CN110423841B - Molecular marker for identifying segregation condition of interspecific hybrids and progeny materials A10 and C09 chromosomes of Chinese cabbages and brassica carinata - Google Patents
Molecular marker for identifying segregation condition of interspecific hybrids and progeny materials A10 and C09 chromosomes of Chinese cabbages and brassica carinata Download PDFInfo
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Abstract
The invention discloses a molecular marker and a method for identifying interspecific hybrids of Chinese cabbages and brassica carinata and tracking the chromosome separation condition of progeny materials A10 and C09 of the interspecific hybrids, and belongs to the field of plant genetic breeding. The marker is a pair of co-dominant molecular markers, and can be used for identifying the authenticity of interspecific hybrids of Chinese cabbages and brassica carinata, and also can be used for identifying and tracking self-bred progeny, backcross progeny, chromosome additional lines of distant hybrids and A10 and C09 chromosome segregation conditions of recombinant segregation populations and plants.
Description
Technical Field
The invention relates to the field of genetic breeding, in particular to a method for identifying and breeding distant hybrid plants.
Background
Distant hybridization is an important means for creating new plant germplasm and expanding breeding resources. Due to species reproductive isolation, distant hybridization often requires technologies such as artificial pollination and embryo rescue, is time-consuming and labor-consuming, and requires certain scientific training. During distant crossing, false hybrid plants may result due to incomplete detasseling, development of female gametes into plants, and other reasons. Therefore, the plants obtained by distant hybridization need to be tested for true hybrids by molecular, cytological and other methods. The self-crossing and backcross progeny of the distant hybrid need to trace the chromosome or chromosome segment by molecular or cytological methods to create genetic materials such as allopolyploid, chromosome additional line, introductive line and the like. Compared with cytology methods, the molecular marker has the advantages of being rapid, simple to operate, low in technical requirements on experiment operators, free of expensive instruments, capable of being used for large-population screening and the like. With the development of sequencing technology, high-throughput sequencing and biochips can also be used for molecular detection of distant hybrids. However, due to the simplicity, rapidness, low cost and the like, the molecular marker still has use value in the rapid screening of distant hybrids and the large-scale population primary screening.
The Chinese cabbage belongs to the cruciferous vegetables originated from China, and has the advantages of multiple varieties, high quality and wide production and sale. The brassica carinata is an oil crop originated in Africa and has excellent disease-resistant and stress-resistant properties. The Chinese cabbage belongs to A genome, and the Arabidopsis thaliana belongs to BC genome; the ABC genome plant synthesized by distant hybridization has important agricultural application prospect. By backcross transformation after hybridization, the chromosomes between A, B, C gene groups are recombined, so that the genetic diversity, the disease-resistant and stress-resistant potentials of the Chinese cabbage and the rape are improved.
The A10 chromosomes of cabbage and the C09 chromosome of Arabidopsis thaliana are partially homologous (homeologous) chromosomes. The two have partial homologous segments and a large number of specific genes generated after species differentiation. The recombination of A02 of Chinese cabbage and C02 chromosome of the mustard in filial generation can obtain new agronomic character, and improve the disease resistance, stress resistance and nutritional quality of vegetable and oil crops.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a molecular marker, a primer and a method for identifying interspecific hybrids of Chinese cabbages and the brassica carinata and tracking the chromosome segregation condition of progeny materials A10 and C09 of the hybrids.
The technical scheme of the invention is as follows:
an SSR molecular marker for identifying interspecific hybrids of Chinese cabbages and the Russian mustard and tracking the chromosome segregation condition of progeny materials A10 and C09 of the Chinese cabbages and the Russian mustard is obtained by amplifying a primer C09D, the forward primer sequence of a primer C09D is C09D-F: 5'-TGACAGCATATGAAGCCTGC-3' (SEQ ID No.1), and the backward primer sequence of the primer C09D is C09D-R: 5'-GATCCTGCCACAAGAATTTGA-3' (SEQ ID No. 2).
The expected length of the molecular marker PCR fragment: 250-270 bp. Indeed, the expected length of the amplified fragment in the genome of cabbage (A) was 267bp, and the expected length of the amplified fragment in the genome of Arabidopsis thaliana (C) was 258 bp. It should be understood that the number of SSR repeats may vary between species, and the length of the amplified fragment may vary between species.
