CN106701926A - Mapping of eggplant fruit color epistatic gene D, and InDel molecular marker development and application thereof - Google Patents

Mapping of eggplant fruit color epistatic gene D, and InDel molecular marker development and application thereof Download PDF

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CN106701926A
CN106701926A CN201611128459.5A CN201611128459A CN106701926A CN 106701926 A CN106701926 A CN 106701926A CN 201611128459 A CN201611128459 A CN 201611128459A CN 106701926 A CN106701926 A CN 106701926A
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fruit color
eggplant
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eggplant fruit
indel
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孙保娟
李植良
黎振兴
罗少波
徐晓美
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Vegetable Research Institute of Guangdong Academy of Agriculture Sciences
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Abstract

The invention discloses a mapping result of an eggplant fruit color epistatic gene D. The gene D is mapped in a position near a nucleotide sequence Sme2.5_00538.1 on a No.10 chromosome of the eggplant. Based on the re-sequencing result of male and female parents, an InDel molecular marker closely linked with the eggplant fruit color epistatic gene D is obtained near the nucleotide sequence Sme2.5_00538.1 of the No.10 chromosome, and is named as InDel22522; the genetic distance of the marker from the epistatic gene D is 0.61 cM. The nucleotide sequence of the molecular marker InDel 22522 in the female parent has one 29-basic-group InDel more than that in the male parent. The mapping result on the eggplant fruit color epistatic gene D provided by the invention lays the foundation for the precise positioning and cloning of the eggplant fruit color epistatic gene; the InDel 22522 molecular marker and a molecular marker amplification primer can be applied to eggplant fruit color breeding practice in a way of simplicity, convenience, high speed and high flux; the eggplant fruit color property improving process is accelerated.

