?1 5′-ATGGGAAGGGTAGGT-3′
Primer?2 5′-GTCGATATGGCGAGTCC-3′
Primer concentration is 20 μ M,-72 ℃/2min polymerization that the condition of pcr amplification is: 93 ℃/1min sex change--55 ℃/1min annealing--, extend 30 circulations, amplified production is electrophoresis on Ago-Gel, ethidium bromide staining, more former OguCMS and new material and maintenance line chloroplast DNA and mitochondrial DNA total amount difference and three parts of mitochondrial DNA and chloroplast DNA differences that supply between the examination material, the chloroplast DNA content of maintenance line is the highest in three parts of materials, is respectively new material and former OguCMS 10 times, 30 times; The chloroplast DNA of new material is between parents, and its content is about 10 times of former OguCMS; From mitochondrial DNA content, former OguCMS material is the highest, is respectively new material and maintenance line 6 times, 8 times, and the mitochondrial DNA content of new material is also between parents; No matter new material and maintenance line are chloroplast DNAs, or mitochondrial DNA all contains RNA; And all do not contain RNA in the chloroplast DNA of former OguCMS material and the mitochondrial DNA.
2. according to the authentication method of the described Chinese cabbage cytoplasmic male sterilty of claim 1 new germ plasm, it is characterized in that: used extraction buffer solution is in esterase, the peroxidase isozyme electrophoretic analysis: the 0.05M borate buffer, include 20.0g/100ml sucrose and 1.0g/100ml polyvinylpyrrolidone, pH8.7.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99114126 CN1119928C (en) | 1999-03-29 | 1999-03-29 | Method for determining cytoplasmic male sterility breed of Chinese cabbage |
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|---|---|---|---|
| CN 99114126 CN1119928C (en) | 1999-03-29 | 1999-03-29 | Method for determining cytoplasmic male sterility breed of Chinese cabbage |
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| CN1119928C true CN1119928C (en) | 2003-09-03 |
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| CN 99114126 Expired - Fee Related CN1119928C (en) | 1999-03-29 | 1999-03-29 | Method for determining cytoplasmic male sterility breed of Chinese cabbage |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100348735C (en) * | 2006-09-06 | 2007-11-14 | 南京农业大学 | Brassica campestris ssp. Chinensis male sterile molecular marker-assisted selection method |
| CN109724977B (en) * | 2018-12-21 | 2021-05-14 | 塔里木大学 | Simple and convenient tree male and female identification method |
| CN110186918A (en) * | 2019-07-08 | 2019-08-30 | 湖北民族大学 | A kind of method of Rapid identification Lauraceae Machilus Nees |
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