The molecular markers are located in the chromosomal location of cabbage and Arabidopsis thaliana: in the cabbage (A) genome, at chromosome 16.04Mb of A10 (according to Chiifu cabbage V1.0 genome; download address:
http:// branched. org/branched/datasets/pub/Brassicaceae genome/Brassica _ rapa/V1.0/), located at Chromosome 39.96Mb of C09 in the Arabidopsis thaliana (C) genome (according to the Brassica oleracea Bol _ Chromosome _ V1.1 genome; downloading an address:
http:// fibrous db. org/read/datasets/pub/Brassicaceae genome/Brassica _ oleracea/Bol _ Chromosome _ V1.1 /). It will be appreciated that since the numbering of chromosomes may be different for different researchers, and the location of the marker may also be different in different versions of the genome assembly, this patent should include the detection of chromosomes and locations that are numbered differently but are substantially identical.
The primer C09D for identifying interspecific hybrids of Chinese cabbages and the Ethiobium aegypti and tracking the segregation condition of the progeny materials A10 and C09 chromosomes of the Chinese cabbages and the Ethiobium aegypti, the forward primer sequence of the primer C09D is shown as SEQ ID No.1, and the backward primer sequence of the primer C09D is shown as SEQ ID No. 2.
The SSR molecular marker or primer C09D of the invention is applied to the identification of interspecific hybrids of Chinese cabbage and the brassica carinata and the tracking of the chromosome separation condition of progeny materials A10 and C09.
Specifically, a primer C09D is used as a primer for PCR amplification, the DNA of a plant to be identified, Chinese cabbage and the parent DNA of the Brassica carinata are used as templates for PCR amplification, and the PCR amplification product is detected for band statistics and genotype analysis.
The method for band statistics and genotype analysis comprises the following steps:
the plant to be identified is a Chinese cabbage and the hybrid F of the Russia carinata1When growing plants, if F1The plants show a cabbage band type and a brassica carinata band type together, so that the plants are true hybrids; if detected F1If the plant only shows the Chinese cabbage banding pattern or only shows the Brassica carinata banding pattern, the plant is a false hybrid.
When the plant to be identified is a self-bred progeny plant of a Chinese cabbage and a distal hybrid of the brassica carinata, if the self-bred progeny plant of the detected distal hybrid shows a cabbage stripe type and a brassica carinata stripe type together, the plant is a whole or partial heterozygous plant of a Chinese cabbage A10 chromosome and a brassica carinata C09 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage stripe type, the plant does not contain the whole or part of the C09 chromosome of the brassica carinata; if the detected distant hybrid selfing progeny plant only shows the Brassica carinata banding pattern, the plant does not contain the whole or part of the Chinese cabbage A10 chromosome.
When the plant to be identified is a backcross progeny taking the Chinese cabbage as a backcross parent, if the detected plant shows a cabbage stripe type and a brassica carinata stripe type, the plant contains the whole or part of the C09 chromosome of the brassica carinata; if only the Chinese cabbage type is shown, the plant does not contain the whole or part of the C09 chromosome of the Arabidopsis thaliana.
When the plant to be identified is a backcross progeny taking the brassica carinata as a backcross parent, if the detected plant shows a cabbage stripe pattern and a brassica carinata stripe pattern together, the plant contains the whole or part of a cabbage A10 chromosome; if only the Arabidopsis thaliana banding pattern is shown, the plant does not contain the cabbage A10 chromosome whole or part.
Reaction system of PCR amplification: 10-50 μ L, which comprises: 1 XPCR with Mg2+ Buffer, 0.5-100ng template DNA, 0.2mM dNTPs, 0.5. mu.M SEQ ID No.1primer, 0.5. mu.M SEQ ID No.2primer, 1U Taq enzyme; reaction procedure for PCR amplification: 94-95 deg.C for 0.5-3 min; 30S at 94-95 ℃, 30S at 55-60 ℃, 30S at 72 ℃ and 35 cycles; 5-10min at 72 ℃.
Methods for detecting PCR amplification products include, but are not limited to, PAGE gel electrophoresis detection, capillary electrophoresis detection, and sequencing detection.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a co-dominant molecular marker and a method for identifying interspecific hybrids of Chinese cabbage and Ethicia carinata and tracking the chromosome separation condition of progeny materials A10 and C09 of the interspecific hybrids. The method identifies recombination and separation of distant hybridization of Chinese cabbage and the brassica carinata and backcross progeny A02 and C02 part homologous (homeologous) chromosomes of the Chinese cabbage and the brassica carinata, can expand genetic resources of the Chinese cabbage and the brassica carinata, transform new characters, enrich vegetable types and enrich daily dietary nutrition of people. Compared with cytology methods, the method has the advantages of being rapid, simple to operate, low in technical requirements on experiment operators, free of expensive instruments, capable of being used for large-population screening and the like. Compared with high-throughput sequencing and biochip methods, the method has the advantages of simplicity, rapidness and low cost. Compared with the method using two sets of molecular markers, the method using one set of codominant molecular marker can reduce the cost of manpower and material resources and can obviously reduce false positive and false negative. The method has use value in rapid screening of distant hybrids and progeny materials and large-scale population primary screening.
