CN106577267B - The gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection - Google Patents
The gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection Download PDFInfo
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- CN106577267B CN106577267B CN201611213375.1A CN201611213375A CN106577267B CN 106577267 B CN106577267 B CN 106577267B CN 201611213375 A CN201611213375 A CN 201611213375A CN 106577267 B CN106577267 B CN 106577267B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
The invention discloses a kind of gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection, step is：(1) a preparing experiment corn inbred line sample, including control male parent sample and maternal sample；(2) mass spectrum of prepared sample is scanned；(3) mass spectrum is compared and analyzed according to existing database；(4) obtained sample metabolic components are subjected to assignment, obtain experiment corn inbred line sample metabolic components matrix；(5) shot chart of experiment corn inbred line sample metabolic components matrix is obtained；(6) it screens and determines and compare sample similar in male parent sample and maternal sample；(7) apolegamy is combined to identified similar sample.The present invention overcomes the non-directiveness that traditional breeding way selects, basis is provided for the selection of parent in follow-up breeding process, this method is simple, science, screening efficiency are high, in high volume, quickly can go out the combination of target corn inbred line in early screening, save a large amount of manpower, material resources cost.
Description
Technical field
The invention belongs to crop seeds selection and breeding fields, join more particularly to a kind of makings of corn inbred line combination selection
Use rapid screening method.
Background technology
Corn is one of three big crop of the world, and seed demand is huge.By crop breeding Crop Improvement hereditary capacity,
It is to improve crop yield, quality, one of the effective way promoted economic development and improved People's livelihood to cultivate fine quality.Current educates
Kind mode includes mainly the methods of crossbreeding, back cross breeding, mostly uses pedigree method to select excellent generation and heterosis, hybrid vigor strong
Parent, spent time is long, and since in field trial, experimental condition is harsh, is highly prone to the influence of environmental condition,
And due to the non-directiveness of traditional breeding way selection, cause the quantities such as collection seed great, extremely wasting manpower and material resources
Resource.Therefore, development one kind can high-volume, in real time, quickly can early screening go out target corn inbred line combination be used for
The analysis method of breeding is very important.
Low-polarity components not only have the advantages that high-throughput, analyze speed is fast, can analyze many kinds of substance simultaneously, but also
Abundant structure of matter information can be provided, not only can be qualitative according to database retrieval, it can be achieved that the identification of component and structure, but also can
It is quantitative.Therefore, which is commonly used for the research in terms of plant aspect metabolism group, is mainly used for amino in Metabolite components
Acid, the detection of the small-molecule substance of the effumabilities such as polysaccharide.But low-polarity components are applied to corn inbred line combination choosing
It is reported with yet there are no.
Invention content
For the deficiencies in the prior art, it is fast that the present invention provides a kind of gas chromatography mass spectrometry of corn inbred line combination selection
Fast screening technique, the non-directiveness for overcoming traditional breeding way to select, the selection for parent in follow-up breeding process provide basis.
This method is simple, science, screening efficiency are high, in high volume, quickly can go out the combination of target corn inbred line, section in early screening
About a large amount of manpower, material resources cost.
The present invention is achieved by the following technical solutions：
A kind of gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection, step are：
(1) multiple corn inbred line samples of preparative gas chromatography-mass spectrometer detection, including control male parent sample
A and maternal sample B；
(2) by the mass spectrum of sample prepared by gas chromatograph-mass spectrometer (GC-MS) scanning step (1), experiment corn is obtained
The mass spectrum of self-mating system sample；
(3) mass spectrum obtained to step (2) according to existing database compares and analyzes, and obtains experiment corn inbred line
The metabolic components of sample；
(4) the main metabolic component for the corn inbred line sample for obtaining step (3) carries out assignment, contains certain metabolism group
Divide and be assigned a value of " 1 ", otherwise be assigned a value of " 0 ", obtains the matrix of the main metabolic component of experiment corn inbred line sample；
(5) it uses Principal Component Analysis to analyze the experiment matrix of corn inbred line sample main metabolic component, obtains reality
It tests and is metabolized key component matrix shot chart with corn inbred line sample；
(6) the experiment corn inbred line sample main metabolic group sub-matrix shot chart obtained according to step (5), screening is simultaneously
It determines and compares sample similar in male parent sample A and maternal sample B；
(7) to determined by step (6) with compare sample similar in male parent sample A and maternal sample B and be combined apolegamy.
