CN110120245A - Method that is a kind of while positioning multiple genes - Google Patents
Method that is a kind of while positioning multiple genes Download PDFInfo
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- CN110120245A CN110120245A CN201910395993.XA CN201910395993A CN110120245A CN 110120245 A CN110120245 A CN 110120245A CN 201910395993 A CN201910395993 A CN 201910395993A CN 110120245 A CN110120245 A CN 110120245A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention relates to a kind of methods for positioning multiple genes simultaneously, specifically construct BC1F1 group and F2 group, screen codominant polymorphic molecular marker, Genotyping is carried out to F2 group using molecular labeling, then the material of phase homogenic type is divided into one group, the phenotypic difference and for statistical analysis between different grouping is investigated, is verified using the F2 single plant offspring of heterozygous genotypes, it can be by a certain phenotypic character assignment of genes gene mapping near specific molecular labeling.The present invention can position quantitative character and qualitative character, and the group of building can position multiple genes, improve the utilization efficiency of group, and the large-scale assignment of genes gene mapping may be implemented in conjunction with phenomics correlation means.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a method of multiple genes are positioned simultaneously.
Background technique
The assignment of genes gene mapping is exactly the position of determining gene on chromosome.In early stage, two o'clock test and three point test are determining
The main method of distance and sequence between gene, indicates the distance between gene with cross-over value.It is non-whole according to chromosome later
Times body, missing and transposition etc. develop the chromosome mapping of gene, utilize the cytological markers structure such as the caryogram of chromosome and banding pattern
Build genetic map.After DNA molecular marker invents, due to its have the advantages that it is a variety of, in genetic map construction, the assignment of genes gene mapping
It is used widely in equal fields.DNA molecular marker can be divided into dominant marker and two kinds of codominant marker, and codominant marker being capable of area
Divide homozygous and heterozygous genotypes, is a kind of more ideal molecular labeling.
Utilize near isogenic lines (Near Isogenic Lines, NIL) and separation of group analytic approach (Bulked
Segregation Analysis, BSA) assignment of genes gene mapping is carried out, it is two kinds of effective ways of gene location.Utilize near isogenic lines
There are many conveniences, but constructing near isogenic lines is a time-consuming and laborious job.Separation of group analytic approach both can be used pure
Close individual segregating population, also can be used heterozygote segregating population carry out the assignment of genes gene mapping, purposes more extensively, economical and effective.
High throughput sequencing technologies are quickly grown in recent years, the method for occurring carrying out the assignment of genes gene mapping using sequencing technologies, by directly comparing
Mutational site is identified compared with mutant and wild type gene group, positioning accuracy is higher, simultaneously because recombination event is not depended on, for
The very low region of recombination frequency is still effective.The above method has played important function in the assignment of genes gene mapping, but every building one
Group can only position a gene, and group's utilization rate is low, higher cost.
Summary of the invention
In view of the above-mentioned problems, utilizing molecule the purpose of the present invention is to provide a kind of method for positioning multiple genes simultaneously
Auxiliary gene parting is marked, the material of phase homogenic type is then divided into one group, the phenotypic difference between investigation different grouping is simultaneously
It is for statistical analysis, it can be positioned simultaneously multiple by a certain phenotypic character assignment of genes gene mapping near specific molecular labeling
Gene, and be easily verified that.
The present invention provides a kind of methods for positioning multiple genes simultaneously, include the following steps:
(1) building group:
Two homozygous inbred lines parent P1 and P2 with a variety of phenotypic differences are chosen, are by nonrecurrent parent, P1 of P2
Recurrent parent is hybridized, and hybridization F1 is obtained, and is continued first backcross generation and obtained BC1F1 group, while F1 selfing obtains F2 group.
(2) molecular labeling auxiliary gene parting is utilized:
A whole set of equally distributed molecular labeling is chosen, using any one BC1F1 single plant and any one F2 single plant as sample
This, screens codominant polymorphism mark.If screening codominance polymorphic marker, illustrate F1 near this marker site
Gene be heterozygosis, will be separated in Offspring F2.F2 group is planted, the codominance polymorphic marker screened is used
Genotyping is carried out to all F2 single plants.