Drawings
FIG. 1 is a polypropylene gel electrophoresis chart of the identification results of Chinese cabbage and Arabidopsis thaliana distant hybrid F1 plants provided by the embodiment of the present invention; lane 4 is Chinese cabbage, lane 5 is Arabidopsis thaliana, lane 6 is Chinese cabbage × Arabidopsis thaliana F1And (4) hybrid. A10 is the Chinese cabbage A10 chromosome characteristic band, and C9 is the Chorispora carinata C09 chromosome characteristic band.
FIG. 2 is the distant hybrid F of cabbage and Arabidopsis thaliana of example 11The phenotype of the plants and their parents is shown in the left graph as Brassica carinata, the middle graph is Brassica oleracea, and the right graph is F1And (5) plant growing.
FIG. 3 is the distant hybrid F of cabbage and Arabidopsis thaliana of example 11Flow cytometric assays of plants and their parents.
FIG. 4 is the distant hybrid BC of cabbage and Arabidopsis thaliana of example 22A polypropylene gel electrophoresis chart of the identification result of the plant; lanes 1-15 are Chinese cabbage × Russia carinata BC having Chinese cabbage as the backcross parent2And (5) plant growing. A10 is the Chinese cabbage A10 chromosome characteristic band, and C9 is the Chorispora carinata C09 chromosome characteristic band. It is noted that the plants in lanes 1, 3, 4, 7, 9, 10, 11, and 13 contain the whole or part of C09 chromosome, and the plants in lanes 2, 5, 6, 8, 12, 14, and 15 do not contain the whole or part of C09 chromosome.
FIG. 5 is a high throughput sequencing chromosome overlay in example 2.
Detailed Description
The molecular markers and methods of the present invention for identifying interspecific hybrids of cabbage and Arabidopsis and for tracking the segregation of chromosomes of progeny A10 and C09 are described in detail below.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
A molecular marker and a method for identifying interspecific hybrids of Chinese cabbage and the brassica carinata and tracking the segregation condition of A10 and C09 chromosomes of progeny materials of the interspecific hybrids comprise the following steps:
extracting genome DNA of a plant to be detected and a parent thereof;
synthesizing a primer:
C09D-F:5’-TGACAGCATATGAAGCCTGC-3’(SEQ ID No.1)；
C09D-R:5’-GATCCTGCCACAAGAATTTGA-3’(SEQ ID No.2)。
and (4) PCR amplification. And (3) carrying out PCR amplification reaction by using the primers by using the DNA of the plant to be detected and the parent DNA thereof as templates. The reaction system is 10-50 mu L, which comprises: 1 XPCR Buffer (containing Mg)2+ ) 0.5-100ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C09D-F, 0.5. mu.M primer C09D-R, 1U Taq enzyme. And (3) PCR reaction conditions: 94-95 deg.C for 0.5-3 min; 30S at 94-95 ℃, 30S at 55-60 ℃, 30S at 72 ℃ and 35 cycles; 5-10min at 72 ℃.
The PCR product is detected by polyacrylamide gel electrophoresis. Preparing 8% polypropylene gel, and performing 180-volt electrophoresis for 1-2 hours until bromophenol blue runs out of the bottom of the electrophoresis tank to finish electrophoresis.
Further, in a preferred embodiment of the present invention, the polyacrylamide gel is taken out, silver-stained and developed,
band statistics and genotype analysis: if F1The plants show a cabbage band type and a brassica carinata band type together, so that the plants are true hybrids; if detected F1If the plant only shows the Chinese cabbage banding pattern or only shows the Brassica carinata banding pattern, the plant is a false hybrid. If the detected self-bred progeny plants of the distant hybrids show the cabbage stripe type and the Arabidopsis thaliana stripe type together, the plants are heterozygous for the whole or part of the cabbage A10 chromosome and the Arabidopsis thaliana C09 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage stripe type, the plant does not contain the whole or part of the C09 chromosome of the brassica carinata; if the detected distant hybrid selfing progeny plant only shows the stripe type of the brassica carinata, the plant does not contain Chinese cabbage A10 dyeThe color bodies are whole or partial. When backcross progeny taking the Chinese cabbage as a backcross parent is detected, if the detected plant shows a cabbage stripe pattern and a Brassica carinata stripe pattern together, the plant contains the whole or part of the Brassica carinata C09 chromosome; if only the Chinese cabbage type is shown, the plant does not contain the whole or part of the C09 chromosome of the Arabidopsis thaliana. When backcross progeny taking the brassica carinata as a backcross parent is detected, if the detected plant shows a cabbage stripe shape and the brassica carinata stripe shape together, the plant contains the whole or part of the cabbage A10 chromosome; if only the Arabidopsis thaliana banding pattern is shown, the plant does not contain the cabbage A10 chromosome whole or part.
