CN117701591B - Molecular marker T33 co-segregating with the gene for petiole thickness in non-heading Chinese cabbage, its primers and their application - Google Patents
Molecular marker T33 co-segregating with the gene for petiole thickness in non-heading Chinese cabbage, its primers and their application Download PDFInfo
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Abstract
The invention discloses a petiole thickness InDel molecular marker T33 co-separated from a non-heading cabbage gene, a primer and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the molecular marker T33 is SEQ ID NO.1 or SEQ ID NO.2. The variation of the molecular marker T33 is stably existing in plants, and whether any nucleotide sequence is detected, the T33-F/R detection contains BrPT petiole thickness genes, the petiole thickness homozygosity can be shown, and the molecular marker can provide auxiliary selection for screening petiole thick individuals in the future. The specific primer T33-F/R can be used for identifying the seedling stage of the plant to be detected, has low detection cost and high accuracy, greatly improves the breeding efficiency and shortens the breeding process.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a petiole thick molecular marker T33 co-separated from a non-heading cabbage gene, a primer and application thereof.
Background
The Chinese cabbage without head is also called as Chinese cabbage, which is widely planted in the south of China, and widely planted in various cities at the middle and downstream of Yangtze river, and accounts for 30% -40% of the total yield of vegetables, and the leaf stalks contain rich dietary fibers which can increase gastrointestinal motility, help digestion, prevent constipation, and also prevent gall stones, hyperlipidemia, cardiovascular diseases, diabetes and the like. The leaf and stem of the non-heading Chinese cabbage are the most visual appearance quality characters and edible organs, the commodity value and the selection of consumers are directly influenced, and the leaf and stem of the leaf have a certain biomass distribution relationship, so that the size of the leaf and stem directly relates to the yield and quality, and the yield mainly comprises scattered leaves because the leaf ball is not produced by the non-heading Chinese cabbage; the maximum She Shebing thickness is found to have larger influence on the yield of the non-heading white menu plant in the relevance ranking between the agronomic traits of the non-heading Chinese cabbage and the yield traits of the single plant, the highest She Shebing thickness and the yield relation are found to still keep higher relevance in the analysis of the relation between a plurality of agronomic traits of the Chinese cabbage and the yield, the influence of the leaf stalk thickness and the width of the maximum leaf on the yield of the single plant is the maximum, and the maximum leaf width and the leaf number are the maximum.
Cheng Yan finds a QTL related to the thickness of the petiole on each of the A1 and A7 linkage groups in the construction of a genetic linkage map of the non-heading cabbage, wherein the contribution rates are respectively 12.29% and 13.83%, and the positions are respectively 35.86 and 56.93cM; zhang Yun in the research of important agronomic traits of Chinese cabbage, QTL of petiole thickness is detected on A06 and A09 linkage groups in multi-field materials, but genetic distance on A09 linkage group is 70.4cM, hanzhong is detected on A03, A06 and A10 in Chinese cabbage, QTL of petiole thickness is found on the QTL positioning of important agronomic traits of non-heading Chinese cabbage, genetic distance is not equal from 32.4 to 187.6cM, wang Qian also utilizes non-heading Chinese cabbage to find QTL of petiole thickness on A02, A04 and A07, the range of interpretable character variation is between 5.28% and 20.86%, geng Jianfeng utilizes non-heading Chinese cabbage DH group to find QTL of rib thickness in control on LG1, LG2, LG3, LG5, LG7, LG8 and LG9, wherein QTL position on LG9 is at 27, the contribution degree is only 4.5%, the quantitative trait of the petiole thickness is regulated by multiple genes, but at present, regulation explanation and fine positioning of the quantitative trait of the petiole thickness are not more, development and application schemes of related molecular markers are few, indel molecular marker patents about the petiole thickness are not searched at present, related genes are few, and the traditional morphological method is time-consuming and labor-consuming in the processes of evaluating the variety of the non-heading Chinese cabbage, classifying groups, identifying consistency, identifying purity and the like, and needs to occupy a large amount of land resources and manpower resources, and the description of the traits has deviation in some traits due to different observers, so that the accuracy of results is not high; therefore, the development of the petiole thickness Indel molecular marker coseparated with the non-heading cabbage gene can rapidly realize the identification and screening of the petiole thickness gene and the characters, and is a technical problem to be solved by those skilled in the biotechnology field in pursuing genetic breeding of the non-heading cabbage.
