CN102816860B - Molecular marker assisted selection method for female line breeding of cucumber - Google Patents
Molecular marker assisted selection method for female line breeding of cucumber Download PDFInfo
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Abstract
The invention discloses a molecular marker assisted selection method for female line breeding of a cucumber. The method includes screening out a simple sequence repeat (SSR) labeled primer which is in close linkage with all-female genes of the cucumber through an all-female variety 230-1-2-2-3-1 inbred line, a weak female variety 3-5-1-3-2-1-1-1-1-2 inbred line and F2-generation segregation population of the cucumber; through extraction of DNA of a control variety and seedling genome of a seed to be detected, obtaining the gel maps of the control variety and the sample to be detected through the screened SSR labeled primer and after polyacrylamide gel electrophoresis and silver staining; and detecting the existence of the total-female genes of the cucumber rapidly and accurately according to a relative position of a stripe on the gel. According to the method, the molecular marker assisted breeding of the female line of the cucumber can be carried on effectively through SSR molecular marker screening of the all-female variety 230-1-2-2-3-1 inbred line and the weak female variety 3-5-1-3-2-1-1-1-1-2 inbred line, the selection efficiency and the standardization degree are improved, and the breeding process is accelerated.
Description
Technical field
What the present invention relates to is a kind of molecular marker-assisted selection method of cucumber female series breeding, belongs to vegetables molecular genetic breeding technical field.
Background technology
Cucumber is one of China vegetables crop, has high economy and social value.Cucumber plant sexual type is various, has the broad varietys such as pure staminiferous plant, hermaphrodite flower strain, monoecism, the strain of male flower hermaphrodite flower and full female plant.The primary factor of restriction cucumber economic yield is plant Proportion of male and female flowers, because except full female plant, that other sexual type plant all shows as is with luxuriant foliage and spreading branches in leafy profusion, large storehouse, source is few, economic coefficient is low.Female line cucumber refers to the only strain of long female flower and not long male flower or long minute quantity male flower of plant.This strain often show precocity, melon close, adopt the melon phase concentrate, the advantage such as yielding ability is strong.Utilize half-blood that female line does maternal preparation also multilist reveal precocity, adopt that the melon phase concentrates and the advantage such as high yield.Meanwhile, utilize female line to prepare the first generation of hybrid without artificial pollination, saved labor force, reduced breeding cost, and the half-blood purity of making is very high.Therefore, concerning breed cucumber, female line has utility value in heterosis utilization and hybrid seeding, and the breeding and application of female line is an important research contents in breed cucumber.
At present, the seed selection of cucumber female series mainly relies on field flowering habit to carry out phenotypic evaluation, the method not only takes very long, can consume a large amount of human and material resources, and cucumber plant sexual type is complicated, being subject to ambient conditions affects and changes, so carry out the efficiency of selection of gynoecy gene from phenotype not high, the seed selection difficulty of female line is large, success ratio is low, and the cucumber female series kind of seed selection mostly exists female unstable, is subject to the drawbacks such as planting environment impact.The appearance of molecule marker, for the seed selection of female line provides new approach, utilize the molecule marker not only can localizing objects gene, also can utilize and the closely linked molecule marker of the target gene gene that follows the trail of the objective, the indirect selection of realization to objective trait, so-called molecular marker assisted selection that Here it is (Molecular Marker Assisted Selection).Compare with traditional Phenotypic Selection, it has the following advantages: (1) is not subject to the impact of genetic expression and environmental factors on the selection of objective trait; (2) can select in any stage of generation morning and plant strain growth, greatly shorten the breeding time limit; (3) at segregating generation, can identify rapidly the genotype of plant, not be subject to gene to show recessive impact; (4) can break the chain of target gene and unfavorable gene.Wherein, SSR molecule marker is to utilize a large amount of micro-satellite repetitive sequence design primer existing in eukaryotic gene group, the tumor-necrosis factor glycoproteins of connecting by pcr amplification, the difference of the multiplicity of the micro-satellite of foundation between same species different genotype, discloses length polymorphism.Because SSR is marked at the polymorphism that detects on single seat far above other several molecule markers, and extensively, at random, be distributed in equably whole genome, tool codominant inheritance, distribute wide, stability and polymorphic rate are high, simple to operate, reproducible, to features such as DNA specification of quality are lower, can differentiate accurately and efficiently a large amount of allelotrope, application is carried out assistant breeding with the mutually chain molecule marker of goal gene and can directly genotype, to offspring's individual plant, be selected, realizing the sexual type in seedling stage identifies, improve efficiency of selection and accuracy, thereby accelerate cucumber female series breeding process, in cucumber female series breeding, there is high using value.