(3) grouping plants and investigates character:
Together by the identical F2 single-strain planting of marker genetype, the phenotypic character of each genotype of probe, with
The plant of the genotype of a certain site homozygous dominant, other site isolations is one group, with corresponding site homozygous recessive, other sites
The plant of isolated genotype is another group, for statistical analysis to each phenotypic character, determines that the character with significant difference is made
For candidate objective trait, then this gene loci can control these candidate traits or participate in associated regulatory process.
(4) it verifies:
Candidate objective trait may have multiple, need to verify using the F2 single plant offspring of heterozygous genotypes.Make spy
The F2 individual plant selfing of anchor point heterozygosis, the genotype of other sites homozygosis obtains F3 group, with the molecular labeling pair of specific site
F3 group carries out Genotyping, and by genotype grouping plantation, investigate candidate trait whether there is significant difference between grouping,
If there is significant difference, then it can be determined that the objective trait of specific site, so that objective trait is located in specific point
Near son label.
In method that is above-mentioned while positioning multiple genes, in the step (2), the molecular labeling is co-dominant molecular
Label；Preferably, the molecular labeling is SSR marker, InDel label or SNP marker.
In method that is above-mentioned while positioning multiple genes, the size of real work amount is considered, in the step (2), choosing
The codominance polymorphic marker number of sites taken is proper with 2~5；Optionally, if in conjunction with phenomics means, not by altogether
Dominant polymorphic marker number of sites limitation.
In method that is above-mentioned while positioning multiple genes, for Control experiment error, in the step (3), grouping kind
When plant, 3 plants or 3 plants of the F2 single-strain planting of every kind of genotype or more.
In method that is above-mentioned while positioning multiple genes, in the step (3), the statistical analysis technique includes t inspection
It tests, variance analysis etc..
In method that is above-mentioned while positioning multiple genes, the step (4) can also substitute using the following method: make specific
Site heterozygosis, the F2 single plant of the genotype of other sites homozygosis and recessive homozygous, other sites homozygosis the F2 single plant of specific site
Test cross, obtains F3 group, and the segregation ratio whether investigation candidate trait meets 1:1 in F3 group can determine if met
It is the objective trait of specific site；Genotyping is carried out to F3 group with the molecular labeling of specific site, obtains each single plant
Candidate trait and marker genetype, and according to the genetic distance between recombination single plant quantity survey gene loci and molecular labeling.
Technical solution of the present invention bring beneficial effect includes:
(1) method that is provided by the invention while positioning multiple genes need to only carry out a character tune after planting F2
It looks into, is then grouped by genotype for statistical analysis, that is, can determine the objective trait of multiple gene locis, the group of building can be with
Multiple genes are positioned, the utilization efficiency of group is improved.
(2) method that is provided by the invention while positioning multiple genes, using on the obtained BC1F1 group theory of backcrossing with
F2 group only exists 25% DNA sequence dna difference, largely reduces the complexity of genome, to reduce non-total
The phenotype interference that dominant marker site generates.
(3) method that is provided by the invention while positioning multiple genes, in addition to being suitable for quantitative character, to qualitative character position
Point is still effective, carries out segregation ratio analysis to the candidate qualitative character of test cross offspring, can verify objective trait, and can basis
Recombinant type single plant quantity determines the genetic distance between target gene and label.
(4) method that is provided by the invention while positioning multiple genes, can be in conjunction with phenomics correlation means, and building is big
The group of scale carries out objective trait screening to more sites, realizes the large-scale assignment of genes gene mapping.
Detailed description of the invention
Fig. 1 is the principle of molecular labeling auxiliary gene parting.
Fig. 2 is the present invention while the Technology Roadmap for positioning multiple genes.
Fig. 3 is the phenotype comparison diagram of new No. 17 of pimento with elephant skin bubble green pepper mutant 242.
Fig. 4 is the amplification of molecular labeling HpmsE016, ge236-708pmc0432C and HpmsE031 in the F2 single plant of part
Situation.
Fig. 5 is the present invention while the F2 group field planting figure for positioning 3 genes.
Specific embodiment
It elaborates below in conjunction with attached drawing to a specific embodiment of the invention, but is not limited to model of the invention
It encloses, made any replacement, improvement and modification should all be included in the protection scope of the present invention on this basis.