Description

The positioning and its InDel molecular markers developments of eggplant fruit color epistatic gene D and application
Technical field
The invention belongs to biology field, and in particular to the positioning and its InDel of eggplant fruit color epistatic gene D point Sub- marker development and application.
Background technology
Eggplant fruit color heredity is the complex character of controlled by multiple genes.Inventor found in eggplant breeding practice 2 pairs it is upper Property effector control eggplant fruit color genetic phenomenon（Fig. 1）.After the i.e. 2 gingko color eggplant hybridization of homozygosis, F1Dai Guose is Aubergine, F2It is 9 for purplish red fruit color and gingko color plant segregation ratio:7, illustrate that its fruit coloration is subject to 2 with epistasis Gene loci control, 2 gingko color eggplants are 2 independences and complementary base due to controlling anthocyanidin biosynthetic process Because of site（D genes and P genes）Inactivation causes respectively.
Relevant eggplant fruit color epistatic gene not yet carries out related molecular marker and Position Research at present.Therefore, eggplant is carried out The molecular labeling of sub- fruit color epistatic gene and Position Research, can be the color molecular marker assisted selection breeding of eggplant fruit and gene Clone lays the foundation.
The content of the invention
It is an object of the invention to provide eggplant fruit color epistatic gene D positioning and its InDel molecular markers developments with Using.
The technical solution used in the present invention is：
Combined with BSA related analysis technologies using SLAF-Seq, 2 genes of eggplant fruit color epistasis heredity will be controlled（D and P） Corresponding two less regions are navigated to respectively.It is that two pairs of independent allele and segregation ratio are 9 due to control proterties: 7, determine two segregation ratio of mixed pond genotype（Purple mixes pond D:Purple mixes pond d：Gingko mixes pond D：Gingko mixes pond d）It is 6:3:2: 5, by Chi-square Test, 784 SNP sites are obtained（P>0.95）；The 784 SNP ED methods that will be filtrated to get are associated Analysis, calculates relating value, and correlation threshold 0.822 is determined according to 99 percentiles.The SNP lists being associated with are shown in Table 1.Can from table 1 See, control the epistatic gene D of eggplant fruit color to be positioned on No. 10 chromosomes of eggplant.And further detect and control eggplant The molecular labeling of the epistatic gene D tight associations of fruit color, it is located on No. 10 chromosomes of eggplant, core where its associated region Nucleotide sequence is as shown in Sme2.5_00538.1.
The application also further developed the InDel molecule marks with the epistatic gene D close linkages of control eggplant fruit color Note, it is the specific insert of length 29bp, and insertion point is at the 22522bp of gene order Sme2.5_00538.1. The nucleotide sequence of the InDel molecular labelings is as shown in SEQ ID NO.1.
Primer pair for expanding InDel molecular labelings described in Claims 2 or 3, its nucleotide sequence is as follows：
22522 F2：5'-ACCGAGCCATTAGGACCTCTTGT -3',
22522 R2: 5' -GGGAGTCCGATGCAAATTCTTGT -3'。
The application of above-described molecular labeling, InDel molecular labelings, primer pair in eggplant fruit color sorting is educated.
The genotype detection method of eggplant fruit color epistatic gene D, comprises the following steps：
（1）Plant DNA to be checked is expanded using above-mentioned primer pair PCR；
（2）If only having 440bp fragments in amplified production, the plant genotype is dd；If only having 469bp in amplified production Fragment, then the plant genotype is DD；The then genotype for having two segments of 469 bp and 440 bp simultaneously is Dd.
The beneficial effects of the invention are as follows：
The smaller association area that the present invention will control 1 epistatic gene D of eggplant fruit color to be positioned on No. 10 chromosomes of eggplant In domain.And there is provided the molecular labeling InDel22522 with eggplant fruit color epistatic gene D close linkages, the marking path target The genetic distance of gene D is 0.61 cM, can be directly used for eggplant fruit color epistatic gene D molecular mark systems Set up.Molecular labeling of the invention and molecular labeling amplimer can with it is easy, quick, be applied to eggplant fruit color sorting with high throughput Educate in putting into practice, while being that clone eggplant fruit color epistatic gene D and its functional study are had laid a good foundation.
Brief description of the drawings
Fig. 1 is that female parent material Female fruits color is white, and in whole breeding time, hypocotyl, vein are spent, and sepal is all The synthesis of anthocyanidin is not observed；Male parent Male fruits color is white, seedling stage hypocotyl micro- purple, the general purple of vein, the micro- purple of pattern, F1 Aubergine is all presented for material hypocotyl, vein, pattern and fruit color.
Fig. 2 is marked at the PCR amplifications in F2 segregating population gingko color individual plants for InDel22522：M is 100bp DNA ladder；Maternal amplification length is 469 bp（Genotype DD）, male parent amplification length be 440 bp（Genotype dd）；F1 generation list Strain amplification banding pattern is 469 bp and the bands of 440 bp two（Genotype Dd）；1-12 be F2 for segregating population individual plant, wherein, 1-6 is Green stem gingko color individual plant（Genotype has DDpp, Ddpp, ddpp）, 7-12 is purplish red stem gingko color individual plant（Genotype have ddPP, ddPp）.