Disclosure of Invention
Aiming at the important influence of petiole thickness on yield, the invention provides a petiole thickness InDel molecular marker T33 which is co-separated from a non-heading cabbage gene, a primer and application for solving the technical problems.
The invention provides a petiole thick InDel molecular marker T33 coseparated with a non-heading cabbage gene, which is characterized in that the nucleotide sequence of the T33 is SEQ ID NO.1 or SEQ ID NO.2, wherein the SEQ ID NO.1 is a molecular marker coseparated with a petiole thin BrPT gene in a petiole thick individual
The invention also provides a PCR specific amplification primer of the petiole thickness molecular marker T33 which is coseparated with the non-heading cabbage gene, comprising
T33-F：5’-CTTTCTCCTTGGCGATAATA-3’(SEQ ID NO.3)
T33-R：5’-GGCAAGACAGTGGTTGATTA-3’。(SEQ ID NO.4)
The invention also provides an application of the InDel molecular marker T33 co-separated from the non-heading cabbage gene in identifying or screening the thickness characteristics of the leaf stalks of the non-heading cabbage.
The invention also provides an application of the InDel molecular marker T33 co-separated from the non-heading Chinese cabbage gene in genetic breeding of the non-heading Chinese cabbage.
The invention also provides an application method of the InDel molecular marker T33 co-separated from the non-heading cabbage gene, which comprises the following steps:
a. extracting genome DNA of the material to be tested
B. PCR amplification
The primer sequence of PCR is
T33-F：5’-CTTTCTCCTTGGCGATAATA-3’
T33-R：5’-GGCAAGACAGTGGTTGATTA-3’；
C. Electrophoresis
Electrophoresis detection is carried out by adopting 12% non-modified polyacrylamide gel, the electrophoresis voltage is 220V, the current is 120mA, the time is 1h 30min, the electrophoresis buffer solution is 1 XTBE, and after electrophoresis, silver staining is carried out and amplification effect is observed;
d. judging
If the nucleotide sequence of the amplified band is SEQ ID NO.1 and 199bp, the amplified band is a thick petiole individual, if the nucleotide sequence of the amplified band is SEQ ID NO.2 and 211bp, the amplified band is a thin petiole individual, and the probability of containing the gene of the petiole thickness BrPT is 100%.
Further, the PCR amplification system was as follows: 1. Mu.L of DNA template, 0.5. Mu.L of each of the upstream and downstream primers, 2X SAN TAQ PCR Mix 5. Mu.L, and 10. Mu.L of sterile distilled water were used to supplement the reaction system;
Further, the PCR amplification procedure was as follows
Pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s; renaturation at 56 ℃ for 30s; extending at 72 ℃ for 30s;34 cycles; finally extending for 5min at 72 ℃ and preserving at 4 DEG C
Compared with the prior art, the invention provides the petiole thickness InDel molecular marker T33 which is co-separated from the non-heading cabbage gene, the primer and the application thereof have the following beneficial effects:
the invention has the beneficial effects that:
1. the research finely locates the petiole thickness gene BrPT and develops an InDel molecular marker T33 closely linked with BrPT, the petiole thickness gene BrPT is located in the 51kb region of the A09 chromosome by InDel markers T31 and T35, the molecular marker T33 is stably present in plants, and when the T33-F/R detects that the petiole thickness gene contains BrPT, the petiole thickness homozygosity is shown, and the molecular marker is used for providing auxiliary selection for screening petiole thick individuals in future.
2. The specific primer T33-F/R can be used for identifying the seedling stage of the plant to be detected, has low detection cost and high accuracy, greatly improves the breeding efficiency and shortens the breeding process.