Summary of the invention
The object of the invention is to: for the conventional field test adopting in existing cucumber female series breeding combine with plant phenotype that the existing workload of method is large, the cycle is grown, the impact, the breeding efficiency that are subject to envrionment conditions are low, high in cost of production defect, provide a kind of in cucumber female series breeding process, for early stage segregating generation and female line evaluation in seedling stage, the method that energy accurately, quick, high efficiency selected goes out the molecular marker assisted selection of female line material.
The object of the invention is achieved by the following technical programs: the molecular marker-assisted selection method of described cucumber female series breeding, and the method described in it is characterized in that is carried out according to the following steps:
(1) collection of cucumber leaves sample to be detected: by cucumber material sowing to be detected, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(2) collection of cucumber control sample blade: by the complete female kind 240-1-2-2-3-1 self-mating system of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material sowing, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(3) sample gene group DNA extraction: extract every strain leaf DNA by CTAB method; Take the blade that 150mg is fresh, together with the precooling in liquid nitrogen of adapter, mill ball, use MM301 cell crushing instrument to be ground into powder; Polish process is set as: milling time 1.5min, grinds frequency 20Hz.After grinding, add 2 * CTAB Extraction buffer, 700 μ l(to be preheated in advance 65 ℃) soak into powder and reverse centrifuge tube, powder is fully scattered, in 65 ℃ of water bath heat preservation 30min, softly mix 2-3 time therebetween; Take out centrifuge tube, be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol, light and slow putting upside down mixes, standing 10min, the centrifugal 15min of 12000rpm; Shift supernatant in another centrifuge tube, add the pre-cold isopropanol of 2/3 volume, light and slow putting upside down mixes, and room temperature is placed 15min, and the centrifugal 10min of 12000rpm, removes supernatant, and centrifuge tube is inverted on thieving paper, controls dry supernatant; By the washing with alcohol of 700 μ l 70%, precipitate 2 times, micro-dry under room temperature, add 300 μ l deionized water dissolving precipitations, add isopyknic chloroform/primary isoamyl alcohol extracting 2 times, draw supernatant, add the NaAc of 1/5 volume, the precooling dehydrated alcohol precipitation DNA of 2.5 times of volumes, place 1h left and right, the centrifugal 10min of 12000rpm, removes supernatant; By the washing with alcohol of 700 μ l 70%, precipitate 2 times air seasoning or seasoning; Add 30 μ l deionized water dissolving DNA, be stored in-80 ℃ of Ultralow Temperature Freezers standby; Making 1% sepharose detects; With SMA3000 trace ultraviolet spectrophotometer, measure concentration and the purity of extracting DNA, DNA is diluted to desired concn, packing, is placed in-20 ℃ and saves backup;
(4) molecule marker of acquisition and cucumber gynoecy genes involved: to the complete female kind 240-1-2-2-3-1 self-mating system of cucumber and weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system and F thereof
2for DNA sample, carry out SSR molecular marker analysis; Select 699 pairs of SSR primers to carry out pcr amplification, pcr amplification reaction is totally 20 μ l, and amplification reaction system composition is: Cucumber germplasm DNA20ng, each 0.4 μ l of 10pmol/ μ l primer, 2.5mM Mg
2+2 μ l, 2mM dNTP1 μ l, 5U/ μ l Taq archaeal dna polymerase 0.1 μ l, 10 * buffer, 2 μ l, add aseptic redistilled water polishing to 20 μ l;
(5) sample survey: utilize the labeled primer obtaining to carry out pcr amplification, amplified production is carried out to polyacrylamide gel electrophoresis compartment analysis, obtain polymorphic bands, according to the relative position of bands of a spectrum on gel, select the electrophoresis banding pattern individual plant identical with donor parents, in conjunction with the selection of field test and other Breeding objectives; Select the fine individual plant with gynoecy feature.
