CN111910013A - InDel molecular marker closely linked with anthocyanin synthetic gene of eggplant, primer and application thereof - Google Patents
InDel molecular marker closely linked with anthocyanin synthetic gene of eggplant, primer and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, particularly relates to the field of molecular genetics, and particularly relates to an InDel molecular marker tightly linked with an eggplant anthocyanin synthetic gene, and a primer and an application thereofwThe gene is positioned in a small related region on the No. 8 chromosome of eggplant, and an eggplant anthocyanin synthesis mutant p is developedwThe genetic closely-linked molecular marker InDel87335789, which has the genetic distance of 0.65cM from the target gene P, is expected to be applied to eggplant anthocyanin synthesis mutant PwMolecular marker assisted breeding of the gene; the molecular marker and the amplification primer of the molecular marker can be simply, conveniently, quickly and high-flux applied to eggplant fruit color breeding practice, and simultaneously clone the eggplant mutant pwGene and functional study thereofAnd a good foundation is laid.
Description
Technical Field
The invention belongs to the field of molecular biology, particularly relates to the field of molecular genetics, and particularly relates to an InDel molecular marker tightly linked with an eggplant anthocyanin synthetic gene, and a primer and application thereof.
Background
Eggplant anthocyanin synthesis is a complex trait controlled by multiple genes. The applicant found in eggplant breeding practice that the genetic phenomenon of eggplant fruit color is controlled by 2 pairs of epistatic effector genes (figure 1). I.e. after crossing 2 homozygous green eggplant, F1The fruit substitute is purple red, F2The segregation ratio of the purple red fruit color plants to the green fruit color plants is 9:7, which indicates that the synthesis of the anthocyanin of the peel is controlled by the up-positioned 2 gene loci, and the 2 green fruit color eggplants are caused by the respective mutation inactivation of 2 independent and complementary gene loci (P gene and Y gene) for controlling the biosynthesis process of the anthocyanin. In the existing references (Tigchelaar E C, Janick J, Erichson H T. the Genetics of the anthocyanidin coloration in eggplants L.). Genetics,1968,60: 475-. Wherein the P gene has P, P and P in dominant orderwThree allelic forms, p inhibition of hypocotyl and pericarp anthocyanin Synthesis, pwThe synthesis of anthocyanin of each part of the plant is inhibited. The P gene mutant solanum glaucophyllum parent material female parent adopted in the research cannot observe synthesis of anthocyanin in hypocotyl, veins, flowers, sepals and fruits in the whole growth period, so that the genotype of a P site is Pwpw。
Mutant p related to anthocyanin synthesis of eggplantwRelated molecular marking and positioning researches on genes are not carried out yet. Therefore, molecular markers for controlling the synthesis of the episomal gene of eggplant anthocyanin are developedThe research on recording and positioning can lay a foundation for the eggplant fruit color molecular marker-assisted selective breeding and gene cloning.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the mutant p for synthesizing the eggplant anthocyaninwInDel molecular marker InDel87335789 with closely linked genes.
The second purpose of the invention is to provide a primer pair for amplifying the molecular marker InDel 87335789.
The third purpose of the invention is to provide the application of the molecular marker InDel87335789 or the primer pair in eggplant fruit color breeding.
The fourth purpose of the invention is to provide a molecular marker-assisted breeding method for eggplant fruit color.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a mutant p synthesized with eggplant anthocyaninwThe gene closely linked InDel molecular marker InDel87335789 is shown as SEQ ID NO.2, the 245pb-294pb position of the SEQ ID NO.2 is an insertion or deletion fragment, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 3.
Preferably, the molecular marker InDel87335789 is a mutant p for inhibiting anthocyanin synthesis of eggplantwThe gene is developed in a related region.
More preferably, the mutant pwThe gene is located on chromosome 8 of eggplant, and the related region is located between 115.311 and 118.302 cM.
Preferably, the genetic distance between the molecular marker InDel87335789 and the episomal gene P is 0.65 cM.
