CN108034752A - InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages - Google Patents
InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages Download PDFInfo
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Abstract
The invention discloses the InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages.InDel molecular labelings and epistatic gene P genetic distances be 0.5cM, in InDel of the male parent nucleotide sequence than special insertion/deletion fragment at female parent 50bp and 35bp more totally 2.The present invention marks the InDel of eggplant fruit color epistatic gene P exploitation close linkages, lays a good foundation for the finely positioning and clone of eggplant fruit color epistatic gene；5513 molecular labelings of InDel and molecular labeling amplimer can it is easy, quick, be applied to high throughput in eggplant fruit color Breeding Practice, accelerate eggplant fruit color character improvement process.
Description
Technical field
The invention belongs to biology field, more particularly to molecular genetics field, and in particular to on eggplant fruit color
The InDel molecular labelings of position property gene P close linkages and application.
Background technology
The fruit color of eggplant directly affects the condition of eggplant, is the important exterior of commodity quality of fruit.The fruit color of eggplant enriches,
Have purple black, purplish red, green and white etc..There are different preferences in different regions since consumption habit is different to the fruit color of eggplant.
Therefore, eggplant fruit color is important breeding objective.
Eggplant fruit color heredity is the complex character of controlled by multiple genes.Pang Wenlong, Liu Fuzhong, Chen Yuhui, wait eggplant fruit colors
Genetic research [J] gardening journals of shape, 2008,35 (7):979-986., with the different eggplant cultigen self-mating system of 3 fruit colors
For test material, fruit color is classified by the method being combined with color difference meter and range estimation, passes through 6 generation of P1, P2, F1, B1, B2 and F2
For unified analysis method, study the genetic development of eggplant fruit color character the result shows that:The F2 fruit color segregating generations of 3 cross combination
In bimodal or unimodal partial velocities, the color heredity of display eggplant fruit is the quantitative character of controlled by multiple genes for colony；Eggplant fruit color character
Heredity meets two pairs of Additive-dominances-epistasis key-gene+Additive-dominance-epistasis polygenic model (E models)；Key-gene is lost
Power transmission is 35.5%~98.4%, and genetic force is higher；Multiple-factor inheritance power is relatively low, is 0~57.7%.
The chromosome mapping in relation to eggplant fruit color epistatic gene site and specifically what gene are still not clear at present,
The research of related molecular marker is not carried out yet.Therefore, carry out the molecule marking research of eggplant fruit color epistatic gene, can be eggplant
Sub- fruit color epistatic gene molecular marker assisted selection breeding and gene finely positioning clone lay the foundation.
The content of the invention
It is an object of the invention to provide a kind of and eggplant fruit color epistatic gene P close linkages InDel molecular labelings
And application.
The technical solution used in the present invention is：
Inventor is found that the eggplant genetic stocks controlled by epistatic gene in the practices of breeding, passes through the depth to the material
Enter research, obtain technical scheme.
Application of the InDel molecular labelings as eggplant fruit color selection markers, the InDel molecular labelings are to be with length
Special insertion/deletion fragment at the 2 of 50bp and 35bp, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the primer sets of recognizable InDel molecular labelings in eggplant fruit color screening reagent is prepared, wherein, InDel
Molecular labeling is as described above.
Further as above application is improved, and the sequence of primer sets is：
5513F1：5'-GTGTTACGAGGGTTGAAATGGAC-3'
5513R1：5'-ATTGGTAAAAGGAAGATTTGAGG-3'.
The insertion of InDel molecular labelings in a kind of method of eggplant fruit color screening, including detection No. 8 chromosomes of eggplant/lack
Mistake situation, eggplant fruit color is determined according to the insertion/deletion situation of InDel molecular labelings, wherein, InDel molecular labelings as above institute
State.
Use the insertion/deletion situation of InDel molecular labelings on PCR methods detection No. 8 chromosomes of eggplant.
The sequence of the primer sets used in PCR methods is：
5513F1：5'-GTGTTACGAGGGTTGAAATGGAC-3'
5513R1：5'-ATTGGTAAAAGGAAGATTTGAGG-3'.
If only having 207bp fragments in amplified production, which is pp；If in amplified production only
292bp fragments, then the plant genotype is PP；The then genotype for having two segments of 292bp and 207bp at the same time is Pp.