In step (3), polish process is set as: milling time 1.5min, grinds frequency 20Hz; After grinding, add and be preheated in advance 2 * CTAB Extraction buffer, the 700 μ l of 65 ℃ and soak into powder; Utilize orthogonal test, set up the optimum augumentation system of cucumber gynoecy gene linkage specific fragment.
In step (4), PCR reaction amplification program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30sec, by specific primer specified temp annealing 1min, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min; In the pcr amplification product of 20 μ l, add 94 ℃ of sex change 5min of 8 μ l Loading Buffer, be placed in rapidly cooled on ice; Every sample is got 6 μ l and is splined on 6% denaturing polyacrylamide gel, and under constant 100W power, electrophoresis to tetrabromophenol sulfonphthalein arrives the gel the other end; After silver dyes, on film illuminator, take a picture, observe, to show the primer of codominance separation between parent, utilize F
2in generation,, strong female and weak female mixing gene pool screened again, thereby obtained and the closely linked SSR polymorphic bands of cucumber gynoecy, and its labeled primer is CSWCT25.
The invention has the beneficial effects as follows:
(1) the present invention has adopted target material DNA has been carried out to PCR and SSR molecular marking technique, and this technology has efficient and feature cheaply, is applicable to great amount of samples material to carry out identification, has greatly improved efficiency of selection;
(2) the present invention can be in office when the phase is detected accurately and rapidly to breed cucumber material, Zhou Nianjun can carry out in laboratory, is not subject to the impact of envrionment conditions, and does not affect the normal growth of plant;
Molecular marker assisted selection cucumber female series breeding method of the present invention, compares with conventional breeding, authentication method, has the advantages such as the cycle is short, cost is low, easy and simple to handle.
Accompanying drawing explanation
Fig. 1 is that CSWCT25 labeled primer is at parent and F
2the gel electrophoresis figure of part individual plant pcr amplification product in colony.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but following description forms the restriction of going up in all senses to protection scope of the present invention.
The present invention is for pcr amplification and the SSR labeled primer (CSWCT25) of cucumber female series molecular marker assisted selection, forward primer sequence wherein: 5 '-AAAGAAATTAAGTCAATCAAACCG-3 ', reverse primer sequence: 5 '-CCCACCAATAGTAAAATTATACAT-3 '.
The molecular marker-assisted selection method of cucumber female series breeding of the present invention, it carries out according to the following steps:
(1) collection of cucumber leaves sample to be detected: by cucumber material sowing to be detected, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(2) collection of cucumber control sample blade: by the complete female kind 240-1-2-2-3-1 self-mating system of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material sowing, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(3) sample gene group DNA extraction: extract every strain leaf DNA by CTAB method; Take the blade that 150mg is fresh, together with the precooling in liquid nitrogen of adapter, mill ball, use MM301 cell crushing instrument to be ground into powder; Polish process is set as: milling time 1.5min, grinds frequency 20Hz.After grinding, add 2 * CTAB Extraction buffer, 700 μ l(to be preheated in advance 65 ℃) soak into powder and reverse centrifuge tube, powder is fully scattered, in 65 ℃ of water bath heat preservation 30min, softly mix 2-3 time therebetween; Take out centrifuge tube, be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol, light and slow putting upside down mixes, standing 10min, the centrifugal 15min of 12000rpm; Shift supernatant in another centrifuge tube, add the pre-cold isopropanol of 2/3 volume, light and slow putting upside down mixes, and room temperature is placed 15min, and the centrifugal 10min of 12000rpm, removes supernatant, and centrifuge tube is inverted on thieving paper, controls dry supernatant; By the washing with alcohol of 700 μ l 70%, precipitate 2 times, micro-dry under room