CN110512022B - Molecular marker and method for identifying segregation condition of intercede hybrids of brassica vegetables and progeny materials A06 and C05 chromosomes - Google Patents
Molecular marker and method for identifying segregation condition of intercede hybrids of brassica vegetables and progeny materials A06 and C05 chromosomes Download PDFInfo
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Abstract
The invention discloses a molecular marker and a method for identifying segregation conditions of hybrid between brassica vegetable species and progeny material A06 and C05 chromosomes, and belongs to the field of plant genetic breeding. The molecular marker can quickly and accurately identify interspecific hybrids of brassica A genome (such as Chinese cabbage, red cabbage moss and the like) and C genome (such as cabbage mustard and brassica carinata (containing C genome)) plants and progeny materials thereof. The method can identify distant hybridization and backcross progeny of brassica A genome and C genome plants, can expand genetic resources of brassica vegetables, transfer new characters, enrich vegetable types and enrich daily dietary nutrition of people.
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 hybridization, false hybrid plants may be produced 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 brassica vegetable comprises A, B, C genomes, wherein the A genome vegetable comprises Chinese cabbage, red cabbage moss and the like, the C genome vegetable comprises cabbage, cabbage and the like, the AB genome vegetable comprises mustard, the BC genome crop comprises the Ethicia carinata, and the AC genome crop comprises rape. These vegetables and oil crops are widely cultivated in China. The genetic bottleneck of the vegetables can be expanded through distant hybridization and backcross transformation between the A and C gene groups, excellent vegetable genetic resources can be created, the scarce resistance genes can be transformed, and the nutritional quality of the vegetables can be improved. The red-vegetable moss belongs to a variety (A genome) of Chinese cabbage, and the quality of the red-vegetable moss is optimal in the flood mountain area of Wuhan city in Hubei province. Is a special bolting vegetable in the Yangtze river basin. The kale belongs to cabbage vegetables (C genome) originated from China, and has the advantages of good resistance, high quality and quick harvesting. Is an important bolting vegetable in south China. Through distant hybridization of the cabbage mustard and the red cabbage moss, the genetic resources of the cabbage mustard and the red cabbage moss can be expanded, new characters can be transferred, the vegetable types can be enriched, and the daily dietary nutrition of people can be enriched.
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 originates from oil crops 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 brassica a06 and C05 chromosomes are partially homologous (homeologous) chromosomes. The two have partial homologous segment and also contain a great deal of species differentiation generated specific gene. The recombination of A06 and C05 chromosomes in filial generations can obtain new agronomic characters, and improve the disease resistance, stress resistance and nutritional quality of vegetables and oil crops.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a molecular marker and a method for identifying segregation conditions of intercede hybrids of brassica vegetables and progeny materials A06 and C05 chromosomes.
The technical scheme of the invention is as follows:
the SSR molecular marker for identifying the segregation condition of the chromosomes of the hybrid between brassica vegetable species and progeny materials A06 and C05 is obtained by amplifying a primer C05B2, the forward primer sequence of the primer C05B2 is C05B2-F: 5'-TTCTTGTCAGTCCTGTCCCC-3' (SEQ ID No.1), and the backward primer sequence of the primer C05B2 is C05B2-R: 5'-TCCCGAGGTACTTCACTTGC-3' (SEQ ID No. 2).
The expected length of the molecular marker PCR fragment: 90-240 bp. Actually, the expected length of the amplified fragment in the genome of vegetable A is 238bp, and the expected length of the amplified fragment in the genome of vegetable C is 94 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 marker is positioned at 8.54Mb of A06 chromosome in a vegetable A genome (according to Chiifu Chinese cabbage V1.0 genome; download address:
http:// fibrous db. org/fiber/datasets/pub/Brassicaceae genome/Brassica _ rapa/V1.0/), located at Chromosome 16.8Mb of C05 in the C genome (according to the cabbage 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 C05B2 of the SSR molecular marker for identifying the segregation condition of the brassica vegetable interspecific hybrid and progeny material A06 and C05 chromosome, the forward primer sequence of the primer C05B2 is shown as SEQ ID No.1, and the backward primer sequence of the primer C05B2 is shown as SEQ ID No. 2.