A kind of method for cultivating different fruit color eggplants, be included in No. 8 chromosomes of eggplant introduce the insertion of InDel molecular labelings/
Missing, wherein, InDel molecular labelings are as described above.
The beneficial effects of the invention are as follows：
1 epistatic gene P for controlling eggplant fruit color is positioned at the smaller association on No. 8 chromosomes of eggplant by the present invention
In region, and provide the molecular labeling InDel 5513 with eggplant fruit color epistatic gene P close linkages, the marking path mesh
The genetic distance for marking gene P is 0.5cM, can be directly used for eggplant fruit color epistatic gene P molecular mark systems
Establish.The molecular labeling and molecular labeling amplimer of the present invention can it is easy, quick, be applied to eggplant fruit color sorting with high throughput
Educate in practice, while be that clone eggplant fruit color epistatic gene P and its functional study are had laid a good foundation.
Brief description of the drawings
Fig. 1 is that female parent material Female fruit colors are white, and in whole breeding time, hypocotyl, vein, flower, sepal is all
The synthesis of anthocyanidin is not observed；Male parent Male fruits color is white, micro- purple, the general purple of vein of seedling stage hypocotyl, the micro- purple of pattern, F1Generation
Aubergine is all presented in material hypocotyl, vein, pattern and fruit color.
Fig. 2 marks the PCR amplification result in the green stem gingko color single plant of F2 segregating populations for InDel 5513：F2 generation separation
The green stem gingko color single plant single plant of colony (genotype has DDpp, Ddpp, ddpp)；M is 100bp DNAladder；Male parent amplification length
It is 207bp (genotype pp) to spend for 292bp (genotype PP), maternal amplification length；F1 generation single plant amplification banding pattern for 292bp and
Two bands (genotype Pp) of 207bp；1-22 is the green stem gingko color single plants of F2.
Fig. 3 marks the PCR amplification result in the purplish red stem gingko color single plant of F2 segregating populations for InDel 5513：Purplish red stem
Gingko color single plant (genotype has ddPP, ddPp)；M is 100bp DNAladder；1-22 is the purplish red stem gingko color single plants of F2.
Fig. 4 marks the PCR amplification result in the purplish red fruit color single plant of F2 segregating populations for InDel 5513：M is 100bp
DNA ladder；1-22 is single to exchange for purplish red fruit color single plant (genotype has DDPP, DDPp, DdPP and DdPp), band " * " for F2
Strain.
Embodiment
In existing bibliography (Tigchelaar E C, Janick J, Erichson H T.The genetics of
anthocyanin coloration in eggplant(Solanum melongena L.).Genetics,1968,60:
Control eggplant fruit color epistatic gene site is respectively defined as D genes and P genes in 475-491), this research is according to parent's material
Material phenotypic characteristic and genetic development continue to use the unnamed gene.
With reference to experiment, the present invention is further illustrated, but is not limited thereto.
Molecular biology experiment technology employed in following embodiments includes DNA extractions, PCR amplification, PAGE gels electricity
The experiment such as swimming, unless otherwise specified, usually conventionally operates, for details, reference can be made to《Molecular Cloning:A Laboratory guide》(the 3rd
Version) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitangs etc. are translated, and 2002, Beijing：Scientific publication
Society), or according to the condition proposed by manufacturer.
First, the structure of genetic group and genetic analysis
1st, material to be tested
Vegetable material：With Vegetables Inst., Guangdong Academy of Agricultural Sciences through more for 2 portions of gingkoes of stringent bagging selfed breeding
The eggplant purifying self-mating system of color is male parent (Male, genotype ddPP) and female parent (Female, genotype DDpp), is prepared miscellaneous
Hand over combination F1, F1Individual plant selfing obtains F2.Female parent material Female fruits color is white, and in whole breeding time, hypocotyl, leaf
Arteries and veins, flower, sepal all do not observe the synthesis of anthocyanidin；Male parent Male fruits color is white, micro- purple, the general purple of vein of seedling stage hypocotyl,
The micro- purple of pattern, F1Aubergine (Fig. 1) is all presented for material hypocotyl, vein, pattern and fruit color.F2Segregating generation is purplish red and gingko
The segregation ratio of color plant meets 9 through Chi-square Test:7.