temperature, add 300 μ l deionized water dissolving precipitations, add isopyknic chloroform/primary isoamyl alcohol extracting 2 times, draw supernatant, add the NaAc of 1/5 volume, the precooling dehydrated alcohol precipitation DNA of 2.5 times of volumes, place 1h left and right, the centrifugal 10min of 12000rpm, removes supernatant; By the washing with alcohol of 700 μ l 70%, precipitate 2 times air seasoning or seasoning; Add 30 μ l deionized water dissolving DNA, be stored in-80 ℃ of Ultralow Temperature Freezers standby; Making 1% sepharose detects; With SMA3000 trace ultraviolet spectrophotometer, measure concentration and the purity of extracting DNA, DNA is diluted to desired concn, packing, is placed in-20 ℃ and saves backup;
(4) molecule marker of acquisition and cucumber gynoecy genes involved: to the complete female kind 240-1-2-2-3-1 self-mating system of cucumber and weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system and F thereof
2for DNA sample, carry out SSR molecular marker analysis; Select 699 pairs of SSR primers to carry out pcr amplification, pcr amplification reaction is totally 20 μ l, and amplification reaction system composition is: Cucumber germplasm DNA20ng, each 0.4 μ l of 10pmol/ μ l primer, 2.5mM Mg
2+2 μ l, 2mM dNTP1 μ l, 5U/ μ l Taq archaeal dna polymerase 0.1 μ l, 10 * buffer, 2 μ l, add aseptic redistilled water polishing to 20 μ l;
(5) sample survey: utilize the labeled primer obtaining to carry out pcr amplification, amplified production is carried out to polyacrylamide gel electrophoresis compartment analysis, obtain polymorphic bands, according to the relative position of bands of a spectrum on gel, select the electrophoresis banding pattern individual plant identical with donor parents, in conjunction with the selection of field test and other Breeding objectives; Select the fine individual plant with gynoecy feature.
In step (3), polish process is set as: milling time 1.5min, grinds frequency 20Hz; After grinding, add and be preheated in advance 2 * CTAB Extraction buffer, the 700 μ l of 65 ℃ and soak into powder; Utilize orthogonal test, set up the optimum augumentation system of cucumber gynoecy gene linkage specific fragment.
In step (4), PCR reaction amplification program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30sec, by specific primer specified temp annealing 1min, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min; In the pcr amplification product of 20 μ l, add 94 ℃ of sex change 5min of 8 μ l Loading Buffer, be placed in rapidly cooled on ice; Every sample is got 6 μ l and is splined on 6% denaturing polyacrylamide gel, and under constant 100W power, electrophoresis to tetrabromophenol sulfonphthalein arrives the gel the other end; After silver dyes, on film illuminator, take a picture, observe, to show the primer of codominance separation between parent, utilize F
2in generation,, strong female and weak female mixing gene pool screened again, thereby obtained and the closely linked SSR polymorphic bands of cucumber gynoecy, and its labeled primer is CSWCT25.
Embodiment 1:(utilizes molecular marker-assisted selection method to detect 1 to the resistance of filial generation material)
(1) collection of cucumber leaves sample to be detected: by cucumber material H174-1-2 sowing to be detected, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(2) collection of cucumber control sample blade: by the complete female kind 240-1-2-2-3-1 self-mating system of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material sowing, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part 0.5, existing using or freezing preservation at-20 ℃ after gathering;
(3) sample gene group DNA extraction: extract every strain leaf DNA by CTAB method.Take the blade that 150mg is fresh, together with the precooling in liquid nitrogen of adapter, mill ball, use MM301 cell crushing instrument to be ground into powder.Polish process is set as: milling time 1.5min, grinds frequency 20Hz.After grinding, add 2 * CTAB Extraction buffer, 700 μ l(to be preheated in advance 65 ℃) soak into powder and reverse centrifuge tube, powder is fully scattered, in 65 ℃ of water bath heat preservation 30min, softly mix 2-3 time therebetween.Take out centrifuge tube, be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol, light and slow putting upside down