Wherein, chromatographiccondition is in the step (2)：Chromatographic column：HP-5MS(30m×250μm×0.25μm)；Into
Sample mouth temperature：250℃；Carrier gas：High-purity helium (99.999%)；Flow velocity：1.0mL/min；Splitless injecting samples；Temperature program is：
60 DEG C of reservation 1min, are warming up to 270 DEG C with the speed of 10 DEG C/min, retain 10min；1 μ L of sample size；Mass spectral analysis condition is：
EI energy：70Ev, transmission line temperature：250℃；Level four bars temperature：150℃；Ion source temperature：230℃；Acquisition mode：It sweeps entirely
Retouch (SCAN) pattern, scanning range：50~500 (m/z), sweep speed：2scan/s,；Solvent delay：4.5min；Tuning side
Formula：Automatic tuning.
Advantages of the present invention and advantageous effect are：
(1) the gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection of the invention, relative to traditional breeding
Process in high volume, quickly can go out the combination of target corn inbred line in early screening.
(2) the gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection of the invention, relative to traditional breeding
Process can save a large amount of human and material resources cost.
(3) method of the invention is simple, science, screening efficiency are high, extends to the self-mating system combination selection of other crops
In.
Description of the drawings
Fig. 1 is the principal component scores figure of 14 corn inbred line sample metabolic components matrixes in the embodiment of the present invention；
Fig. 2 is the principal component load diagram of 14 corn inbred line sample metabolic components matrixes in the embodiment of the present invention.
Specific implementation mode
The present invention is described in further detail by following embodiment.It should be noted that：Following embodiments are illustrative, are not
It is limited, protection scope of the present invention cannot be limited with following embodiments.
A kind of gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection, step are：
(1) 14 corn inbred line samples (serial number No.1-No.14) of preparative gas chromatography-mass spectrometer detection,
Including compareing male parent sample No.4 and maternal sample No.7；
(2) it by the mass spectrum of 14 corn inbred line samples of gas chromatograph-mass spectrometer (GC-MS) scanning step (1), obtains
The mass spectrum of experiment corn inbred line sample；
(3) 14 corn inbred line sample mass spectrums that step (2) obtains are compared and analyzed according to existing database, is obtained
To the experiment metabolic components of corn inbred line sample；
(4) 14 corn inbred line sample metabolic components for obtaining step (3) carry out assignment, contain certain metabolic components
It is assigned a value of " 1 ", otherwise is assigned a value of " 0 ", obtains experiment corn inbred line sample metabolic components matrix；
(5) the experiment corn inbred line sample metabolic components matrix for using Principal Component Analysis analytical procedure (4) to obtain,
Obtain the shot chart of experiment corn inbred line sample metabolic components matrix；
(6) the experiment corn inbred line sample metabolic components matrix shot chart obtained according to step (5) is screened and is determined
With compare sample similar in male parent sample No.4 and maternal sample No.7；
(7) to determined by step (7) with compare sample similar in male parent sample No.4 and maternal sample No.7 and carry out group
Close apolegamy.
14 corn inbred line samples provide for Hainan seeds company in the present embodiment, and each jade is ground under liquid nitrogen environment
Rice self-mating system sample, and 80 mesh sieve is crossed, pack standby survey.