2nd, genetic analysis
In F2It is divided into purplish red fruit color (having anthocyanidin) and gingko color (no anthocyanidin) for segregating population fruit color.486 single plants
The F of composition2In colony, purplish red fruit color single plant and gingko color single plant are respectively 259 plants and 227 plants, block square (X2) whether examine it
Meet 9：7 ratios, as a result X2=1.73 (P=0.19), less than X2 0.05=3.84 (df=1), so purplish red fruit color single plant is total
Meet 9 with gingko color single plant sum：7 ratios.Therefore, it specify that eggplant material fruit color heredity used in this research is by 2
Epistatic gene (the D and P) control of interaction.
2nd, eggplant fruit color epistatic gene positions
1st, prepared by parent and the purplish red fruit color ponds of F2 and gingko color pond DNA
2 white eggplant fathers, female parent respectively take 10 plants of seedling leafs, extract leaf DNA respectively, and every plant takes equivalent DNA to mix, and forms
Male parent pond and maternal pond；F2Segregating population, seedling stage single plant take blade, extract DNA respectively, and the phase of bearing fruit carries out fruit tone and looks into, then
100 plants of fruit colors are taken to form purplish red fruit color pond for purplish red single plant DNA mixed in equal amounts, it is white single plant DNA equivalent to take 100 plants of fruit colors
Mixing forms gingko color pond.
2nd, SLAF-seq combinations BSA analysis methods positioning eggplant fruit color epistatic gene
Early period is entrusted SLAF-seq technology of the Beijing Biomarker Technologies Co., Ltd. using its independent research in this research
Simplify gene order-checking (Sun et al., 2013), and it is upper to eggplant fruit color to combine cluster method for separating and analyzing (BSA)
Property gene is positioned.Utilize eggplant genome SME_r2.5.1 (http disclosed in September, 2014://
Eggplant.kazusa.or.jp) sequence information, by totally 231490 SNP are corresponded on genome on 1117 Scaffold
Corresponding Scaffold.Then the SNP site of low depth is filtered out, filters out that parent genotype is identical and the SNP of heterozygosis positions
Point, finally filters out the SNP site for not meeting segregation ratio, and 784 SNP sites are obtained.Be obtained by filtration 784 SNP are used
ED methods are associated analysis, calculate relating value, correlation threshold 0.822 is determined according to 99 percentiles.According to Chi-square Test and pass
Online is calculated as a result, 1 epistatic gene P for controlling eggplant fruit color is positioned on No. 8 chromosomes of eggplant genome.
3rd, molecular labeling InDel5513 is developed
Sequence is resurveyed based on father, female parent gene group as a result, finding InDel marks near associated region, have found 1
InDel is marked, it is in male parent than more insertions of 1 50 base in female parent.One group is devised according to above-mentioned InDel marks
For specific primer to (SEQ ID NO.3 and SEQ ID NO.4), it is 207bp's which can amplify length in female parent
Fragment (genotype pp), sequence are：GTGTTACGAGGGTTGAAATGGACAAGCTGTCAAGGAGGGCTCGATCTTACCTTAT
CAATCGTAATTATACCAAACCTCTACCAATTAACTACCATGAAGAAAATAAGAGTTAGAGAGAGAATTTTATTATGA
TATTACAGATGAAGGCAAAATTATAAAATCAAGAACAAAGTTGAATTATCAACCTCAAATCTTCCTTTTACCAAT
(SEQ ID NO.5)；The fragment (genotype PP) that length is 292bp can be amplified in male parent, sequence is：
There are two segment amplifications of 292bp and 207bp, genotype Pp at the same time in F1.Further analysis shows, Parent
Between there are InDel at 2, length is respectively 50bp and 35bp, and sequence is respectively：CTTACCTTATCAATCGTAATTGTACCAAAC
CTCTACCAGTTAACTACCAT (SEQ ID NO.1) and ACCGTCAAAATTTATCACACACTGTTATGATATTT (SEQ ID
NO.2)。
The nucleotide sequence of the primer pair is as follows：
5513F1：5'-GTGTTACGAGGGTTGAAATGGAC-3'(SEQ ID NO.3),
5513R1：5'-ATTGGTAAAAGGAAGATTTGAGG-3'(SEQ ID NO.4).