The SSR molecular marker or primer C05B2 is applied to identification of the segregation condition of the chromosomes of brassica vegetable interspecific hybrids and progeny materials A06 and C05.
Specifically, a primer C05B2 is used as a primer for PCR amplification, a plant to be identified and parent DNA are used as templates for PCR amplification, and a PCR amplification product is detected for band statistics and genotype analysis; the parents are brassica vegetables containing A genomes and brassica plants containing C genomes.
The method for band statistics and genotype analysis comprises the following steps:
the plant to be identified is F of the hybridization of Brassica A genome vegetable and C genome or BC genome plant1When growing plants, if F1The plant shows both A genome banding pattern and C genome banding pattern, so that the plant is a true hybrid; if the tested F1 plant shows only A genome banding pattern or only C genome banding pattern, the plant is false hybrid.
When the plant to be identified is an inbred progeny plant of a distant hybrid of a brassica A genome vegetable and a C genome or BC genome plant, if the detected inbred progeny plant of the distant hybrid shows both an A genome banding pattern and a C genome banding pattern, the plant is a whole or partial heterozygous plant of the A genome vegetable A06 chromosome and the C genome plant C05 chromosome; if the detected distant hybrid selfing progeny plant only shows the A genome banding pattern, the plant does not contain the whole or part of the C05 chromosome of the C genome plant; if the detected distant hybrid self-bred progeny plant only shows the C genome banding pattern, the plant does not contain the whole or part of the A genome vegetable A06 chromosome.
When the plant to be identified is a backcross progeny taking brassica A genome vegetables as backcross parents, if the detected plant shows both A genome banding patterns and C genome banding patterns, the plant contains the whole or part of C05 chromosome of the C genome plant; if only the A genome banding pattern is shown, the plant does not contain the whole or part of C05 chromosome of the C genome plant.
When the plant to be identified is a backcross progeny taking a C (including BC) genome plant as a backcross parent, if the detected plant shows both the A genome banding pattern and the C genome banding pattern, the plant contains the whole or part of the A genome vegetable A06 chromosome; if only the C genome banding pattern is shown, the plant does not contain the whole or part of the A genome vegetable A06 chromosome.
Reaction system of PCR amplification: 10-50 μ L, which comprises: 1 XPCR with Mg+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 an SSR molecular marker and a method for identifying segregation conditions of brassica vegetable interspecific hybrids and progeny materials A06 and C05 chromosomes. The method identifies distant hybridization of genome A and genome C of the brassica vegetable and recombination and separation of chromosomes A06 and C05 of backcross offspring of the genome A and the genome C, can expand genetic resources of the brassica vegetable, transfer 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.
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FIG. 1 is the distant hybrid F of cabbage and Arabidopsis thaliana provided in example 11A polypropylene gel electrophoresis chart of the identification result of the plant; lane 4 is Chinese cabbage, lane 5 is Arabidopsis thaliana, lane 6 is Chinese cabbage × Arabidopsis thaliana F1Hybrid seed. A6 is the Chinese cabbage A06 chromosome characteristic band, and C5 is the Arabidopsis thaliana C05 chromosome characteristic band.
FIG. 2 is the distant hybrid F of cabbage and Arabidopsis thaliana provided in example 11Phenotype of the plant and its parent.
FIG. 3 is the distant hybrid F between cabbage mustard and red bolts as provided in example 21A polypropylene gel electrophoresis chart of the identification result of the plant; lane 1 is red moss, lane 2 is cabbage mustard, lane 3 is cabbage mustard × red moss F1And (4) hybrid. C05 is the characteristic band of cabbage mustard chromosome 5, A06 is the characteristic band of cabbage moss chromosome 6.
FIG. 4 is the distant hybrid F of cabbage mustard and red moss provided in example 21Phenotype of the plant and its parent.