Mass spectrum acquisition uses the gas chromatograph-mass spectrometer (GC-MS) (GC7890A- of Aglient companies of the U.S. in the present invention
MSD5975C).To the sample after above-mentioned sieving, 150mg/ parts are weighed respectively, and goes to 5mL glass centrifuge tubes, and 1mL extractions are added
Agent is (according to volume ratio, acetonitrile：B propanol：Water=1:1:1), under the conditions of 4 DEG C and 40KHZ, ultrasonic extraction 35min；It has extracted
Supernatant is transferred in 2mL centrifuge tubes by Cheng Hou, under 4 DEG C and 10000rpm of rotating speed, centrifuges 1min；Take 100 microlitres of supernatants
For liquid to sample introduction bottle, nitrogen drying (uses ice water hybrid mode, control temperature to be no more than 10 DEG C)；Again respectively through persalt methoxy
Amine and trimethyl N-methyl trifluoroacetamide (MSTFA) derivatization treatment twice, and solubilizer normal heptane (includes 20 micrograms/ml
Internal standard hendecane) constant volume is to 1mL；It is filtered finally by 0.22 μm of mocromembrane, is transferred to GC sample introduction bottles, examination with computer obtains 14
The mass spectrum of a corn inbred line sample.
It establishes on the basis of existing database, to the matter of the 14 corn inbred line samples acquired under these conditions
Spectrum
It compares and analyzes, finds out 128 kinds of metabolism small molecule components altogether, wherein including mainly carbohydrate (melibiose, grape
Sugar, fructose etc.), fats (oleic acid, linoleic acid and hard fatty acids etc.) and amino acids (Valine, l-Alanine, L- junket ammonia
Acid etc.).
Confirm each corn inbred line be metabolized small molecule component on the basis of, according to metabolism small molecule component with and without
Carry out qualitative analysis, i.e.,：1 is assigned a value of to the sample containing certain metabolic components, otherwise is 0, if metabolic components are phenol,
Sample No.1 metabolic components include phenol, then are assigned a value of 1, and reactive phenol in sample No.2 metabolic components, then are assigned a value of 0, pass through
The above method can be obtained 14 corn inbred line sample metabolic components matrixes and be shown in Table 1.
Table 1
Principal component analysis is carried out to 14 corn inbred line sample metabolic components matrixes.PC1 and PC2 points of the first two principal component
Do not explain the 23.76% and 11.27% of total variable, Fig. 1 be 14 corn inbred line sample metabolic components matrix the first two it is main at
Divide the shot chart of PC1 and PC2.From figure 1 it appears that 14 corn inbred lines are divided into two major classes, one kind is located at the areas Fu PC1
Domain, it is in addition a kind of to be located at the positive regions PC1.It compares male parent sample No.4 and is located at the negative regions PC1, No.1, No.13 and No.3 samples
Product all in its vicinity, can be initially believed that these samples can be as new male parent；And maternal sample No.7 is compareed positioned at positive
The regions PC1, and the sample away from nearest neighbours is：No.6, No.8 and No.9 can be initially believed that these samples can be as new
It is maternal.It is selected with compare male parent sample No.4 similar sample No.1, No.13 and No.3, can with compare maternal sample
It is mutually assembled between No.7 similar sample No.6, No.8 and No.9.
In order to prove the correctness of the method for the invention the selection result, we are further confirmed by principal component load diagram
Molecular marked compound, and quantitative analysis is carried out to it.Fig. 2 give 14 corn inbred line sample metabolic components matrixes it is main at
Separate loading figure, the bigger metabolic components of load value are bigger to the screening contribution of corn inbred line sample, and the component is as potential
Molecular marked compound.Figure it is seen that larger to the screening contribution of corn inbred line sample has phenol, DL-malic acid, L-
The metabolic components such as valine, l-Alanine, stearic acid and PEARLITOL 25C, these components are potential molecular marked compound.
On the basis of above-mentioned determination potential molecular marked compound, small molecule metabolites content is analyzed using inner mark method ration,
The result is shown in tables 2.No.1, No.13, No.3 sample all contain phenol metabolic components with compareing in male parent sample No.4, do not contain
DL-malic acid metabolic components；And No.6, No.8, No.9 contain DL-malic acid metabolism group with compareing in maternal sample No.7
Point, do not contain phenol (content 0).This just illustrates No.1, No.13, No.3 sample with to compare male parent sample No.4 similar, can be with
As new male parent；And No.6, No.8, No.9 with to compare maternal sample No.7 similar, can be as new female parent.The above results
Further demonstrate the correctness that method through the invention carries out screening acquired results.