The invention adopts the SLAF-seq technology and the HighMap software to carry out E4453F treatment on eggplants2Carrying out high-density molecular marker development on a genetic segregation population (2 parent re-sequencing and 154 offspring SLAF simplified genome sequencing), constructing a genetic map, carrying out QTL positioning analysis on related characters, and carrying out 2 mutant genes (y) for controlling synthesis of eggplant pericarp anthocyaninAnd pw) Respectively to the corresponding two smaller areas. Found an epistatic gene p for simultaneously inhibiting the synthesis of the anthocyanin in the stems, veins, flowers and fruits of eggplant plantswLocated on the same region on chromosome 8 of eggplant. Combining with the result of genome re-sequencing, the episomal gene p of the No. 8 eggplant chromosomewThe InDel molecular marker InDel87335789 is developed in the related region (between 115.311-118.302 cM).
The molecular marker InDel87335789 is applied to female parent (genotype is p)wpwYY) has a nucleotide sequence shown as SEQ ID NO. 1; the male parent (the genotype is PPyy) has a nucleotide sequence shown as SEQ ID NO.2, and the female parent and the male parent lack 50 bpInDel. According to F2The purplish red fruit color single plant (the genotype is PPYY and Pp)wYY, PPYy and PpwYy) and F2Group green fruit single plant (genotype has p)wpwYY、pwpwYy, PPyy and Ppwyy) and calculating the exchange law of the InDel87335789 molecular marker, and determining that the genetic distance of the InDel marker from the target gene P is 0.65 cM. Can be directly used for synthesizing mutant p by eggplant anthocyaninwThe establishment of a molecular marker-assisted breeding system of the gene is expected to be applied to eggplant fruit color breeding practice; meanwhile, the mutant p is synthesized from eggplant anthocyanin by the inventionwThe positioning result of the method lays a foundation for fine positioning and cloning of the related genes for synthesizing the anthocyanin of the eggplant.
The invention also provides a primer pair for amplifying the molecular marker InDel 87335789.
Preferably, the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.
The invention also provides application of the molecular marker InDel87335789 or a primer pair thereof in eggplant fruit color breeding.
The invention also provides a molecular marker assisted breeding method for eggplant fruit color, namely, the specific primer of the molecular marker InDel87335789 is used for PCR amplification of DNA of a plant to be detected, and if the amplified product only has a 278bp fragment, the genotype of the plant is pwpwThe fruit color is green;if only the 328bp fragment is present, the genotype of the plant is PP, and if the two fragments are present, the genotype of the plant is PpwThe genotype is PP and PpwIn this case, the fruit color depends on the genotype of another episomal locus Y.
Preferably, the specific primers include, but are not limited to, the primer pairs shown in SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the female parent and the male parent of the plant to be detected are both fruit color epistatic inherited parent types.
Compared with the prior art, the invention has the beneficial effects that:
the invention synthesizes 1 mutant p for inhibiting eggplant anthocyaninwThe gene is positioned in a small related region on the No. 8 chromosome of eggplant, and an eggplant anthocyanin synthesis mutant p is developedwThe genetic distance between the molecular marker and a target gene P is 0.65cM, and the molecular marker can be directly used for synthesizing an eggplant anthocyanin mutant PwEstablishing a molecular marker-assisted breeding system of the gene; the molecular marker and the amplification primer of the molecular marker can be simply, conveniently, quickly and high-flux applied to eggplant fruit color breeding practice, and simultaneously clone the eggplant mutant pwThe gene and the function research thereof lay a good foundation.
Drawings
FIG. 1 shows female parent, male parent, E4453F1The main characteristics of each tissue site;
FIG. 2 shows InDel87335789 labeled E4453F2Male parent, female parent and E4453F1And E4453F2Distribution among different individuals.
In FIG. 1, the fruit color of the mother material was green, and no anthocyanin synthesis was observed in the cotyledons, hypocotyls, veins, flowers, sepals and pericarps throughout the growth period; the fruit color of the male parent material is green, the synthesis of anthocyanin can not be observed in hypocotyl, vein, flower, sepal and pericarp in the whole growth period, and the hypocotyl, vein, flower color and fruit color of the F1 generation material are all purple red.