mixes, standing 10min.The centrifugal 15min of 12000rpm.Shift supernatant in another centrifuge tube, add the pre-cold isopropanol of 2/3 volume, light and slow putting upside down mixes, and room temperature is placed 15min.The centrifugal 10min of 12000rpm, removes supernatant, and centrifuge tube is inverted on thieving paper, controls dry supernatant.By the washing with alcohol of 700 μ l 70%, precipitate 2 times, micro-dry under room temperature.Add 300 μ l deionized water dissolving precipitations.Add isopyknic chloroform/primary isoamyl alcohol extracting 2 times.Draw supernatant, add the NaAc of 1/5 volume, the precooling dehydrated alcohol precipitation DNA of 2.5 times of volumes, places 1h left and right, and the centrifugal 10min of 12000rpm, removes supernatant.By the washing with alcohol of 700 μ l 70%, precipitate 2 times air seasoning or seasoning.Add 30 μ l deionized water dissolving DNA, be stored in-80 ℃ of Ultralow Temperature Freezers standby.Making 1% sepharose detects.With SMA3000 trace ultraviolet spectrophotometer, measure concentration and the purity of extracting DNA, DNA is diluted to desired concn, packing, is placed in-20 ℃ and saves backup;
(4) pcr amplification and amplified production detect: the DNA sample to the complete female kind 240-1-2-2-3-1 self-mating system of cucumber and weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system and H174-1-2 carries out SSR molecular marker analysis, and labeled primer is CSWCT25.Pcr amplification reaction is totally 20 μ l, and amplification reaction system composition is: Cucumber germplasm DNA20ng, each 0.4 μ l of 10pmol/ μ l primer, 2.5mM Mg
2+2 μ l, 2mM dNTP1 μ l, 5U/ μ l Taq archaeal dna polymerase 0.1 μ l, 10 * buffer, 2 μ l, add aseptic redistilled water polishing to 20 μ l.
PCR reaction amplification program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30sec, by specific primer specified temp annealing 1min, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min; In the pcr amplification product of 20 μ l, add 94 ℃ of sex change 5min of 8 μ lLoading Buffer, be placed in rapidly cooled on ice.Every sample is got 6 μ l and is splined on 6% denaturing polyacrylamide gel, and under constant 100W power, electrophoresis to tetrabromophenol sulfonphthalein arrives the gel the other end.After silver dyes, on film illuminator, take a picture, observe and record result;
(5) sample survey: utilize the labeled primer obtaining to carry out pcr amplification, amplified production is carried out to polyacrylamide gel electrophoresis compartment analysis, obtain polymorphic bands, the relative position of bands of a spectrum on H174-1-2 gel, the fragment amplifying with 240-1-2-2-3-1 self-mating system is identical, illustrate that this kind is gynoecy kind, selection in conjunction with field test and other Breeding objectives, meet completely with Field inoculation qualification result, illustrate that the detection method that the present invention sets up can identify cucumber gynoecy fine individual plant accurately and rapidly.
Embodiment 2:(utilizes molecular marker-assisted selection method to detect 2 to the resistance of filial generation material)
(1) collection of cucumber leaves sample to be detected: by cucumber material H158-1-1-2-2 sowing to be detected, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(2) collection of cucumber control sample blade: by the complete female kind 240-1-2-2-3-1 self-mating system of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material sowing, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(3) sample gene group DNA extraction: extract every strain leaf DNA by CTAB method.Take the blade that 150mg is fresh, together with the precooling in liquid nitrogen of adapter, mill ball, use MM301 cell crushing instrument to be ground into powder.Polish process is set as: milling time 1.5min, grinds frequency 20Hz.After grinding, add 2 * CTAB Extraction buffer, 700 μ l(to be preheated in advance 65 ℃) soak into powder and reverse centrifuge tube, powder is fully scattered, in 65 ℃ of water bath heat preservation 30min, softly mix 2-3 time therebetween.Take out centrifuge tube, be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol, light and slow putting upside down mixes, standing 10min.The centrifugal 15min of 12000rpm.Shift supernatant in another centrifuge