CN107312870B - Molecular marker closely linked with pepper sterility restoring gene, method and application - Google Patents
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Abstract
The invention belongs to the technical field of molecular biology, and discloses a molecular marker closely linked with a pepper sterility restoring gene, a method and application thereof, wherein the molecular marker is scd06-17 and comprises a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2. The molecular marker provided by the invention has stable and reliable results, is a co-dominant molecular marker, realizes the identification of whether the pepper material contains cytoplasmic male sterile restoring genes or not in the seedling stage of the pepper, improves the breeding efficiency, accelerates the breeding process, and has important significance in three-line hybrid seed production of the pepper and auxiliary selection breeding of the restoring material molecular marker compared with other published pepper restoring gene molecular markers at home and abroad.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker closely linked with a pepper sterility restoring gene, a method and application thereof.
Background
The pepper is bred by using CMS fertility gene (Cytoplasmic male sterility) three-line matching, so that artificial emasculation can be omitted, the seed purity is improved, and the protection of intellectual property rights is facilitated. However, if the fertility restoring force of the male parent is not strong, the fruit shape and yield of F1 are seriously affected. Therefore, the selective breeding of a restorer line (male parent) with strong restorer, high coordination and excellent agronomic characters becomes one of the main problems to be solved urgently in heterosis utilization of the pepper CMS. Traditional selection of CMS fertility genes for pepper is based on the fertility performance of the test cross combination. The molecular marker assisted selection can directly reflect the polymorphism difference of DNA of different materials, and the selection is not influenced by time and environment. The selection of fertility genes is directly carried out through the molecular markers with closely linked fertility, so that the screening process of the restorer can be effectively accelerated. At present, the molecular marker assisted breeding technology is applied to the breeding of CMS restoring lines of various crops, individuals with Rf genes are identified in the seedling stage of filial generations (or anther regeneration plants), a large number of samples can be detected indoors in a short time, and the selection efficiency of breeding is obviously improved. Many studies suggest that restoration of fertility of capsicum is controlled by a major restorer gene Rf and is additionally influenced by a modifier gene and environmental factors.
In the prior art, the research on molecular markers related to pepper traits mainly comprises the following steps: (1) markers OP131400(0.37cM) and OW19800(8.12cM) flanking the Rf gene were found using RAPD techniques; researchers have transformed the molecular marker OPP131400 into an easier-to-manipulate OPP13-CAPS (Hinf I) marker, and the OPP13-CAPS marker is a dominant marker and has strong universality. (2) An STS marker CRF-SCAR was developed using the RAPD marker OPT-02/570(5cM) linked to the Rf gene. (3) The AFLP marker, AFRF8, was transformed into a co-dominant CAPS marker, AFRF8CAPS (1.8cM), closely linked to the Rf gene. (4) Partial fertility (PR) phenomenon exists in pepper, and 1 molecular marker PR-CAPS (1.8cM) linked with the PR gene is developed by using the AFLP-BSA method and can be used for removing partial fertility materials. (5) The PCR marker S4181515 was obtained by screening using BSA assay and was confirmed to be useful for primary screening of candidate fertility materials. (6) 3 markers AFRF1, AFRF3 and AFRF4 linked to the Rf gene were developed using AFLP technology and converted into types of markers usable for PCR. (7) The AF208834 marker was found to be linked to the Rf gene at a genetic distance of 20.8 cm. (8) The dwarf morning glory Rf gene is used for screening a pepper BAC library to develop a linkage marker BAC13T7SCAR (1.4 cM). (9) Sweet pepper is taken as a test material, a molecular marker related to sweet pepper restoring genes is obtained by screening through an SRAP molecular marker technology, and the SRAP marker is successfully converted into a simple and stable SCAR marker. (10) The applicability test of 11 reported pepper restorer gene linked markers is carried out by using 36 parts of pepper inbred lines with known genotypes (RfRf or RfRf), and the applicability of dominant markers CRF3S1S and CRF-SCAR is found to be the widest, and the accuracy rates are 91.67% and 88.89% respectively; the CaRf-FL-M2 has the widest applicability among 6 pairs of co-dominant markers, and the accuracy rate is 80.56%. In practical application of breeding, firstly, a dominant marker CRF3S1S or CRF-SCAR is used for carrying out primary identification on pepper materials, and a co-dominant marker CaRf-FL-M2 is used for carrying out secondary identification on pepper materials containing Rf genes. (11) The 9 reported markers linked with the pepper restorer gene are used for testing the backcross population of the CMS sterile line 83-3A, the restorer line 812, the F1(83-3A x 812) and the F1' (F1 x 83-3B) of the processing pepper and the maintainer line 83-3B, and the CRF-SCAR, PR-CAPS and OPP13-CAPS markers are found to be closely linked with the CMS fertility restorer gene Rf, but the CRF-SCAR markers do not need enzyme digestion, so that whether the processing pepper material contains the restorer gene can be identified through PCR product electrophoresis, and the pepper fertility identification accuracy of the backcross population can reach 100%.