FIG. 5 is the distant hybrid BC of cabbage and Arabidopsis thaliana of example 32A polypropylene gel electrophoresis chart of the identification result of the plant; lanes 1-24 are Chinese cabbage × Russia carinata BC having Chinese cabbage as the backcross parent2And (5) plant growing. A06 is the characteristic band of cabbage A06 chromosome, and C05 is the characteristic band of Arabidopsis thaliana C05 chromosome. It is known that, while all of the chromosomes a06 are contained, plants corresponding to lanes 2, 3, 7, 9, 11, 12, 13, 16, 18 and 23 contain the whole or part of the chromosome C05, and plants corresponding to lanes 1, 4, 5, 6, 8, 10, 14, 15, 17, 19, 20, 21, 22 and 24 do not contain the whole or part of the chromosome C05.
FIG. 6 is a high throughput sequencing chromosome overlay of example 3.
FIG. 7 is a polypropylene gel electrophoresis image of the results of the identification of plants of the BC1 generation from the backcross of the distant hybrids of cabbage mustard and red moss with red moss as provided in example 4; lane a is red moss; lane C is cabbage mustard; lanes 15-22 are (cabbage mustard. times. red bolts). times. red bolts BC1And (4) planting. C05 is a feature band of cabbage mustard chromosome 5, A06 is a feature band of red cabbage moss chromosome 6.
FIG. 8 is a high throughput sequencing chromosome overlay in example 4.
Detailed Description
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.
An SSR molecular marker and a method for identifying segregation conditions of hybrid between brassica vegetable species and progeny material A06 and C05 chromosomes, wherein the identification method comprises the following steps:
extracting genome DNA of a plant to be detected and a parent thereof;
synthesizing a primer:
C05B2-F:5’-TTCTTGTCAGTCCTGTCCCC-3’(SEQ ID No.1)；
C05B2-R:5’-TCCCGAGGTACTTCACTTGC-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)+) 0.5-100ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C05B2-F, 0.5. mu.M primer C05B2-R, 1U Taq enzyme. And (3) PCR reaction conditions: 94-95 deg.C for 0.5-3 min; circulating at 94-95 deg.C for 30S,55-60 deg.C for 30S, and 72 deg.C for 30S, 35; 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: the plants to be identified are F of Brassica A genome vegetables and C (containing BC) genome plants1When growing plants, if F1The plant shows both A genome banding pattern and C genome banding pattern, so that the plant is a true hybrid; if detected F1If the plant only shows the A genome banding pattern or only shows the C genome banding pattern, the plant is a false hybrid.
When the plant to be identified is a distant hybrid selfed progeny plant of the brassica A genomic vegetable and the C (containing BC) genomic plant, if the detected distant hybrid selfed progeny plant shows both the A genomic band type and the C genomic band type, the plant is a whole or partial heterozygous of the chromosome of the brassica A genomic vegetable A06 and the C (containing BC) genomic plant C05; if the detected distant hybrid self-bred progeny plant only shows the A genome banding pattern, the plant does not contain the whole or part of the C05 chromosome of the C (including BC) genome plant; if the detected distant hybrid inbred plant only shows the C genome banding pattern, the plant does not contain the whole or part of the A genome vegetable A06 chromosome.
When the plant to be identified is a backcross progeny taking brassica A genome vegetables as backcross parents, if the detected plant shows both A genome banding patterns and C genome banding patterns, the plant contains the whole or part of C05 chromosome of the C (including BC) genome plant; if only the A genome banding pattern is shown, then the plant does not contain the whole or part of the C (BC containing) genome plant C05 chromosome.
When the plant to be identified is a backcross progeny which takes brassica C (including BC) genome plants as backcross parents, if the detected plant shows both A genome banding patterns and C genome banding patterns, the plant contains the whole or part of A genome vegetable A06 chromosome; if only the C genome banding pattern is shown, the plant does not contain the whole or part of the A genome vegetable A06 chromosome.