tube, add the pre-cold isopropanol of 2/3 volume, light and slow putting upside down mixes, and room temperature is placed 15min.The centrifugal 10min of 12000rpm, removes supernatant, and centrifuge tube is inverted on thieving paper, controls dry supernatant.By the washing with alcohol of 700 μ l 70%, precipitate 2 times, micro-dry under room temperature.Add 300 μ l deionized water dissolving precipitations.Add isopyknic chloroform/primary isoamyl alcohol extracting 2 times.Draw supernatant, add the NaAc of 1/5 volume, the precooling dehydrated alcohol precipitation DNA of 2.5 times of volumes, places 1h left and right, and the centrifugal 10min of 12000rpm, removes supernatant.By the washing with alcohol of 700 μ l 70%, precipitate 2 times air seasoning or seasoning.Add 30 μ l deionized water dissolving DNA, be stored in-80 ℃ of Ultralow Temperature Freezers standby.Making 1% sepharose detects.With SMA3000 trace ultraviolet spectrophotometer, measure concentration and the purity of extracting DNA, DNA is diluted to desired concn, packing, is placed in-20 ℃ and saves backup;
(4) pcr amplification and amplified production detect: the DNA sample to the complete female kind 240-1-2-2-3-1 self-mating system of cucumber and weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system and H158-1-1-2-2 carries out SSR molecular marker analysis, and labeled primer is CSWCT25.Pcr amplification reaction is totally 20 μ l, and amplification reaction system composition is: Cucumber germplasm DNA20ng, each 0.4 μ l of 10pmol/ μ l primer, 2.5mM Mg
2+2 μ l, 2mM dNTP1 μ l, 5U/ μ l Taq archaeal dna polymerase 0.1 μ l, 10 * buffer, 2 μ l, add aseptic redistilled water polishing to 20 μ l.
PCR reaction amplification program is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30sec, by specific primer specified temp annealing 1min, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min; In the pcr amplification product of 20 μ l, add 94 ℃ of sex change 5min of 8 μ lLoading Buffer, be placed in rapidly cooled on ice.Every sample is got 6 μ l and is splined on 6% denaturing polyacrylamide gel, and under constant 100W power, electrophoresis to tetrabromophenol sulfonphthalein arrives the gel the other end.After silver dyes, on film illuminator, take a picture, observe and record result;
(5) sample survey: utilize the labeled primer obtaining to carry out pcr amplification, amplified production is carried out to polyacrylamide gel electrophoresis compartment analysis, obtain polymorphic bands, the relative position of bands of a spectrum on H158-1-1-2-2 gel, the fragment amplifying with 3-5-1-3-2-1-1-1-1-2 self-mating system is identical, illustrate that this kind is not gynoecy kind, the selection in conjunction with field test and other Breeding objectives, meets completely with Field inoculation qualification result.
<110> Zhejiang Academy of Agricultural Science
The molecular marker-assisted selection method of a <120> cucumber female series breeding
<160>2
<210>1
<211>24
<212>DNA
<213> artificial sequence
<222>（1）…（24）
<400>1
aaagaaatta agtcaatcaa accg 24
<210>2
<211>24
<212>DNA
<213> artificial sequence
<222>（1）…（24）
<400> 2
cccaccaata gtaaaattat acat 24
Claims (1)
1. a molecular marker-assisted selection method for cucumber female series breeding, is characterized in that described method carries out according to the following steps:
(1) collection of cucumber leaves sample to be detected: by cucumber material sowing to be detected, when 1-2 sheet leaf period, get the comparatively young tender blade in plant top, every part of 0.5g, existing using or freezing preservation at-20 ℃ after gathering;
(2) select the complete female kind 240-1-2-2-3-1 self-mating system of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material and F thereof
2generation;
(3) utilize improved method of CTAB to extract cucumber leaves DNA, respectively to the complete female kind 240-1-2-2-3-1 self-mating system material of cucumber, weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system material, F