With the publication of a pepper genome reference sequence database, the method provides convenience for developing pepper restorer gene molecular markers and map-based cloning pepper restorer genes. Although so many molecular markers have been disclosed, no molecular marker that is closely linked to the cytoplasmic male sterility restorer gene of capsicum has been found so far.
Disclosure of Invention
The molecular marker, the method and the application which are closely linked with the pepper sterility restoring gene can effectively identify the cytoplasmic male sterility restoring gene in pepper materials.
The first purpose of the invention is to provide a molecular marker of a molecular marker closely linked with a pepper sterility restoring gene, wherein the molecular marker is scd06-17 and comprises a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2.
The second purpose of the invention is to provide a method for utilizing the molecular marker which is closely linked with the sterility restoring gene of the pepper, comprising the following steps:
s1, extracting the genome DNA of the pepper material to be detected;
s2, PCR amplification:
a. reaction system: 10 mu L system, the contents of each component substance are respectively 1 mu L genome DNA, 0.5 mu L upstream primer, 0.5 mu L downstream primer, 5 mu L2 xTaq Master Mix and ddH2Supplementing 10 μ L of O, and mixing;
wherein, the sequence of the upstream primer is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO. 4;
b. and (3) amplification procedure:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45 s; annealing at 55 ℃ for 45 s; extension at 72 ℃ for 30 s; 35 cycles; extending for 10min at 72 ℃; finally storing at 4 ℃;
s3, electrophoresis
Performing polyacrylamide electrophoresis;
s4, silver staining and developing
After electrophoresis is finished, silver staining and developing are carried out on the gel sheet, and observation is carried out;
s5, judging the result
If a 342bp band can be observed in the electrophoretogram, the pepper material to be detected contains a cytoplasmic male sterility restoring gene; if the band of 342bp can not be observed in the electrophoretogram, but only the band of 321bp can be observed, the pepper material to be detected does not contain the cytoplasmic male sterility restoring gene.
Preferably, in the method for identifying the hot pepper male sterility restoring gene, the concentration of the genomic DNA is 30 ng/mu L, and the concentrations of the upstream primer and the downstream primer are both 10 mu mol/L.
The third purpose of the invention is to provide the application of the molecular marker closely linked with the pepper sterile restoring gene in the molecular marker-assisted breeding of the pepper cytoplasmic male sterile restoring material.
The fourth purpose of the invention is to provide the application of the method for identifying the hot pepper male sterility restoring gene in molecular marker-assisted breeding of hot pepper cytoplasmic male sterility restoring materials.
Compared with the prior art, the invention provides a molecular marker closely linked with a pepper sterility restoring gene, an identification method and application, and has the following beneficial effects:
on the basis of the existing research, the invention further develops the molecular marker scd06-17 closely linked with the hot pepper cytoplasmic male sterile restoring gene, verifies the effectiveness of the molecular marker scd06-17 in separate populations with different genetic backgrounds, has the identification success rate of more than 90 percent, stable and reliable results, is a co-dominant molecular marker, realizes the identification of whether the hot pepper material contains the cytoplasmic male sterile restoring gene in the seedling stage of the hot pepper, improves the breeding efficiency and accelerates the breeding process. Compared with other published pepper restorer gene molecular markers at home and abroad, the molecular marker is used as an SSR co-dominant molecular marker, can simultaneously detect three genotypes of filial generations, reveals a recessive relation, does not need to perform an enzyme digestion link, directly utilizes PCR to perform large-scale batch detection, and is quicker and more convenient in pepper three-line hybrid seed production and molecular marker-assisted selective breeding of restorer materials, thereby having important significance. The method lays a foundation for the capsicum three-line hybrid seed production by using molecular markers to assist in selecting the transferred capsicum restoring gene.