Drawings
FIG. 1 shows the fine positioning of the leaf stalk thickness gene BrPT of non-heading Chinese cabbage; and (3) injection: the right end is the genetic distance, the left end is the primer name and the physical position, the number of recombinant single plants is in parentheses, the dotted line frame is the final candidate interval, and the size is 51kb.
FIG. 2 shows cloning results in BrPT parents ZY11 and XS 38; and (3) injection: the leftmost sequences of REF-gdna, REF-CDS, XS38 and ZY11 are sequentially arranged from top to bottom, and compared with the full length of a reference genome and CDS, the XS38 does not find a frame shift mutation, and the frame shift at a blue frame line is the frame shift mutation existing on a Marker T33; the four common sequences are arranged on the black background, and the base with difference is arranged on the white background;
FIG. 3 is a graph of primer T33 population verification results; and (3) injection: lanes 6-7 are F 1, thick, lanes 8-9 are ZY11, thin, and lanes 10-12 are XS38, thick; lanes 1-5 and 13-27 are thin petiole individuals, and lanes 28-40 are thick petiole individuals; m is MAKER, and the middle band is 50bp above and below 200 bp.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Example 1 sample acquisition/construction
The test materials adopted by the application are provided by cruciferous subject groups of the university of northwest agriculture and forestry science and technology, wherein the test materials comprise thin-stalk non-heading Chinese cabbage which is 'ZY-11' and thick-stalk non-heading Chinese cabbage which is '14XS38'.
The cabbage '14XS38' is obtained by using the leaf margin split variant strain in the cabbage inbred line 'dwarf resistance blue' as an original material, and through continuous selfing and combined phenotype observation, breeding the cabbage leaf margin split near isogenic line.
The thin-stalk non-heading Chinese cabbage ZY-11 is a purple non-heading Chinese cabbage high-generation inbred line with stable properties, which is bred by taking a variant strain introduced into non-heading Chinese cabbage PK11 (Beijing Zhongnong Lvheng seed technology Co., ltd.) as an original material through continuous selfing separation and purification.
Note that: PK11 properties: the plant is semi-upright, has strong growth vigor, flat and flat leaves, purple red and glossy pages, green petioles, rich anthocyanin and rich nutrition, and is an ideal variety for cooking or fresh salad.
F 2:4 segregating populations obtained by multi-generation selection selfing after hybridization of the two materials. And selecting extremely thin-stalk plants and thick-stalk plants in the segregating group to construct an F 2 segregating group, and constructing a parent and F 2 mixed pool to carry out BSA-seq and genetic positioning.
The hybrid population is planted in autumn 2020, the positioning population is planted in spring 2022, and the rest populations are planted in spring 2021. The materials required for the test were planted in Yang Lingou Cao Xinzhuang test farms and too white test bases.
The petiole thickness property observation of the two parents, the F 1 and the F 2 groups is carried out in the adult plant period, namely, when the petiole thickness is obviously separated, the thickness of the whole petiole is counted by utilizing a vernier caliper measurement method.
Example 2InDel molecular marker development
1. DNA extraction sequencing
Respectively selecting 20 strains of thick-stalk and thin-stalk parents, and selecting 30 strains of single extremely thin-stalk and thick-stalk from F 2 separation groups
The extraction of leaf genomic DNA was performed by the modified CTAB method. The concentration is detected after DNA is extracted, and the parent gene pool and the F 2 gene pool are built by equal amount of evenly mixing after dilution and adjustment, and the obtained product is sent to Beijing Baimeike biotechnology Co. The reference genome is Brara _ Chiifu _v3.0.
2. Indel primer design
Based on the BSA sequencing results, indel markers were designed at sites with large fragment differences in the peak region, and PRIMERPREMIER 5.0.0 software was used to design primers, which were all synthesized by Shanghai Biotechnology Inc.