In addition to polyacrylamide gel electrophoresis, PCR amplification products can be detected by capillary electrophoresis or high throughput sequencing. The method of determining the genotype is the same.
The present invention will be described in further detail with reference to examples.
EXAMPLE 1 this example identifies intercropping hybrid F of cabbage and Arabidopsis thaliana1Plant, its production method and use
1.1 extraction of F to be detected1Genomic DNA of the plant and its parent.
1.2 primer synthesis:
C09D-F:5’-TGACAGCATATGAAGCCTGC-3’(SEQ ID No.1)；
C09D-R:5’-GATCCTGCCACAAGAATTTGA-3’(SEQ ID No.2)。
1.3PCR amplification. To be detected F1The plant and the parent DNA thereof are used as templates, and the primers are used for carrying out PCR amplification reaction. The reaction system is 10 μ L, which comprises: 1 XPCR Buffer (containing Mg)2+ ) 0.5ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C09D-F, 0.5. mu.M primer C09D-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 94 ℃; 30S at 94 ℃, 30S at 56.1 ℃ and 30S at 72 ℃ for 35 cycles; 5min at 72 ℃.
1.4 the PCR product was detected by polyacrylamide gel electrophoresis. Preparing 8% polypropylene gel, and carrying out 180V electrophoresis for 1.5 hours until bromophenol blue runs out of the bottom of the electrophoresis tank to finish electrophoresis.
1.5 taking out the polyacrylamide gel, and carrying out silver staining and color development.
1.6 band statisticsAnd genotyping: the leucoderma and the eruca carinata are shown together in FIG. 1, and F is1The plants are true hybrids.
1.7 phenotypic characterization
The phenotype of the F1 plant and the parent is shown in figure 2, and the phenotype of the F1 plant is obviously different from that of the parent, which proves that the F1 plant is a true hybrid.
1.8 flow cytometric characterization (FIG. 3) showed that the plant nucleus chromatin content was between the mean values of parents, demonstrating that F1The plants are true hybrids.
Therefore, the molecular marker can identify interspecific hybrid F of Chinese cabbage and Arabidopsis thaliana1And (5) plant growing.
Example 2 this example identifies interspecific hybrid backcross progeny (BC) of cabbage and Arabidopsis thaliana2) Material
1.1 extracting the genome DNA of the plant to be detected and the parent thereof.
1.2 primer synthesis:
C09D-F:5’-TGACAGCATATGAAGCCTGC-3’(SEQ ID No.1)；
C09D-R:5’-GATCCTGCCACAAGAATTTGA-3’(SEQ ID No.2)。
1.3PCR amplification. And (3) carrying out PCR amplification reaction by using the primers by using the DNA of the plant to be detected and the parent DNA thereof as templates. The reaction system is 20 μ L, which comprises: 1 XPCR Buffer (containing Mg)2+ ) 1ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C09D-F, 0.5. mu.M primer C09D-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 95 ℃; 30S at 95 ℃, 30S at 56.1 ℃ and 30S at 72 ℃ for 35 cycles; 10min at 72 ℃.
1.4 the PCR product was detected by polyacrylamide gel electrophoresis. Preparing 8% polypropylene gel, carrying out 180V electrophoresis for 2 hours, and finishing electrophoresis until bromophenol blue runs out of the bottom of the electrophoresis tank.
1.5 the polyacrylamide gel was removed and developed by silver staining, see FIG. 3.
1.6 band statistics and genotyping: the tested plants No.1, 3, 4, 7, 9, 10, 11, 13 showed both the brassica oleracea and the brassica carinata banding patterns, indicating that these plants contain the whole or part of the cabbage A10 and the brassica carinata C09 chromosomes; the plants tested 2, 5, 6, 8, 12, 14, 15 showed only the Chinese cabbage A10 chromosome banding pattern, indicating that these plants did not contain the whole or part of the C09 chromosome of the Arabidopsis thaliana.
1.7 sequencing identification
Carrying out whole genome illumina sequencing on the single plant No. 9 with positive molecular marker identification, wherein the sequencing depth is 10X; comparing the reads with reference genomes A, B and C, and judging whether the target chromosome exists in the plant according to the coverage degree.