Drawings
FIG. 1 shows the electrophoresis results of 32 pepper materials;
FIG. 2 shows the electrophoresis results of the genetically isolated population of 32 cytoplasmic male sterility restorer genes of Capsicum annuum.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The molecular marker of the molecular marker closely linked with the pepper sterility restoring gene provided by the invention is scd06-17 and comprises a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2.
The molecular marker Scd06-17 is amplified by an upstream primer (Scd06-17F) sequence shown in SEQ ID NO.3 and a downstream primer (Scd06-17R) sequence shown in SEQ ID NO.4, wherein a specific 342bp fragment can be amplified in the pepper material containing the cytoplasmic male sterility restoring gene, but cannot be amplified in the pepper material without the cytoplasmic male sterility restoring gene.
The molecular marker scd06-17 is obtained by the following steps:
firstly, after obtaining the sequence of a pepper restorer gene molecular marker which is widely applied at present and has higher accuracy, performing blast search on a pepper reference genome database to find that the markers are all positioned on the sixth chromosome of pepper, and then synthesizing a published primer (https:// vegmarks.nivot.affrc.go.jp /) on a pepper Chr06 chromosome genetic map to identify the molecular marker linked with the pepper CMS restorer gene; and a three-line hybrid new family 8 No. F2 population consisting of 384 strains is constructed. The field phenotype identification shows that in 384F 2 offspring, 93 male sterile plants, 291 fertile plants and chi fang test show that the ratio of fertile to sterile accords with the Mendelian genetic rule of 3:1, which indicates that the pepper male sterility restoring gene is controlled by a pair of dominant genes. On the basis of primary positioning of the pepper restoring genes, more SSR molecular markers are developed by utilizing a pepper variety according to the reference genome sequence of peppers No.2 for positioning the pepper restoring genes. As a result, the SSR primer Scd06-17F/Scd06-17R is closely linked with the cytoplasmic male sterility restoring gene of the hot pepper, and can be used for molecular marker identification and selection of hot pepper restoring materials.
The molecular marker Scd06-17 is a codominant molecular marker, so that a small amount of DNA of a plant can be extracted and then PCR amplification can be directly carried out, and PAGE (polyacrylamide gel electrophoresis) gel detection is carried out on a PCR product, so that the operation is convenient, the experiment cost is low, and the accurate genotype of a single pepper plant can be obtained, thereby effectively accelerating the screening process of a pepper restorer.
The following provides a method for identifying whether the pepper material to be detected contains cytoplasmic male sterility restoring genes by using the molecular marker scd06-17, which comprises the following steps:
s1, extracting the genome DNA of the pepper material to be detected by adopting a CTAB method.
S2, PCR amplification:
a. reaction system: 10 μ L system, the contents of each component material are 1 μ L genome DNA, 0.5 μ L upstream primer (Scd06-17F), 0.5 μ L downstream primer (Scd06-17R), 5 μ L2 XTaq Master Mix, ddH2Supplementing 10 μ L of O, and mixing;
wherein the sequence of the upstream primer (Scd06-17F) is shown as SEQ ID NO.3, and the sequence of the downstream primer (Scd06-17R) is shown as SEQ ID NO. 4;
wherein the genomic DNA is the genomic DNA of the pepper material to be detected, the concentration is 30 ng/mu L, and the concentrations of the upstream primer and the downstream primer are both 10 mu mol/L.