3. Indel primer polymorphism screening and genotyping
(1) 30 Plants of thick and thin stalk plants were selected from the F 2 population for DNA pool sequencing (BSA-seq). And (3) analyzing the sequencing data of the mixed pool by using an ED algorithm and an SNP-index algorithm respectively, and finally obtaining 1 candidate region related to the thickness property of the petioles, wherein the total length of the candidate region is 3.01Mb, and the candidate region is positioned on an A09 chromosome.
(2) Designing a molecular marker for Indel sites with the base difference number between two parents being more than 5bp on an A09 chromosome, verifying the markers by utilizing the isolated population parents and the F 1 single plant, screening polymorphism, reducing the interval to 51kb by combining the recombinant single plant, and obtaining 1 Indel marker T33 for controlling the co-isolation of thick stalk character genes.
Example 3 method for identifying the Capacity of non-heading Chinese cabbage Using Indel molecular markers
Step one extraction method of genome DNA of leaf of non-heading Chinese cabbage
The improved CTAB method is used for extracting the DNA of the leaf of the non-heading cabbage, and the specific operation is as follows:
(1) Taking about 0.1g of non-heading cabbage leaves, quickly freezing with liquid nitrogen, grinding into powder, transferring to a 2ml centrifuge tube, adding 700 mu l of DNA CTAB extract, uniformly mixing, placing in a 65 ℃ incubator for about 1h, and vibrating and uniformly mixing once in 20 min; then adding 700 μl of chloroform for full extraction, centrifuging to obtain 500 μl of supernatant, and transferring to a 1.5ml centrifuge tube; adding an equal volume of ethanol and supernatant, gently mixing, refrigerating at-20 ℃ for 1h, centrifuging, discarding supernatant, retaining DNA sediment at the bottom of the tube, and washing twice with 70% ethanol; after the DNA was dried, it was dissolved in 150. Mu.l of ddH2O.
(2) Detecting the concentration and quality of DNA, if necessary, detecting by 1% agarose gel electrophoresis, measuring the concentration of DNA by ultraviolet spectrophotometer, and storing in a refrigerator at-20deg.C for use.
Step two, the labeled primer T33 is amplified in the non-heading cabbage DNA
Wherein, 1. Mu.L of DNA template, 0.5. Mu.L of each of the upstream and downstream primers, 2X SAN TAQ PCR Mix 5. Mu.L, and the reaction system was supplemented with sterile distilled water to 10. Mu.L; the reaction procedure is: 94 ℃ for 5min;94 ℃ for 30s; 30s at 56 ℃; 30s at 72 ℃;34×; and at 72℃for 5min. The primer sequences are shown in Table 1.
Step trimeric acrylamide gel electrophoresis detection
After the PCR is finished, electrophoresis detection is carried out by adopting 12% non-denaturing polyacrylamide gel, the electrophoresis voltage is 220V, the current is 120mA, the time is 1h and 30min, the electrophoresis buffer solution is 1 XTBE, and after the electrophoresis is finished, silver staining and color development are carried out, and observation and photographing are carried out.
Step four genotyping statistics
The primer T33 separation strip only contains 199bp DNA fragment, which shows that the leaf of the material is represented as a thick handle, and is marked as B, and the sequence of the leaf is SEQ ID NO.1, and the specific sequence is as follows:
GGCAAGACAGTGGTTGATTACCTAACGGGAGAGTTATCTAGAAAGCCACACTCAGTTGTGTTCCTCGAAAACGTGGAGAAGTCAGAGTTCCCGGATCAGAGGAGATTGTCTGAAGCTGTGAGTACAGGGAGACTCCGTGACTCGCATGGGAGAGTGATCAGTATGAAGAATGTAATTGTTATTATCGCCAAGGAGAAAG.