In FIG. 2, M is DNAmarker; lanes 1-24 are all E4453F2And (4) single plants.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
The molecular biology experimental techniques used in the following examples include DNA extraction, PCR amplification, PAGE gel electrophoresis, etc., and unless otherwise specified, they are performed according to conventional methods, specifically, see molecular cloning, A.D. (third edition) (SambrookJ, Russell DW, Janssen K, Argentine J. Huang Peyer, 2002, Beijing, science publishers), or according to the methods suggested by manufacturers.
Example 1 acquisition of molecular marker InDel87335789
(1) Construction of genetic populations and genetic analysis
1. Test material
Plant material: 2 parts of green eggplant purified inbred line (variety number is E4453) which is bred by strictly bagging and inbreeding for multiple generations at vegetable research institute of agricultural academy of sciences of Guangdong province is taken as female parent (genotype is p)wpwYY) and male parent (the genotype is PPyy), and preparing a hybrid combination F1(E4453 F1)，F1Selfing the individual plant to obtain F2(E4453 F2). The fruit colors of the female parent material and the male parent material are green, and the synthesis of anthocyanin can not be observed in hypocotyl, vein, flower and sepal in the whole growth period; f1Hypocotyls, veins, colors and fruit colors of the substitute material are all purple red; f2The segregation ratio of the purple red plants and the green fruit plants of the segregation generation is proved to be 9:7 by chi square test (figure)1)。
2. Genetic analysis
F2The fruit color of the generation segregating population can be divided into purple red (with anthocyanin) and green (without anthocyanin). F consisting of 154 individuals2In the population, the number of purple fruit individual plants and green fruit individual plants is 92 and 62, respectively, chi square (X)2) Check if it meets 9:7 ratio, result X20.76(P ═ 0.38), less than X2 0.053.84(df ═ 1), indicating that the total number of purplish red and green fruit individual plants is 9:7 in the same ratio. Thus, it was clarified that the fruit color inheritance of eggplant material used in this study is controlled by 2 interacting episomal genes (P and Y). The fruit color of the study is closely linked with flower color, stem color and leaf vein color, the fruit does not contain anthocyanin and is green, the flower color is white (does not contain anthocyanin), the stem color and the leaf vein are green (does not contain anthocyanin), and if the fruit color is purple red (contains anthocyanin), the flower, stem and leaf vein are all synthesized into anthocyanin and are purple red.
(2) Upper gene location of eggplant fruit color
1. Parent and F2Individual DNA preparation
Respectively extracting leaf DNA from 2 male parent and female parent of solanum glaucophyllum in 10 seedling stage, and mixing equivalent DNA of each leaf to form a male parent pool and a female parent pool; f2Separating the colony, taking leaves of a single plant, and respectively extracting DNA. The cotyledon, hypocotyl, stem color, flower color and fruit color were investigated at seedling stage, flowering stage and fruit setting stage.
2. Genetic map construction and eggplant fruit color epistatic gene positioning
In the early stage of the research, the Beijing Baimai biological technology Limited company is entrusted to carry out simplified genome sequencing by utilizing the SLAF-seq technology independently developed by the Beijing Baimai biological technology Limited company, SNP is divided into 12 linkage groups by positioning with a reference genome, MLOD value is calculated by recombination rate between every two markers, the markers with the MLOD value lower than 3 of other SNP are filtered, 2,017 markers are added in total, and the markers are positioned as top-map markers (Marker). And analyzing by using HighMap software to obtain linear arrangement of the markers in the linkage group, and estimating genetic distance between adjacent markers to finally obtain a genetic map with the total map distance of 1,176.5 cM.QTL positioning analysis is carried out by adopting lciMapping, the threshold value is 2.5, and the eggplant anthocyanin synthesis mutant p is inhibitedwGene mapping on chromosome 8 of eggplant, mutant pwThe basic information of the region of association of the gene on chromosome 8 (115.311-118.302cM) is shown in Table 1.