Unless otherwise specified, the conventional hybridization skill that hybridization technique used in embodiment is well known to those skilled in the art
Art.
In method that is provided by the invention while positioning multiple genes, the principle of molecular labeling auxiliary gene parting is as schemed
Shown in 1:
DNA molecular marker can be divided into dominant marker and two kinds of codominant marker, when screening polymorphism mark, polymorphic position
The bands of a spectrum of point are likely to occur situation as shown in Figure 1.For individual molecule marker site, if P1 is identical as P2 banding pattern,
The bands of a spectrum then marked are as shown in M1, no matter type is dominant or codominance, using BC1F1 and F2 as screening sample molecule mark
Note, label display is without polymorphism；If P1 is different from P2 banding pattern and type is dominant, the bands of a spectrum marked such as M2 and M3
Shown, using BC1F1 and F2 as screening sample molecular labeling, label display is without polymorphism or dominant banding pattern；If P1 and P2 banding pattern
Different and type is codominance, then the bands of a spectrum marked are as shown in M4, and using BC1F1 and F2 as sample, label may show nothing
Polymorphism, dominant banding pattern or codominance banding pattern, and the probability that codominance banding pattern occurs is 1/2 × 1/4=1/8.It can be seen that
Only when P1 and P2 is different (i.e. F1 is heterozygosis in this site) in the banding pattern of a certain marker site, with BC1F1 and F2
It is likely to codominance banding pattern occur for screening sample molecular labeling.When using BC1F1 and F2 as screening sample polymorphism mark, such as
There is dominant marker type in some label of fruit, then counter can release, F1 is heterozygosis in this marker site, and F2 is in this marker bit
Point can separate, and carry out band analysis to F2 group with this molecular labeling, then can distinguish homozygous individual and heterozygous individual,
To complete the genotyping in this site.
The technology path of method that is provided by the invention while positioning multiple genes is as shown in Figure 2.
Embodiment 1
Method that is a kind of while positioning multiple genes, includes the following steps:
(1) building group:
It is female parent with the elephant skin bubble green pepper mutant 242 of an acaulescence hair, it is miscellaneous for male parent progress with Sweet Pepper Varieties new No. 17
It hands over, obtains F1, and continue to obtain BC1F1 group with new No. 17 backcrossings, while F1 selfing obtains F2 group.
New No. 17 of pimento and the phenotype of elephant skin bubble green pepper mutant 242 are shown in Fig. 3.
(2) molecular labeling auxiliary gene parting is utilized:
The molecular labeling in capsicum KL-DH genetic map is chosen, document " Development of simple is specifically shown in
sequence repeat markers and construction of a high-density linkage map of
Capsicum annuum " arbitrarily selects a BC1F1 single plant B-7 and any one F2 single plant F2-6, using B-7 and F2-6 as sample
This, screens codominant polymorphism mark.As a result 3 codominant SSR polymorphism marks: HpmsE016, ge236- are filtered out
708pmc0432C and HpmsE031.200 plants of F2 group is planted, in seedling stage using the codominance polymorphic marker pair for screening 3
All F2 single plants carry out genotyping, and the analysis result of plant part is as shown in Figure 4.
Assuming that label HpmsE016, ge236-708pmc0432C and HpmsE031 respectively with Gene A, gene B and gene C
It is chain, then will appear 27 kinds of genotype in F2 group: AABBCC, AABbCC, AAbbCC, AaBBCC, AaBbCC, AabbCC,
AaBBCC, aaBbCC, aabbCC, AABBCc, AABbCc, AAbbCc, AaBBCc, AaBbCc, AabbCc, aaBBCc, aaBbCc,
AabbCc, AABBcc, AABbcc, AAbbcc, AaBBcc, AaBbcc, Aabbcc, aaBBcc, aaBbcc, aabbcc.Such as Fig. 4 institute
Showing, the marker genetype of the F2 plant marked as 1 is AAbbCc, and the marker genetype of the F2 plant marked as 2 is AaBBcc, with
This analogizes, and can obtain the marker genetype of each F2 single plant.
The primer sequence of above-mentioned codominant polymorphic molecular marker is shown in Table 1.