If only a 211bp DNA fragment is contained, the leaf of the material is shown as a thin handle, which is marked as A, and the sequence is SEQ ID NO.2, and the specific sequence is as follows:
GGCAAGACAGTGGTTGATTACCTAACGGGAGAGTTGTCAAGGAAGCCACACTCAGTTGTGTTCCTTGAAAACGTGGAGAAGTCAGAGTTCCCTGATCAGAGCAGGTTGTCCGAAGCTATAACTACAGGGAGACTCCGTGACTCGCATGGGAGAGTGATCAGTATGAAGAACGTGATTGTTCTTGCAGCTTCTAGTATCGACAAGGAGAAAG.
if both 199bp and 211bp fragments were included, the petiole phenotype was indicated as medium thickness and was designated as H.
Step five feasibility verification
To verify the feasibility and accuracy of the markers, 93F 2 samples were identified by the method, respectively, all sample materials were from the university of North-North agriculture and forestry science and technology crucifer laboratory. The corresponding degree can reach 1:1, from Table 1, the leaf stalk thin corresponding genotypes are all 'A', while the thick plants are 'B' and 'H', wherein lanes 6-7 are F 1 and lanes 8-9 are ZY11 and the leaf stalk phenotype is thin; lanes 10-12 are XS38, the petiole phenotype is thick; lanes 1-5 and 13-27 are thin petiole individuals, and lanes 28-40 are thick petiole individuals; m is MARKER, the middle band is 50bp above and below 200 bp; see in particular table 1 and the partial results in figure 3.
TABLE 1 primer T33 population validation results
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.
Claims (5)
1. The application of the petiole thickness InDel molecular marker T33 coseparated with the non-heading Chinese cabbage genes in identifying or screening the petiole thickness characteristics of the non-heading Chinese cabbage is characterized in that the nucleotide sequence of the molecular marker T33 is SEQ ID NO.1 or SEQ ID NO.2, wherein the SEQ ID NO.1 exists in a thick petiole individual, and the SEQ ID NO.2 is a molecular marker coseparated with the petiole thickness BrPT genes.
2. The use of the InDel molecular marker T33 co-isolated from a non-heading cabbage gene as claimed in claim 1 in genetic breeding of non-heading cabbage.
3. The method for identifying the thickness of the petiole by adopting the InDel molecular marker T33 co-separated from the non-heading cabbage gene as claimed in claim 1, which is characterized by comprising the following steps:
a. Extracting genome DNA of a material to be detected;
b. PCR amplification
The primer sequence of PCR is
T33-F：5’-CTTTCTCCTTGGCGATAATA-3’
T33-R：5’-GGCAAGACAGTGGTTGATTA-3’；
C. Electrophoresis
Electrophoresis detection is carried out by adopting 12% non-denaturing polyacrylamide gel, the electrophoresis voltage is 220V, the current is 120mA, the time is 1h and 30min, the electrophoresis buffer solution is 1 XTBE, and after electrophoresis, silver staining is carried out and amplification effect is observed;
d. judging
If the amplified band has a nucleotide sequence of SEQ ID NO. 1 and a sequence length of 199bp, the band is a thick petiole individual, and if the amplified band has a nucleotide sequence of SEQ ID NO. 2 and a sequence length of 211bp, the band is a thin petiole individual.
4. A method according to claim 3, characterized in that the PCR amplification system is as follows: 1. Mu.L of DNA template, 0.5. Mu.L of each of the upstream and downstream primers, 2X SAN TAQ PCR Mix 5. Mu.L, and sterile distilled water were used to supplement the reaction system to 10. Mu.L.
5. The method of claim 3, wherein the PCR amplification procedure is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s; renaturation at 56 ℃ for 30s; extending at 72 ℃ for 30s;34 cycles; extension was carried out at 72℃for 5min and storage at 4 ℃.
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| CN115261501A (en) * | 2022-06-27 | 2022-11-01 | 浙江省农业科学院 | Indel molecular marker closely linked with flowering morning and evening of non-heading Chinese cabbage and application thereof |
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| CN115261501A (en) * | 2022-06-27 | 2022-11-01 | 浙江省农业科学院 | Indel molecular marker closely linked with flowering morning and evening of non-heading Chinese cabbage and application thereof |
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