TABLE 1 mutant pwBasic information of Gene-related region
3. Development of molecular marker InDel87335789
Based on the results of the parent and maternal genome re-sequencing, an InDel marker is searched near the region associated with the chromosome 8, and an eggplant anthocyanin synthesis mutant p is found at the 87335789 th site of the chromosome 8wThe InDel molecular marker with closely linked genes is named InDel 87335789. The molecular marker is in female parent (genotype is p)wpwYY) is as follows:ACTTCAAACAAAGCATCAAACTCAAATTCCAGTCGTGTTGAATACTGAAAAAATGCAAGTGATGAATTTGAAATCCCATATTGCACGCCAGGGTCACTCAGTGACCCTGCCATAAGAGGAACGATAGTCATATGTTTGGCAGGTGGTGGTGTTACGAGGGTTGAAATGGACAAGCTGTCGAGGAGGGCTCGATCTTACCTTATCAATCGTAATTGTACCAAACCTCTACCAATTAACTACCATCAAGAAAATAAGAGTTAGAGAGAGAATTTTTTTCT(ii) a The nucleotide sequence in the male parent (genotype is PPyy) is
It is deleted 50 bases in the female parent compared to the male parent (TTACCTTATCAATCGTAATTGTACCAAACCTCTACCAGTTAACTACCATG). According to the F2 group, purplish red fruit color single plant (the genotype is PPYY and Pp)wYY, PPYy and PpwYy) and F2 population green fruit single plant (genotype has p)wpwYY、pwpwYy, PPyy and Ppwyy), calculating the exchange law of the molecular marker, and determining the genetic distance between the molecular marker and the target gene PThe separation was 0.65 cM.
Based on the sequence information of the InDel molecular marker developed above, specific primers were designed using Primer5.0 software:
87335789F：5'-ACTTCAAACAAAGCATCAAACTC-3'；
87335789R：5'-AGAAAAAAATTCTCTCTCTAACTC-3'。
example 2 application of molecular marker InDel87335789
1. Plant material: 2 parts of green eggplant purified inbred line (variety number is E4453) which is bred by multiple-generation strict bagging inbred breeding at vegetable research institute of Guangdong province academy of agricultural sciences is used as a female parent (genotype is pwwYY) and a male parent (genotype is PPyy), a hybrid combination F1 (E4453F 1) is prepared, and F2 (E4453F 2) is obtained by single plant inbreeding of F1 and is used as an experimental material.
2. Extraction and detection of genomic DNA
Taking young leaves of eggplant male parent, female parent, F1 and F2, and extracting by a CTAB method. The method comprises the following specific steps: taking 0.5g of fresh tender leaves, quickly grinding the leaves in liquid nitrogen into powder, and placing the powder in a 1.5mL centrifuge tube; adding 0.5mL of extraction buffer solution, and carrying out water bath at 65 ℃; after cooling, adding 0.5mL of phenol-chloroform-isoamyl alcohol (volume ratio of 25: 24: 1), uniformly mixing by using a pipette, then centrifuging for 10min under the condition of 12000rpm, extracting supernatant, transferring into a new tube, adding cold-treated isopropanol, slightly and uniformly mixing, and carrying out ice bath for 30 min; centrifuging at 8000rpm for 10min at 4 deg.C; pouring off the supernatant, washing twice with 70% alcohol, drying, adding buffer solution for dissolving, adding RNase to remove RNA, carrying out water bath at 37 ℃ for 30min, and carrying out agarose electrophoresis to detect the quality of DNA.
3. PCR amplification
The specific primer pair designed in the example 1 is used for carrying out PCR amplification in the isolated population single strains of the male parent, the female parent, the F1 and the F2, and the PCR reaction system is as follows: PCRmix (2X Taq PCR Green mix, Dingguo) 10.0. mu.L, R (10. mu. mol. L)-1)0.5μL，F(10μmol·L-1) 0.5. mu.L, DNA template 1.0. mu.L, RNase Free dH2O8.0. mu.L, Total 20. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 35 times; further extension was carried out at 72 ℃ for 5 min.
4. PAGE gel electrophoresis and color development
After the reaction is finished, the PCR product is detected by 8% modified polyacrylamide gel electrophoresis, the loading amount is 1 mu L, and 160V constant voltage electrophoresis is adopted for 2 h. The PAGE gel dyeing adopts a rapid silver dyeing method, which specifically comprises the following steps: (1) removing the glue from the glass plate, rinsing the glue in distilled water, and repeating the rinsing step once; (2) 0.1% AgNO3Dyeing in the solution for 10 min; (3) rinsing with distilled water for 2 times; (4) developing in 1 Xdeveloping solution 200mL + 800. mu.L formaldehyde for 10 minutes; (5) the band was read after 2 rinses with tap water. The electropherogram obtained after silver staining is shown in FIG. 2.