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:
C05B2-F:5’-TTCTTGTCAGTCCTGTCCCC-3’(SEQ ID No.1)；
C05B2-R:5’-TCCCGAGGTACTTCACTTGC-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)+) 0.5ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C05B2-F, 0.5. mu.M primer C05B2-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 95 ℃; the temperature of the mixture is 95 ℃ for 30s,30S at 59.8 ℃, 30S at 72 ℃ and 35 cycles; 10min 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 the polyacrylamide gel was removed and developed by silver staining, see FIG. 1.
1.6 band statistics and genotyping: shows both the leucorrhea type and the eruca sativa banding type, this F1The plants are true hybrids.
1.7 phenotypic characterization
The F1The phenotype of the plant and the parent is shown in FIG. 2, in which F is shown1The phenotype of the plant is obviously different from that of the parent, and F is proved1The 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 an interspecific hybrid F of cabbage mustard and red cabbage moss1Plant, its production method and use
1.1 extraction of F to be detected1Genomic DNA of the plant and its parent.
1.2 primer synthesis:
C05B2-F:5’-TTCTTGTCAGTCCTGTCCCC-3’(SEQ ID No.1)；
C05B2-R:5’-TCCCGAGGTACTTCACTTGC-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 15 μ L, which comprises: 1 XPCR Buffer (containing Mg)+) 1ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C05b2-F, 0.5. mu.M primer C05b2-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 94 ℃; 30S at 94 ℃, 30S at 59.8 ℃ 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 the polyacrylamide gel was removed and developed by silver staining, see FIG. 3.
1.6 band statistics and genotyping: f1The plants show the cabbage mustard zone type and the red cabbage moss zone type together, and the plants are true hybrids. If only the maternal banding pattern is shown, the hybrid is false. If neither maternal nor paternal banding is indicated, the test fails. In this example, F1The plants show both a cabbage mustard zone pattern and a red cabbage moss zone pattern, the F1The plants are true hybrids.
1.7 phenotypic characterization
The phenotype of the F1 plant and the parent is shown in FIG. 4, where F is shown1The phenotype of the plant is obviously different from that of the parent, and the F1 plant is proved to be a true hybrid.
Therefore, the molecular marker of the invention can identify the interspecific hybrid F of cabbage mustard and red cabbage moss1And (5) plant growing.
Example 3 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:
C05B2-F:5’-TTCTTGTCAGTCCTGTCCCC-3’(SEQ ID No.1)；
C05B2-R:5’-TCCCGAGGTACTTCACTTGC-3’(SEQ ID No.2)。
1.3PCR amplification. And (3) carrying out PCR amplification reaction by using the primers by using 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)+) 10ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C05B2-F, 0.5. mu.M primer C05B2-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 94 ℃; 30S at 94 ℃, 30S at 59.8 ℃ 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 hour until bromophenol blue runs out of the bottom of the electrophoresis tank to finish electrophoresis.
1.5 the polyacrylamide gel was removed and developed by silver staining, see FIG. 5.
1.6 band statistics and genotyping: detecting distant hybrid BC of Chinese cabbage and brassica carinata by taking the Chinese cabbage as backcross parent2Plants all containWith a06 chromosome, plants No.2, 3, 7, 9, 11, 12, 13, 16, 18, 23 were tested to contain the whole or part of the C05 chromosome, while plants No.1, 4, 5, 6, 8, 10, 14, 15, 17, 19, 20, 21, 22, 24 were tested to contain no whole or part of the C05 chromosome.
1.7 sequencing identification
Carrying out whole genome illumina sequencing on the No. 9 plant after molecular marker identification, wherein the sequencing depth is 10 times; 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 high-throughput sequencing chromosome coverage map of the 9 th plant is shown in fig. 6, and shows that the a06 and the C05 chromosomes are densely and uniformly covered, which indicates that the 2 chromosomes exist in the detected plant, and proves that the molecular marker identification result is reliable. The B04 chromosome coverage was low, which served as a negative control indicating that the chromosome was not present in the plants tested.