Fig. 3 is marked at the PCR amplifications in the purplish red fruit color individual plant of F2 segregating populations for InDel22522：M is 100bp DNA ladder；Maternal amplification length is 469 bp（Genotype DD）, male parent amplification length be 440 bp（Genotype dd）； F1 It is 469 bp and the bands of 440 bp two for individual plant amplification banding pattern（Genotype Dd）；1-12 is F2 for purplish red fruit color individual plant（Genotype There are DDPP, DDPp, DdPP and DdPp）.
Specific embodiment
With reference to embodiment, the present invention is further illustrated, but is not limited thereto.
Molecular biology experiment technology employed in following examples includes that DNA is extracted, PCR is expanded, PAGE gels electricity The experiment such as swimming, unless otherwise specified, generally conventionally operates, and 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, 2002, Beijing：Science Publishing house), or according to the condition proposed by manufacturer.
Embodiment 1
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 many 2 parts of gingko colors for strict bagging selfed breeding Eggplant purifying self-mating system is male parent（Male, genotype is ddPP）With maternal (Female, genotype is DDpp), preparing hybrid group Close F1, F1Individual plant selfing obtains F2.Female parent material Female fruits color is white, and in whole breeding time, hypocotyl, vein, Flower, sepal does not all observe the synthesis of anthocyanidin；Male parent Male fruits color is white, seedling stage hypocotyl micro- purple, the general purple of vein, pattern Micro- purple, F1Aubergine is all presented for material hypocotyl, vein, pattern and fruit color（Fig. 1）.F2Segregating generation is purplish red and gingko color is planted The segregation ratio of strain meets 9 through Chi-square Test:7.
2nd, genetic analysis
In F2It is divided into purplish red fruit color for segregating population fruit color（There is anthocyanidin）With gingko color（Without anthocyanidin）.486 individual plant compositions F2In colony, purplish red fruit color individual plant and gingko color individual plant are respectively 259 plants and 227 plants, card square（X2）Check whether it accords with Close 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 individual plant is total Meet 9 with gingko color individual plant sum：7 ratios.Therefore, specify that eggplant material fruit color heredity used in this research is by 2 The epistatic gene of interaction（D and P）Control.
2nd, eggplant fruit color epistatic gene positioning
1st, prepared by parent and the purplish red fruit color ponds of F2 and gingko color pond DNA
2 white eggplant fathers, female parents respectively take 10 plants of seedling leafs, and leaf DNA is extracted respectively, and every plant takes equivalent DNA mixing, constitutes male parent Pond and maternal pond；F2Segregating population, seedling stage individual plant takes blade, and DNA is extracted respectively, and the phase of bearing fruit carries out fruit tone and looks into, and then takes 100 Strain fruit color is that purplish red individual plant DNA mixed in equal amounts constitutes purplish red fruit color pond, and it is white individual plant DNA mixed in equal amounts to take 100 plants of fruit colors Constitute gingko color pond.
2nd, SLAF-seq combinations BSA analysis methods positioning eggplant fruit color epistatic gene
This research early stage entrusts Beijing Biomarker Technologies Co., Ltd. to be carried out using the SLAF-seq technologies of its independent research Simplify gene order-checking（Sun et al., 2013）, and combine cluster method for separating and analyzing（BSA）To eggplant fruit color epistasis Gene is positioned.Using eggplant genome SME_r2.5.1 disclosed in September, 2014（http:// eggplant.kazusa.or.jp）Sequence information, by totally 231490 SNP are corresponded on genome on 1117 Scaffold Corresponding Scaffold.The SNP site of low depth is then filtered off, parent genotype is filtered out identical and heterozygosis SNP Point, finally filters out the SNP site for not meeting segregation ratio, and 784 SNP sites are obtained.784 SNP that will be filtrated to get are used ED methods are associated analysis, calculate relating value（Table 1）, correlation threshold 0.822 is determined according to 99 percentiles.According to Chi-square Test With associate result of calculation, will control eggplant fruit color 1 epistatic gene D be positioned on No. 10 chromosomes of eggplant, its association Nucleotide sequence such as Sme2.5_00538.1 where region（http://eggplant. kazusa.or.jp）It is shown.（Table 1）.
The D gene locis of table 1 are associated with significant SNP lists
Sacffold titles Position Correspondence linkage group Correspondence Marker ED5 relating values
Sme2.5_00538.1 18818 E10 gg3107_1719 1.120
Sme2.5_00538.1 18874 E10 gg3107_1719 1.052
Sme2.5_00538.1 18886 E10 gg3107_1719 1.052
3rd, molecular labeling InDel22522 exploitations
Based on father, female parent gene group weight sequencing result, sought near the Scaffold Sme2.5_00538.1 where associated region Look for InDel to mark, an InDel mark is have found in the 22522nd site of sequence Sme2.5_00538.1, it compares in female parent Many 1 29 insertions of base in male parent.Sequence is as shown in SEQ ID NO.1：5’- TTGCTGATTCACTCATATGATTTTTTGGA-3’（SEQ ID NO.1）.