b. And (3) amplification procedure:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45 s; annealing at 55 ℃ for 45 s; extension at 72 ℃ for 30 s; 35 cycles; extending for 10min at 72 ℃; finally storing at 4 ℃;
s3. electrophoresis
Performing polyacrylamide gel electrophoresis (PAGE gel) with a small vertical plate at a gel concentration of 9% and a voltage of 2-10V/cm for 1 h;
s4, silver staining and developing
After electrophoresis is finished, silver staining and developing are carried out on the gel sheet, and observation is carried out, specifically:
after the electrophoresis was completed, the two glass plates were peeled off, and the gel pieces were placed in 1000ml of a fixative (100 ml of pure alcohol +900ml of distilled water +5ml of glacial acetic acid) and gently shaken on a shaker for 10 min. After the fixation is complete, the gel is quickly placed in 1000ml of silver staining solution (1000ml distilled water +2g silver nitrate) and stained on a shaker for 10 min. After the staining was complete, the gel was placed in 1000mL of developer (3mL of formaldehyde +9g of NaOH +1000mL of distilled water) and placed on a shaker with gentle shaking until the bands were clear. Pouring off the developing solution, and carefully washing the gel with distilled water for 2-3 times;
reading the film under the film viewing lamp, inputting the reading result into a computer for storage, and taking a picture of the film for storage;
s5, judging the result
If a 342bp band can be observed in the electrophoretogram, the pepper material to be detected contains a cytoplasmic male sterility restoring gene; if the band of 342bp can not be observed in the electrophoretogram, but only the band of 321bp can be observed, the pepper material to be detected does not contain the cytoplasmic male sterility restoring gene.
The molecular Marker Scd06-17 and corresponding upstream and downstream primers Scd06-17F/R are used for identifying whether 32 pepper materials contain cytoplasmic male sterility restoring genes, the electrophoresis chart of a PCR product is shown in figure 1, a lane M in figure 1 is Marker, and other lanes are pepper materials, wherein samples No.1, 3, 5, 15, 16, 17, 18, 20, 21, 22, 25, 26, 27 and 31 can detect a 342bp band which contains the cytoplasmic male sterility restoring genes; while the remaining samples could not detect the 342bp band, but could detect only the 321bp band, which does not contain the cytoplasmic male sterile restoring gene.
In addition, the molecular Marker Scd06-17 and corresponding upstream and downstream primers Scd06-17F/R are used for detecting a genetic segregation population of 32 pepper cytoplasmic male sterility restoring genes, the result is shown in FIG. 2, a lane M in FIG. 2 is Marker, other lanes are pepper materials, a band of 321bp is detected in 17, 19, 23 and 24 samples, a band of 342bp is detected in 18, 25, 29 and 30 samples, and two bands of 321bp and 342bp are simultaneously detected in the other samples, which indicates that Scd06-17 is used as a co-dominant molecular Marker and is tightly linked with the cytoplasmic male sterility restoring genes.
It should be noted that the preferred embodiments of the present invention have been described for the purpose of preventing redundancy, but that additional variations and modifications to these embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<120> molecular marker closely linked with pepper sterility restoring gene, method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 321
<212> DNA
<213> Pepper
<400> 1
tcaacacaag cagaatcaca acaaacacaa gtataaagtt ggcgcccata cactaactgg 60
tgcataaaat aaaggaaccc atcacggcgt ccaataccac caatgattac acctaaggaa 120
ataataataa taataataat aataataata ataataataa taataataat aataatacaa 180
ctattacacc atcaatgtgt ccaaatctca tcattatttg accatattcc acttttatca 240
tcgatcggga attttaacca aatttcatta ggtaccaata tcaaccatat aaacaagtcc 300
atactattac catcattagc c 321
<210> 2
<211> 342
<212> DNA
<213> Pepper
<400> 2
tcaacacaag cagaatcaca acaaacacaa gtataaagtt ggcgcccata cactaactgg 60
tgcataaaat aaaggaaccc atcacggcgt ccaataccac caatgattac acctaaggaa 120
ataataataa taataataat aataataata ataataataa taataataat aataataata 180
ataataataa taataataca actatttcac catcaatgtg tccaaatctc atcattattt 240
gaccatattc cacttttatc atcgatcggg aattttaacc aaatttcatt aggtaccaat 300
atcaaccata taaacaagtc catactatta ccatcattag cc 342
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence
<400> 4
ggctaatgat ggtaatagta tggac 25
Claims (5)
1. A molecular marker closely linked with a pepper sterility restoring gene is characterized in that the molecular marker is scd06-17, the sequence of the molecular marker is a nucleotide sequence shown as SEQ ID number 2, and the pepper variety is a New family No. 8.