The application of molecular labeling InDel 5513
1st, prepared by F2 single plants DNA
With Vegetables Inst., Guangdong Academy of Agricultural Sciences through pure more for the eggplant of 2 parts of gingko colors of stringent bagging selfed breeding
Change self-mating system is male parent (Male, genotype ddPP) and maternal (Female, genotype DDpp), preparing hybrid combination F1, F1
Individual plant selfing obtains F2, F2Segregating population, totally 400 plants.Male parent, female parent, F2Single plant seedling stage takes blade, extracts DNA respectively, bears fruit
Phase carries out fruit tone and looks into.
2nd, the PCR amplifications in male parent, female parent, F2 segregating population single plants of molecular labeling InDel 5513 detect
Amplification system is 20 μ L：ddH2O 5 μ L, PCR mix10 μ L (Guangzhou Zhan Chen bio tech ltd), 5513F1
Primer (10 μm of ol/L) 1.5 μ L, 5513R1 primer (10 μm of ol/L) 1.5 μ L, DNA profiling (500ng/ μ L) 2 μ L.PCR reaction intervals
Sequence is:94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 30s, are circulated 35 times；72 DEG C re-extend
5min.After reaction, 20 μ L products electrophoresis on 1.2% agarose is taken respectively, with gel imaging system after Goldview dyeing
Overall view, which is examined, takes pictures.
3rd, PAGE glue electrophoresis and colour developing
PCR product detection utilizes 8% modacrylic acyl ammonia detected through gel electrophoresis, and applied sample amount is 1 μ L, using the permanent electricity of 160V
Piezoelectricity swimming 2h.PAGE glue dyeing uses rapid silver staining, is specially：(1) glue is pulled down from glass plate it is placed in distilled water and rinses,
And it is repeated once；(2) 0.1%AgNO310min is dyed in solution；(3) distilled water rinses 2 times；(4) 1x nitrite ions 200ml+800
Develop the color 10 minutes in μ L formaldehyde；(5) read tape after tap water rinse 2 times.
4th, testing result
Testing result is as shown in Figures 2 and 3.Specific primer is to the fragment (gene to maternal amplification length for 207bp
Type pp), the fragment (genotype PP) that the amplification length of male parent is 292bp；The amplification banding pattern of F1 generation single plant is 292bp and 207bp
(genotype Pp)；F2 is considered for amplification 2 kinds of situations of banding pattern point of gingko color single plant：A kind of situation is green stem gingko color single plant,
Its possible genotype has DDpp, Ddpp, ddpp, therefore 5513 amplifications of P gene molecule markers InDel only have a kind of situation,
207bp (genotype pp) (Fig. 2)；Another situation is purplish red stem gingko color single plant, its possible genotype has ddPP, ddPp, because
5513 amplifications of this P gene molecule marker InDel are 292bp (genotype PP) single slice, 292bp and 207bp (genotype
Pp) heterozygosis band (Fig. 3).
Amplification banding pattern to F2 for purplish red fruit color single plant：F2 for purplish red fruit color single plant may genotype have DDPP, DDPp,
DdPP and DdPp, therefore 5513 amplifications of P gene molecule markers InDel have 2 kinds of situations, both only 292bp (genotype
PP), 292bp and 207bp heterozygosis (genotype Pp), as shown in Figure 4.
InDel mark testing results meet F2The separation of fruit color and the correspondence of genotype of segregating generation, illustrate we
Method accuracy is high, can be applied to the assistant breeding of eggplant fruit color epistatic gene P molecular labelings.