The results are shown in FIG. 4, which shows that the A10 and C09 chromosomes are densely and uniformly highly covered, indicating that the 2 chromosomes exist in the detected plants, and proving that the identification result of the molecular marker is reliable. The B07 chromosome coverage was low, which served as a negative control indicating that the chromosome was not present in the tested plants.
In summary, the molecular marker and the identification method for identifying interspecific hybrids of cabbage and brassica carinata and progeny materials thereof provided by the embodiment of the invention can identify interspecific hybrid true hybrids of cabbage and brassica carinata through a pair of codominant SSR molecular markers, and can also be used for identifying cabbage A10 chromosomes and brassica carinata C09 chromosome episomes or introgression lines of backcrossed progeny of distant hybrids.
Sequence listing
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> molecular marker for identifying segregation condition of interspecific hybrid and progeny material A10 and C09 chromosomes of Chinese cabbage and Erysia carinata
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgacagcata tgaagcctgc 20
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatcctgcca caagaatttg a 21
Claims (7)
1. The primer C09D for identifying interspecific hybrids of Chinese cabbages and the Ethiobium aegypti and tracking the segregation condition of the progeny materials A10 and C09 chromosomes of the Chinese cabbages and the Ethiobium aegypti, the forward primer sequence of the primer C09D is shown as SEQ ID No.1, and the backward primer sequence of the primer C09D is shown as SEQ ID No. 2.
2. The application of the primer C09D in identifying interspecific hybrids of cabbage and Arabidopsis thaliana and tracking the segregation of chromosomes of progeny materials A10 and C09 of cabbage and Arabidopsis thaliana according to claim 1, wherein the primer C09D is used as a primer for PCR amplification, the DNA of a plant to be identified and parent DNAs of cabbage and Arabidopsis thaliana are used as templates for PCR amplification, and the PCR amplification products are detected for band statistics and genotype analysis, wherein the band type of the cabbage is 267bp of the amplification fragment, and the band type of the Arabidopsis thaliana is 258bp of the amplification fragment.
3. The use according to claim 2, wherein the method of band statistics and genotyping is: the plant to be identified is a Chinese cabbage and the hybrid F of the Russia carinata1When growing plants, if F1The plants show a cabbage band type and a brassica carinata band type together, so that the plants are true hybrids; if detected F1If the plant only shows the Chinese cabbage banding pattern or only shows the Brassica carinata banding pattern, the plant is a false hybrid.
4. The use according to claim 2, wherein the method of band statistics and genotyping is: when the plant to be identified is a self-bred progeny plant of a Chinese cabbage and a distal hybrid of the brassica carinata, if the self-bred progeny plant of the detected distal hybrid shows a cabbage stripe type and a brassica carinata stripe type together, the plant is a whole or partial heterozygous plant of a Chinese cabbage A10 chromosome and a brassica carinata C09 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage stripe type, the plant does not contain the whole or part of the C09 chromosome of the brassica carinata; if the detected distant hybrid selfing progeny plant only shows the Brassica carinata banding pattern, the plant does not contain the whole or part of the Chinese cabbage A10 chromosome.
5. The use according to claim 2, wherein the method of band statistics and genotyping is: when the plant to be identified is a backcross progeny taking the Chinese cabbage as a backcross parent, if the detected plant shows a cabbage stripe type and a brassica carinata stripe type, the plant contains the whole or part of the C09 chromosome of the brassica carinata; if only the Chinese cabbage type is shown, the plant does not contain the whole or part of the C09 chromosome of the Arabidopsis thaliana.
6. The use according to claim 2, wherein the method of band statistics and genotyping is: when the plant to be identified is a backcross progeny taking the brassica carinata as a backcross parent, if the detected plant shows a cabbage stripe pattern and a brassica carinata stripe pattern together, the plant contains the whole or part of a cabbage A10 chromosome; if only the Arabidopsis thaliana banding pattern is shown, the plant does not contain the cabbage A10 chromosome whole or part.
7. The use according to any one of claims 2 to 6, wherein the reaction system for PCR amplification is: 10-50 μ L, which comprises: 1 XPCR with Mg2+ Buffer, 0.5-100ng template DNA, 0.2mM dNTPs, 0.5. mu.M SEQ ID No.1primer, 0.5. mu.M SEQ ID No.2primer, 1U Taq enzyme; reaction procedure for PCR amplification: 94-95 deg.C for 0.5-3 min; 30S at 94-95 ℃, 30S at 55-60 ℃, 30S at 72 ℃ and 35 cycles, and 5-10min at 72 ℃.
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