The primer sequence of table 1, the codominance polymorphic molecular marker filtered out
| Mark title | Forward primer sequence (5 ' -3 ') | Reverse primer sequences (5 ' -3 ') |
| HpmsE016 | CCAAGTTCAGGCCCAGGAGTAA | TGCAGAGAAGACTCACCAGTCC |
| ge236-708pmc0432C | ATCTTACTCCGACGTCTGCTTCAC | GTTTGGAGAAGCATGGGCAAAGTGATAC |
| HpmsE031 | CCCTAAATCAACCCCAAATTCAA | CCCCCATTACCTGACTGCAAAA |
(3) grouping plants and investigates character:
According to the genotypic results of F2 group, the genotype AaBbCc that 3 genes are all heterozygosis is removed, other 26 kinds of bases
Because the single plant of type carries out field planting according to Fig. 5, every kind of genotype plants 3 plants, plants 78 plants altogether.According to " hot pepper germ plasm resource
Quality control specifications " each plant of investigation phenotypic character.As shown in table 2, with frequency of genotypes AA _ _ _ _ plant be one group, gene
The plant of type aa____ is one group, carries out variance analysis to two groups of phenotypic characters, two groups long thick with pulp in fruit as the result is shown
On there are significant differences；Similarly with the plant of genotype _ _ BB__ and _ _ bb__ for two groups of samples, the results of analysis of variance shows two
There are significant differences on strain width for group；With the plant of genotype _ _ _ _ CC and _ _ _ _ cc for two groups of samples, phenotypic evaluation shows stem
Hair only occurs in _ _ _ _ CC, the results of analysis of variance show two groups with there are significant differences on single plant yield.
Table 2, F2 group phenotypic character and fractional analysis
Note: numerical value is the average value for counting single plant in table；* indicate that there are significant differences in 0.05 level for packet data.
(4) it verifies:
It is selfed the F2 single plant F2-4 of genotype Aabbcc, I group of F3- is obtained, uses HpmsE016 pairs of SSR marker
I group's single plant of F3- carries out genotyping, then will appear 3 kinds of genotype: AA, Aa and aa.According to genotypic results, respectively
Fruit length and the fruit of each plant are investigated in 30 plants of AA and aa genotype of plantation according to " hot pepper germ plasm resource quality control specifications "
Meat is thick, and pulp thickness long to the fruit of two kinds of genotypes carries out variance analysis, the results are shown in Table 3.Two kinds of genotype as the result is shown
Plant is not significantly different on fruit is long, but there are significant differences in pulp thickness, then can determine that pulp thickness is
The objective trait in the site HpmsE016, so that objective trait is located near label HpmsE016.Capsicum fruit will temporarily be controlled
The unnamed gene of meat thickness is Ppt1 (Pepper pulp thickness 1), then Ppt1 Primary Location is attached in label HpmsE016
Closely.
Table 3, I group of F3- phenotypic character and Gene A fractional analysis
Note: numerical value is the average value for counting single plant in table；* indicate that there are significant differences in 0.05 level for packet data.
It is selfed the F2 single plant F2-7 of genotype AABbcc, II group of F3- is obtained, uses SSR marker ge236-
708pmc0432C carries out genotyping to II group's single plant of F3-, it may appear that 3 kinds of genotype: BB, Bb and bb.According to gene point
Type as a result, plant 30 plants of BB and bb genotype, according to each plant of " hot pepper germ plasm resource quality control specifications " investigation respectively
The strain width of strain carries out variance analysis to the strain width of two kinds of genotypes, the results are shown in Table 4.Two kinds of genotypes as the result is shown
It is not significantly different on strain width, then gene B is not the gene for controlling strain width, may be in label ge236-708pmc0432C
Neighbouring gene not phene, the difference showed on strain width is as caused by test error, so in label ge236-
708pmc0432C does not navigate to phenotypic character gene nearby.
Table 4, the phenotypic character of II group of F3- and gene B fractional analysis
Note: numerical value is the average value for counting single plant in table.
Make the F2 single plant F2-14 of genotype AAbbCc and F2-8 single plant test cross that genotype is AAbbcc, obtains F3- III
Group carries out genotyping to F3-3 group single plant using label HpmsE031, will appear 2 kinds of genes in III group of F3-
Type: CC and cc.It is grouped and plants by genotype, every kind of genotype plants 100 plants, and whether there is or not produce with single plant for the stem hair of every plant of investigation
Amount, whether the segregation ratio of analysis stem hair meets the ratio of 1:1, and carries out variance analysis to single plant yield, and the results are shown in Table 5.