SEQUENCE LISTING
<110>Vegetables Inst., Guangdong Academy of Agricultural Sciences
<120>InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213>Eggplant
<400> 1
cttaccttat caatcgtaat tgtaccaaac ctctaccagt taactaccat 50
<210> 2
<211> 35
<212> DNA
<213>Eggplant
<400> 2
accgtcaaaa tttatcacac actgttatga tattt 35
<210> 3
<211> 23
<212> DNA
<213>Artificial primer
<400> 3
gtgttacgag ggttgaaatg gac 23
<210> 4
<211> 23
<212> DNA
<213>Artificial primer
<400> 4
attggtaaaa ggaagatttg agg 23
<210> 5
<211> 207
<212> DNA
<213>Eggplant
<400> 5
gtgttacgag ggttgaaatg gacaagctgt caaggagggc tcgatcttac cttatcaatc 60
gtaattatac caaacctcta ccaattaact accatgaaga aaataagagt tagagagaga 120
attttattat gatattacag atgaaggcaa aattataaaa tcaagaacaa agttgaatta 180
tcaacctcaa atcttccttt taccaat 207
<210> 6
<211> 292
<212> DNA
<213>Eggplant
<400> 6
gtgttacgag ggttgaaatg gacaagctgt cgaggagggc tcgatcttac cttatcaatc 60
gtaattgtac caaacctcta ccaattaact accatcttac cttatcaatc gtaattgtac 120
caaacctcta ccagttaact accatgaaga aaataagagt tagagagaga atttttttct 180
accgtcaaaa tttatcacac actgttatga tatttgatat tacagatgaa ggcaaaatta 240
taaaatcaag aacaaagttg aattatcaac ctcaaatctt ccttttacca at 292
Claims (8)
- Application of the 1.InDel molecular labelings as eggplant fruit color selection markers, the InDel molecular labelings are to be with length Special insertion/deletion fragment at the 2 of 50bp and 35bp, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
- 2. application of the primer sets of recognizable InDel molecular labelings in eggplant fruit color screening reagent is prepared, wherein, InDel points Son mark is as claimed in claim 1.
- 3. application according to claim 2, it is characterised in that：The sequence of primer sets is：5513 F1：5'-GTGTTACGAGGGTTGAAATGGAC-3'5513 R1：5'-ATTGGTAAAAGGAAGATTTGAGG-3'.
- 4. a kind of method of eggplant fruit color screening, including detect the insertion/deletion of InDel molecular labelings on No. 8 chromosomes of eggplant Situation, eggplant fruit color is determined according to the insertion/deletion situation of InDel molecular labelings, wherein, InDel molecular labelings such as right will Ask described in 1.
- 5. according to the method described in claim 4, it is characterized in that：Use InDel points on PCR methods detection No. 8 chromosomes of eggplant The insertion/deletion situation of son mark.
- 6. according to the method described in claim 5, it is characterized in that：The sequence of the primer sets used in PCR methods is：5513 F1：5'-GTGTTACGAGGGTTGAAATGGAC-3'5513 R1：5'-ATTGGTAAAAGGAAGATTTGAGG-3'.
- 7. the method according to claim 5 or 6, it is characterised in that：, should if only having 207bp fragments in amplified production Plant genotype is pp；If only having 292bp fragments in amplified production, which is PP；At the same time have 292bp and The then genotype of two segments of 207bp is Pp.
- 8. a kind of method for cultivating different fruit color eggplants, is included in No. 8 chromosomes introducing InDel molecular labelings of eggplant and is inserted into/lacks Lose, wherein, InDel molecular labelings are as claimed in claim 1.
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| CN111454973A (en) * | 2020-04-24 | 2020-07-28 | 广东省农业科学院蔬菜研究所 | Eggplant fruit color epistatic P gene locus candidate gene and application method thereof |
| CN111454973B (en) * | 2020-04-24 | 2021-04-20 | 广东省农业科学院蔬菜研究所 | Application of candidate gene of eggplant fruit color epistatic P gene locus |
| CN111926098A (en) * | 2020-08-17 | 2020-11-13 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with epistatic gene Y of eggplant fruit color and application |
| CN111910013A (en) * | 2020-08-20 | 2020-11-10 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with anthocyanin synthetic gene of eggplant, primer and application thereof |
| CN111910013B (en) * | 2020-08-20 | 2021-11-19 | 广东省农业科学院蔬菜研究所 | InDel molecular marker closely linked with anthocyanin synthetic gene of eggplant, primer and application thereof |
| CN116426684A (en) * | 2023-04-24 | 2023-07-14 | 上海市农业科学院 | InDel molecular marker closely linked with eggplant peel color traits and application |
| CN116426684B (en) * | 2023-04-24 | 2024-05-28 | 上海市农业科学院 | InDel molecular marker closely linked with eggplant peel color traits and application |
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