One group-specific primers pair is devised according to above-mentioned InDel marks, the primer pair can amplify length in female parent It is the fragment of 469 bp（Genotype DD）, sequence is as shown in SEQ ID NO.2；It is 440 bp that length can be amplified in male parent Fragment（Genotype dd）, sequence is as shown in SEQ ID NO.3.The base if thering are two segments of 469 bp and 440 bp to expand simultaneously Because type is Dd.
The nucleotide sequence of shown primer pair is as follows：
22522 F2：5' -ACCGAGCCATTAGGACCTCTTGT -3'（SEQ ID NO.4）,
22522 R2: 5' -GGGAGTCCGATGCAAATTCTTGT -3'（SEQ ID NO.5）.
The application of the molecular labeling InDel22522 of embodiment 2
1st, prepared by F2 individual plants DNA
It is purified from through many eggplants for 2 parts of gingko colors of strict bagging selfed breeding with Vegetables Inst., Guangdong Academy of Agricultural Sciences It is male parent to hand over system（Male, genotype is ddPP）With maternal (Female, genotype is DDpp), preparing hybrid combination F1, F1Individual plant Selfing obtains F2, F2Segregating population, totally 207 plants.Male parent, female parent, F2Individual plant seedling stage takes blade, and DNA is extracted respectively, and the phase of bearing fruit enters Row fruit tone is looked into.
2nd, molecular labeling InDel22522 PCR augmentation detections in male parent, female parent, F2 segregating population individual plants
Amplification system is 20 μ L：ddH2The μ L of 5 μ L, PCR mix (Genstar) of O 10,22522 F2 primers（10 μmol/ L）1.5 μ L, 22522 R2 primers（10 μmol/L）1.5 μ L, DNA profiling（500 ng/μL）2 μL.PCR response procedures are: 94 DEG C of predegeneration 3min；94 DEG C of denaturation 30 s, 55 DEG C of annealing 30 s, 72 DEG C of 30 s of extension, circulate 35 times；72 DEG C re-extend 7min.After reaction terminates, 20 μ L products electrophoresis on 1.2% agarose is taken respectively, gel imaging system is used after Goldview dyeing Overall view is examined and taken pictures.
3rd, PAGE glue electrophoresis and colour developing
PCR primer detection utilizes 8% modacrylic acyl ammonia detected through gel electrophoresis, and applied sample amount is 1 μ L, using 160V constant voltages The h of electrophoresis 2.5 ~ 3.PAGE glue dyeing uses rapid silver staining, specially：（1）Floated from pulling down glue on glass plate and being placed in distilled water Wash, and be repeated once；（2）0.1%AgNO310 min are dyeed in solution；（3）Distilled water is rinsed 2 times；（4）1x nitrite ions 200ml Developed the color 10 minutes in+800 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 being 469 bp to maternal amplification length（Genotype DD）, father This amplification length is 440 bp（Genotype dd）；The amplification banding pattern of F1 generation individual plant is 469 bp and 440 bp（Genotype Dd）； F2 is considered for 2 kinds of situations of amplification banding pattern point of gingko color individual plant：A kind of situation is green stem gingko color individual plant, its possible genotype There are DDpp, Ddpp, ddpp, therefore D gene molecule marker InDel22522 amplifications there are 3 kinds of situations, both 469 bp（Genotype DD）、440 bp（Genotype dd）, 469 bp and 440 bp（Genotype Dd）, in Fig. 2（Swimming lane 1-6）It is the band of rear 2 kinds of situations Type；Another situation is purplish red stem gingko color individual plant, and its possible genotype has ddPP, ddPp, therefore D gene molecule markers InDel22522 amplifications are 440 bp（Genotype dd）, such as Fig. 2 swimming lane 7-12 results.
To F2 for purplish red fruit color individual plant amplification banding pattern：F2 for purplish red fruit color individual plant may genotype have DDPP, DDPp, DdPP and DdPp, therefore D gene molecule marker InDel22522 amplifications have 2 kinds of situations, both 469 bp（Genotype DD）、 469 bp and 440 bp（Genotype Dd）, as shown in Figure 3.
InDel mark testing results meet F2The fruit color of segregating generation separates the corresponding relation with genotype, illustrates we Method accuracy is high, can be applied to the assistant breeding of eggplant fruit color epistatic gene D molecular labelings.
SEQUENCE LISTING
<110>Vegetables Inst., Guangdong Academy of Agricultural Sciences
<120>The positioning and its InDel molecular markers developments of eggplant fruit color epistatic gene D and application
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213> Solanum melongena L.
<400> 1
ttgctgattc actcatatga ttttttgga 29
<210> 2
<211> 469
<212> DNA
<213> Solanum melongena L.
<400> 2
accgagccat taggacctct tgtcgagctc gttgctgatt cactcatatg attttttgga 60
ttgcggattc actcatatga ttttttggaa atattgcatc ggaccccctg cagcgccaga 120
attcgtgggc taacacccat agaaaactgc tgaaatcgtt tgtttcctgt tccttggtcc 180
ataaaatcct atcaaacatc cccccgccgc cgctgggaca catacgcctc ttgccaggct 240
cgttttggat ccactcatat aattttttgg gaatgttgcg ccagaccccc tcagcgccag 300
aatccgtggg gtaataccca tagaaaactg tcgaaatcat ttgtttcctg ttccttggcc 360
cataaaatcc taccaagcgt cccccgccgc cacaaggcca cctaagcctc ttgctgagct 420
cgttacggac ccattcatat gattttacaa gaatttgcat cggactccc 469
<210> 3
<211> 440
<212> DNA
<213> Solanum melongena L.
<400> 3
accgagccca taggacctct tgtcgagctc gttgcggatt cactcatatg attttttggg 60
aatattgcgt cggaccccct gcagcgccag aattcgtggg ctaacaccca tagaaaactg 120
ctgaaatcgt ttgtttcctg ttccttggtc cataaaatcc tatcaaacat ccccccgccg 180
ccgctgggac acatacgcct cttgccaggc tcgttttgga tccactcata taattttttg 240
ggaatgttac gccagacccc ctcagcgcca gaatccgtgg ggtaataccc atagaaaact 300
gtcgaaatca tttgtttcct gttccttggc ccataaaatc ctaccaagcg tccccctccg 360
ccacaaggcc acctaaacct cttgttgagc tcgttgcgga cccattcata tgattttaca 420
agaatttgca tcggactccc 440
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
accgagccat taggacctct tgt 23
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
gggagtccga tgcaaattct tgt 23