2generation, testing sample extract DNA: take the blade that 150mg is fresh, together with the precooling in liquid nitrogen of adapter, mill ball, use MM301 cell crushing instrument to be ground into powder; Polish process is set as: milling time 1.5 min, grind frequency 20Hz; After grinding, add 2 * CTAB Extraction buffer, the 700 μ l that are preheated in advance 65 ℃, soak into testing sample powder the centrifuge tube that reverses, powder is fully scattered, in 65 ℃ of water bath heat preservation 30min, softly mix 2-3 time therebetween; Take out centrifuge tube, be chilled to room temperature, add isopyknic chloroform/primary isoamyl alcohol, light and slow putting upside down mixes, standing 10min; Centrifugal 15 min of 12000rpm; Shift supernatant in another centrifuge tube, add the pre-cold isopropanol of 2/3 volume, light and slow putting upside down mixes, and room temperature is placed 15 min; The centrifugal 10min of 12000rpm, removes supernatant, and centrifuge tube is inverted on thieving paper, controls dry supernatant; By 70% washing with alcohol of 700 μ l, precipitate 2 times, micro-dry under room temperature; Add 300 μ l deionized water dissolving precipitations; Add isopyknic chloroform/primary isoamyl alcohol extracting 2 times; Draw supernatant, add the NaAc of 1/5 volume, the precooling dehydrated alcohol precipitation DNA of 2.5 times of volumes, places 1h left and right, and the centrifugal 10min of 12000rpm, removes supernatant; By 70% washing with alcohol of 700 μ l, precipitate 2 times air seasoning or seasoning; Add 30 μ l deionized water dissolving DNA, be stored in-80 ℃ of Ultralow Temperature Freezers standby; Making 1% sepharose detects; With SMA3000 trace ultraviolet spectrophotometer, measure concentration and the purity of extracting DNA, DNA is diluted to desired concn, packing, is placed in-20 ℃ and saves backup;
(4)) the molecule marker of acquisition and cucumber gynoecy genes involved: to the complete female kind 240-1-2-2-3-1 self-mating system of cucumber and weak female kind 3-5-1-3-2-1-1-1-1-2 self-mating system and F thereof
2for DNA sample, carry out SSR molecular marker analysis; Select 699 pairs of SSR primers to carry out pcr amplification, pcr amplification reaction is totally 20 μ l, and amplification reaction system composition is: Cucumber germplasm DNA20ng, each 0.4 μ l of 10 pmol/ μ l primers, 2.5 mM Mg
2+2 μ l, 2mM dNTP 1 μ l, 5 U/ μ lTaq DNA polysaccharase 0.1 μ l, 10 * buffer2 μ l, adds aseptic redistilled water polishing to 20 μ l;
PCR reaction amplification program is: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 30sec, by specific primer specified temp 1 min that anneals, 72 ℃ are extended 1 min, 35 circulations, 72 ℃ are extended 10min; In the pcr amplification product of 20 μ l, add 94 ℃ of sex change 5min of 8 μ lLoading Buffer, be placed in rapidly cooled on ice; Every sample is got 6 μ l and is splined on 6% denaturing polyacrylamide gel, and under constant 100W power, electrophoresis to tetrabromophenol sulfonphthalein arrives the gel the other end; After silver dyes, on film illuminator, take a picture, observe, to show the primer of codominance separation between parent, utilize F
2in generation,, strong female and weak female mixing gene pool screened again, thereby obtain and the closely linked SSR polymorphic bands of cucumber gynoecy, its labeled primer is CSWCT25, its forward primer sequence: 5 '-AAAGAAATTAAGTCAATCAAACCG-3 ', reverse primer sequence: 5 '-CCCACCAATAGTAAAATTATACAT-3 ';
Utilize the labeled primer obtaining to carry out pcr amplification to testing sample DNA sample, amplified production is carried out to polyacrylamide gel electrophoresis compartment analysis, obtain testing sample polymorphic bands, according to the relative position of bands of a spectrum on gel, select the electrophoresis banding pattern individual plant identical with donor parents, in conjunction with the selection of field test and other Breeding objectives, select the fine individual plant with gynoecy feature.
Priority Applications (1)
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| CN104313020B (en) * | 2014-10-15 | 2017-10-10 | 广东省农业科学院蔬菜研究所 | Indel molecular labelings and its application with hua ' nan-type cucumber gynoecy shape close linkage |
| CN115058538B (en) * | 2022-06-30 | 2023-03-21 | 广东省农业科学院设施农业研究所 | SNP molecular marker closely linked with bitter gourd full female shape and application thereof |
| CN117106968A (en) * | 2023-10-19 | 2023-11-24 | 北京市农林科学院 | Primer group and kit for identifying female strength of cucumber and application of primer group and kit |
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