Table 2
It is above-mentioned with reference to embodiment to the detailed description of the gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection, be
It is illustrative without being restrictive, it should explanation, in the case where not departing from core of the invention, any simple change
Shape, modification or other skilled in the art can not spend the equivalent replacement of creative work to each fall within protection of the invention
Range.
Claims (3)
1. a kind of gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection, includes the following steps：
(1) multiple corn inbred line samples of preparative gas chromatography-mass spectrometer detection, including control male parent sample A and
Maternal sample B；
(2) it by the mass spectrum of sample prepared by gas chromatograph-mass spectrometer (GC-MS) scanning step (1), obtains testing and be selfed with corn
It is the mass spectrum of sample；
(3) mass spectrum obtained to step (2) according to existing database compares and analyzes, and obtains experiment corn inbred line sample
Metabolic components；
(4) the main metabolic component for the corn inbred line sample for obtaining step (3) carries out assignment, is assigned containing certain metabolic components
Value is " 1 ", otherwise is assigned a value of " 0 ", obtains the matrix of experiment corn inbred line sample main metabolic component；
(5) matrix for using Principal Component Analysis analysis experiment corn inbred line sample main metabolic component obtains experiment and uses
Corn inbred line sample main metabolic group sub-matrix shot chart；
(6) the experiment corn inbred line sample main metabolic group sub-matrix shot chart obtained according to step (5) is screened and is determined
With compare sample similar in male parent sample A and maternal sample B；
(7) to determined by step (6) with compare sample similar in male parent sample A and maternal sample B and be combined apolegamy.
2. gas chromatography mass spectrometry rapid screening method according to claim 1, it is characterised in that：Chromatography point in above-mentioned steps (2)
Analysis condition is：Chromatographic column：HP-5MS(30m×250μm×0.25μm)；Injector temperature：250℃；Carrier gas：High-purity helium
(99.999%)；Flow velocity：1.0mL/min；Splitless injecting samples；Temperature program is：60 DEG C of reservation 1min, with the speed of 10 DEG C/min
270 DEG C are warming up to, 10min is retained；1 μ L of sample size.
3. gas chromatography mass spectrometry rapid screening method according to claim 1, it is characterised in that：Mass spectrum point in above-mentioned steps (2)
Analysis condition is：EI energy：70Ev, transmission line temperature：250℃；Level four bars temperature：150℃；Ion source temperature：230℃；Acquisition
Mode：Full scan pattern, scanning range：50~500 (m/z), sweep speed：2scan/s,；Solvent delay：4.5min；Tuning
Mode：Automatic tuning.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331474A (en) * | 2011-06-20 | 2012-01-25 | 天津大学 | Method for seeking quality control index of raw material corn steep liquor for fermentation |
| CN102778436A (en) * | 2011-12-20 | 2012-11-14 | 金华市农业科学研究院 | Method for breeding freshly eaten corn with eye health-care effect |
| CN103020864A (en) * | 2012-12-07 | 2013-04-03 | 南通大学 | Corn fine breed breeding method |
| CN103404430A (en) * | 2013-08-16 | 2013-11-27 | 广西壮族自治区农业科学院玉米研究所 | Method for breeding high-pro-vitamin A content maize inbred lines |
| CN106153800A (en) * | 2016-06-22 | 2016-11-23 | 中国农业科学院生物技术研究所 | The HPLC MS/MS detection method of transgenic corns secondary metabolites |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102331474A (en) * | 2011-06-20 | 2012-01-25 | 天津大学 | Method for seeking quality control index of raw material corn steep liquor for fermentation |
| CN102778436A (en) * | 2011-12-20 | 2012-11-14 | 金华市农业科学研究院 | Method for breeding freshly eaten corn with eye health-care effect |
| CN103020864A (en) * | 2012-12-07 | 2013-04-03 | 南通大学 | Corn fine breed breeding method |
| CN103404430A (en) * | 2013-08-16 | 2013-11-27 | 广西壮族自治区农业科学院玉米研究所 | Method for breeding high-pro-vitamin A content maize inbred lines |
| CN106153800A (en) * | 2016-06-22 | 2016-11-23 | 中国农业科学院生物技术研究所 | The HPLC MS/MS detection method of transgenic corns secondary metabolites |
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