5. Fruit color identification results
As shown in FIG. 2, the amplification length of the InDel87335789 marker in the female parent was 278bp (genotype p)wpw) The amplification length in the male parent is 328bp (genotype PP), and the two are hybridized F1The generation amplification band type is 278bp and 328bp (genotype Pp)w)，E4453 F2The green fruit individual plants 1, 16, 19, 22, 23 and 24 only have 278bp of amplified bands, and the genotype is pwpw(ii) a The genotypes of the E4453F 2 individuals 4, 5, 12, 13, 14, 15, 18 and 21 are PP, and the fruit color is determined by another gene locus Y (the genotypes PPYY are purple red fruit color and PPYy is green fruit color); genotypes Pp of E4453F 2 individuals 2, 3, 6, 7, 8, 9, 10, 11, 17 and 20wThe fruit color depends on another gene locus Y (genotype Pp)wYY，PpwYy is purplish red fruit color, Ppwyy green fruit).
It can be seen from the combination of examples 1 and 2 that the mutant p of the present invention is synthesized from eggplant anthocyanidinwThe positioning result lays a foundation for fine positioning and cloning of the related genes for synthesizing the anthocyanin of the eggplant; the InDel87335789 molecular marker and the amplification primer thereof can be simply, quickly and high-flux applied to eggplant fruit color breeding practice, and accelerate the process of eggplant fruit color character improvement.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> vegetable research institute of academy of agricultural sciences of Guangdong province
<120> InDel molecular marker tightly linked with anthocyanin synthetic gene of eggplant, primer and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Claims (10)
1. Mutant p synthesized with eggplant anthocyaninwThe InDel molecular marker InDel87335789 tightly linked with genes is characterized in that the nucleotide sequence of the molecular marker InDel87335789 is shown as SEQ ID NO.2, an insertion or deletion fragment is arranged at 245pb-294pb of the SEQ ID NO.2, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 3.
2. Mutant p for synthesizing eggplant anthocyanidin according to claim 1wThe gene closely linked InDel molecular marker InDel87335789 is characterized in that the molecular marker InDel87335789 is a mutant p for inhibiting the synthesis of anthocyanin of eggplantswThe gene is developed in a related region.
3. Mutant p for synthesizing eggplant anthocyanidin according to claim 2wAn InDel molecular marker InDel87335789 with closely linked genes, which is characterized in that the mutant pwThe gene is located on chromosome 8 of eggplant, and the related region is located between 115.311 and 118.302 cM.
4. Mutant p for synthesizing eggplant anthocyanidin according to claim 1wInDe with closely linked genesl molecular marker InDel87335789, wherein the genetic distance between the molecular marker InDel87335789 and the episomal gene P is 0.65 cM.
5. Primer pair for amplifying the molecular marker InDel87335789 according to any one of claims 1 to 4.
6. The primer pair of claim 5, wherein the nucleotide sequences of the primer pair are shown as SEQ ID No.4 and SEQ ID No.5, respectively.
7. Use of the molecular marker InDel87335789 of any one of claims 1-4 or the primer pair of any one of claims 5-6 for eggplant fruit color breeding.
8. A molecular marker-assisted breeding method for eggplant fruit color is characterized in that a specific primer of a molecular marker InDel87335789 is used for PCR amplification of DNA of a plant to be detected, and if an amplification product only has a 278bp fragment, the genotype of the plant is pwpwThe fruit color is green; if only the 328bp fragment is present, the genotype of the plant is PP, and if the two fragments are present, the genotype of the plant is PpwThe genotype is PP and PpwIn this case, the fruit color depends on the genotype of another episomal locus Y.
9. The molecular marker-assisted breeding method for eggplant fruits according to claim 8, wherein the specific primers include but are not limited to the primer pair of any one of claims 5 to 6.
10. The molecular marker-assisted breeding method for eggplant fruit color according to claim 8, wherein the female parent and the male parent of the plant to be detected are both of the parent type of fruit color epistasis inheritance.
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| CN111910013B (en) | 2021-11-19 |
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