By Chi-square test, the segregation ratio of stem hair meets the ratio (χ of 1:12 0.05It is 0.5, is less than critical value 3.84), and two kinds of genotype
Plant is not significantly different on single plant yield, then can determine gene C be control caulis capsici hair whether there is or not gene, thus by it
It is located near label HpmsE031.Temporarily by control caulis capsici hair whether there is or not unnamed gene be Psp1 (Pepper stem
Pilose 1), it is [(5+10)/(100+ according to the exchange rate that the quantity of recombinant type single plant calculates Psp1 and marks HpmsE031
100)] × 100%=7.5%, so the genetic distance of Psp1 and label HpmsE031 are 7.5cM.
Table 5, III group of F3- phenotypic character and gene C fractional analysis
Note: numerical value is the average value for counting single plant in table.
In summary as a result, eventually by verifying, the gene Ppt1 Primary Location for controlling capsicum pulp thickness is being marked
Near HpmsE016, by control caulis capsici hair whether there is or not gene Psp1 be located in distance label HpmsE031 about 7.5cM position.
Claims (6)
1. a kind of method for positioning multiple genes simultaneously, includes the following steps:
(1) building group:
Two homozygous inbred lines parent P1 and P2 with a variety of phenotypic differences are chosen, are circulation by nonrecurrent parent, P1 of P2
Parent hybridizes, and obtains hybridization F1, and continues first backcross generation and obtain BC1F1 group, while F1 selfing obtains F2 group；
(2) molecular labeling auxiliary gene parting is utilized:
A whole set of equally distributed molecular labeling is chosen, using any one BC1F1 single plant and any one F2 single plant as sample, sieve
Select codominant polymorphism mark；F2 group is planted, all F2 single plants are carried out using the codominance polymorphic marker screened
Genotyping；
(3) grouping plants and investigates character:
Together by the identical F2 single-strain planting of marker genetype, the phenotypic character of each genotype is investigated, with a certain site
The plant of the genotype of homozygous dominant, other site isolations is one group, with corresponding site homozygous recessive, the base of other site isolations
Because the plant of type is another group, for statistical analysis to each phenotypic character, determining has the character of significant difference as candidate's
Objective trait；
(4) it verifies:
Make the F2 individual plant selfing of specific site heterozygosis, the genotype of other sites homozygosis, obtain F3 group, with point of specific site
Son label carries out Genotyping to F3 group, and by genotype grouping plantation, investigation candidate trait whether there is between grouping
Significant difference then can be determined that the objective trait of specific site if there is significant difference, so that objective trait is located in
Near specific molecular labeling.
2. method that is according to claim 1 while positioning multiple genes, which is characterized in that described in the step (2)
Molecular labeling is codominant marker；Preferably, the molecular labeling is SSR marker, InDel label or SNP marker.
3. method that is according to claim 1 while positioning multiple genes, which is characterized in that in the step (2), choose
Codominance polymorphic marker number of sites be 2~5；Optionally, if in conjunction with phenomics means, not by the polymorphic mark of codominance
Remember number of sites limitation.
4. method that is according to claim 1 while positioning multiple genes, which is characterized in that in the step (3), grouping
When plantation, 3 plants or 3 plants of the F2 single-strain planting of every kind of genotype or more.
5. method that is according to claim 1 while positioning multiple genes, which is characterized in that described in the step (3)
Statistical analysis technique includes t inspection, variance analysis etc..
6. method that is according to claim 1 while positioning multiple genes, which is characterized in that the step (4) can be with
Using the following method substitute: make specific site heterozygosis, the F2 single plant of the genotype of other sites homozygosis and specific site recessiveness homozygosis,
The F2 single plant test cross of other sites homozygosis, obtains F3 group, and whether investigation candidate trait meets the separation of 1:1 in F3 group
Than can be determined that the objective trait of specific site if met；Base is carried out to F3 group with the molecular labeling of specific site
Because of parting, the candidate trait and marker genetype of each single plant are obtained, and according to recombination single plant quantity survey gene loci and is divided
Genetic distance between son label.
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