2. A method for identifying a pepper male sterility restoring gene by using the molecular marker of claim 1, comprising the steps of:
s1, extracting the genome DNA of the pepper material to be detected, wherein the pepper variety is New family No. 8;
s2, PCR amplification:
a. reaction system: a 10 mu L system, wherein the contents of each component substance are respectively 1 mu L genome DNA, 0.5 mu L upstream primer, 0.5 mu L downstream primer, 5 mu L2 XTaq Master Mix and ddH2Supplementing 10 mu L of O, and uniformly mixing;
wherein, the sequence of the upstream primer is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO. 4;
b. and (3) amplification procedure:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45 s; annealing at 55 ℃ for 45 s; extension at 72 ℃ for 30 s; 35 cycles; extending for 10min at 72 ℃; finally storing at 4 ℃;
s3, electrophoresis
Performing polyacrylamide electrophoresis;
s4, silver staining and developing
After electrophoresis is finished, silver staining and developing are carried out on the gel sheet, and observation is carried out;
s5, judging the result
If a 342bp band can be observed in the electrophoretogram, the pepper material to be detected contains a cytoplasmic male sterility restoring gene; if the band of 342bp can not be observed in the electrophoretogram, but only the band of 321bp can be observed, the pepper material to be detected does not contain the cytoplasmic male sterility restoring gene.
3. The method for identifying the pepper male sterility restoring gene according to claim 2, wherein the concentration of the genomic DNA is 30ng/μ L, and the concentrations of the upstream primer and the downstream primer are both 10 μmol/L.
4. The use of a reagent for detecting the molecular marker tightly linked to the sterility restoring gene of capsicum according to claim 1 in the molecular marker assisted breeding of a cytoplasmic male sterility restoring material of capsicum.
5. The use of the method for identifying a pepper male sterility restoring gene as claimed in claim 2 in molecular marker-assisted breeding of pepper cytoplasmic male sterility restoring material.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108411027A (en) * | 2018-05-24 | 2018-08-17 | 武汉市农业科学院 | It is a kind of detection capsicum CMS fertility restorer genes CAPS molecular labeling primers and application |
| CN108893555B (en) * | 2018-07-23 | 2019-05-17 | 青岛农业大学 | A method of based on InDel molecular markers for identification hot pepper male sterile three series mating cenospecies authenticity and purity |
| CN109913575B (en) * | 2019-04-09 | 2022-07-19 | 河南省农业科学院园艺研究所 | KASP molecular marker for identifying CMS male sterility restoring gene of pepper, kit and application thereof |
| CN110129481B (en) * | 2019-06-19 | 2020-04-14 | 青岛农业大学 | Method for simultaneously breeding hot pepper male sterile line and homozygous restoring gene line by restoring gene linked markers |
| CN113637791B (en) * | 2021-08-25 | 2023-05-30 | 青岛农业大学 | Molecular marker for simultaneously identifying restorability and authenticity of pepper male sterile three-line hybrid and identification method thereof |
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| CN103981177A (en) * | 2014-04-22 | 2014-08-13 | 西北农林科技大学 | Molecular marker for cabbage cytoplasm male sterile line restore gene and application thereof |
| CN104561297A (en) * | 2014-12-29 | 2015-04-29 | 浙江省农业科学院 | Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker |
| CN106048012A (en) * | 2016-06-06 | 2016-10-26 | 中国农业科学院蔬菜花卉研究所 | Molecular marker for aiding selection of Rf (fertility restorer) gene, specific primers and application |
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| CN103981177A (en) * | 2014-04-22 | 2014-08-13 | 西北农林科技大学 | Molecular marker for cabbage cytoplasm male sterile line restore gene and application thereof |
| CN104561297A (en) * | 2014-12-29 | 2015-04-29 | 浙江省农业科学院 | Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker |
| CN106048012A (en) * | 2016-06-06 | 2016-10-26 | 中国农业科学院蔬菜花卉研究所 | Molecular marker for aiding selection of Rf (fertility restorer) gene, specific primers and application |
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