Claims (7)

1., with the molecular labeling of the epistatic gene D tight associations of control eggplant fruit color, it is located on No. 10 chromosomes of eggplant, Nucleotide sequence is as shown in Sme2.5_00538.1 where its associated region.
2., with the InDel molecular labelings of the epistatic gene D close linkages of control eggplant fruit color, it is that the special of length 29bp inserts Enter fragment, insertion point is at the 22522bp of gene order Sme2.5_00538.1.
3. InDel molecular labelings according to claim 2, it is characterised in that the nucleotides sequence of the InDel molecular labelings Row are as shown in SEQ ID NO.1.
4. it is used to expand the primer pair of InDel molecular labelings described in Claims 2 or 3.
5. primer pair according to claim 4, its nucleotide sequence is as follows：
22522 F2：5'-ACCGAGCCATTAGGACCTCTTGT -3',
22522 R2: 5' -GGGAGTCCGATGCAAATTCTTGT -3'。
6. molecular labeling described in claim 1, the InDel molecular labelings described in Claims 2 or 3 or claim 4 or 5 Application of the described primer pair in eggplant fruit color sorting is educated.
7. the genotype detection method of eggplant fruit color epistatic gene D, comprises the following steps：
（1）Plant DNA to be checked is expanded using the primer pair PCR described in claim 4 or 5；
（2）If only having 440bp fragments in amplified production, the plant genotype is dd；If only having 469bp in amplified production Fragment, then the plant genotype is DD；The then genotype for having two segments of 469 bp and 440 bp simultaneously is Dd.
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CN108034752A (en) * 2018-01-09 2018-05-15 广东省农业科学院蔬菜研究所 InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages
CN109338009A (en) * 2018-12-11 2019-02-15 广东省农业科学院蔬菜研究所 InDel molecular labeling and its application with pumpkin taro aroma characteristics close linkage
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
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
CN112176091A (en) * 2020-10-26 2021-01-05 武汉市农业科学院 CAPS molecular marker closely linked with eggplant sepal color character gene and preparation method thereof
CN113999844A (en) * 2021-11-30 2022-02-01 中国农业科学院蔬菜花卉研究所 Primer pair for identifying dominant parthenocarpic gene of eggplant by using InDel molecular marker and application of primer pair
CN114480704A (en) * 2022-01-07 2022-05-13 中国热带农业科学院南亚热带作物研究所 SNP combined marker for identifying eggplant seed resources

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Publication number Priority date Publication date Assignee Title
CN108034752A (en) * 2018-01-09 2018-05-15 广东省农业科学院蔬菜研究所 InDel molecular labelings and application with eggplant fruit color epistatic gene P close linkages
CN108034752B (en) * 2018-01-09 2018-10-02 广东省农业科学院蔬菜研究所 InDel molecular labelings with eggplant fruit color epistatic gene P close linkages and application
CN109338009A (en) * 2018-12-11 2019-02-15 广东省农业科学院蔬菜研究所 InDel molecular labeling and its application with pumpkin taro aroma characteristics close linkage
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
CN112176091A (en) * 2020-10-26 2021-01-05 武汉市农业科学院 CAPS molecular marker closely linked with eggplant sepal color character gene and preparation method thereof
CN113999844A (en) * 2021-11-30 2022-02-01 中国农业科学院蔬菜花卉研究所 Primer pair for identifying dominant parthenocarpic gene of eggplant by using InDel molecular marker and application of primer pair
CN113999844B (en) * 2021-11-30 2023-11-03 中国农业科学院蔬菜花卉研究所 Primer pair for identifying dominant parthenocarpy gene of eggplant by utilizing InDel molecular marker and application of primer pair
CN114480704A (en) * 2022-01-07 2022-05-13 中国热带农业科学院南亚热带作物研究所 SNP combined marker for identifying eggplant seed resources
CN114480704B (en) * 2022-01-07 2023-06-30 中国热带农业科学院南亚热带作物研究所 SNP combined marker for eggplant seed resource identification

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