EXAMPLE 4 this example identifies progeny (BC) of interspecific hybrid backcrosses between cabbage mustard and red cabbage moss1) Material
1.1 extracting the genome DNA of the plant to be detected and the parent thereof.
1.2 primer synthesis:
C05B2-F:5’-TTCTTGTCAGTCCTGTCCCC-3’(SEQ ID No.1)；
C05B2-R:5’-TCCCGAGGTACTTCACTTGC-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 15 μ L, which comprises: 1 XPCR Buffer (containing Mg)+) 5ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C05b2-F, 0.5. mu.M primer C05b2-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 94 ℃; 30S at 94 ℃, 30S at 59.8 ℃ 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, 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 the polyacrylamide gel was removed and developed by silver staining, see FIG. 7.
1.6 band statistics and genotyping: detecting backcross progeny taking red cabbage moss as backcross parents, wherein the detected No. 16, 17, 18, 19, 20 and 22 plants show a cabbage belt type and a red cabbage moss belt type together, which indicates that the plants contain the whole or part of the cabbage C05 chromosome; the plants tested, No. 15 and 21, showed only the red laver lace pattern, indicating that these plants did not contain the whole or part of the cabbage C05 chromosome.
1.7 sequencing assay
Carrying out illumina sequencing on the 16 # plant with positive molecular marker identification, wherein the sequencing depth is 10 x; 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.
High throughput sequencing chromosome coverage of plant 16 is shown in FIG. 8, which shows dense and uniform coverage of both chromosomes A06 and C05, with an average coverage depth of 8 × or more; the 2 chromosomes are shown to exist in the detected plant, and the molecular marker identification result is proved to be reliable.
In summary, the SSR molecular markers and the method for identifying the segregation of the brassica vegetable interspecific hybrids and progeny materials A06 and C05 chromosomes can identify the interspecific hybrid true hybrids of Chinese cabbage and the Ethiobium aegyuense or red cabbage moss and cabbage mustard, and can also be used for identifying the Chinese cabbage or red cabbage moss A06 chromosomes and the Ethiobium aegyuense or cabbage mustard C05 chromosome addition lines or introgression lines of distant hybrid backcrossed progeny.
Sequence listing
<110> the name of the applicant, vegetable and flower institute of academy of agricultural sciences
<120> molecular marker and method for identifying segregation condition of brassica vegetable interspecific hybrid and progeny material A06 and C05 chromosomes
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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Claims (7)
1. The primer C05B2 of the SSR molecular marker for identifying the segregation condition of the chromosomes of the interspecific hybrid and progeny material A06 and C05 of the brassica vegetables, the forward primer sequence of the primer C05B2 is shown as SEQ ID No.1, the backward primer sequence of the primer C05B2 is shown as SEQ ID No.2, the brassica vegetables are Chinese cabbages or red cabbage sprouts of A genome, and the cabbage of C genome or the Arabidopsis thaliana of BC genome.
2. The application of the primer C05B2 in identifying the segregation condition of the chromosomes of the interspecific hybrids and progeny materials A06 and C05 of the brassica vegetables in claim 1, wherein the primer C05B2 is used as a primer for PCR amplification, the DNA of a plant to be identified and a parent is used as a template for PCR amplification, and the PCR amplification products are detected for strip statistics and genotype analysis; the A genome has an amplified fragment with the banding pattern of 238bp, and the C genome has an amplified fragment with the banding pattern of 94 bp; the brassica vegetable is Chinese cabbage or red cabbage moss with A genome, and mustard blue with C genome or eruca carinata with BC genome.
3. The use according to claim 2, wherein the method of band statistics and genotyping is:
the plants to be identified are F of the A genome and the C genome or the BC genome1When growing plants, if F1The plant shows both A genome banding pattern and C genome banding pattern, so that the plant is a true hybrid; if detected F1If the plant only shows the A genome banding pattern or only shows the C genome 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 distant hybrid selfing progeny plant of the A genome vegetable and the C genome or the BC genome plant, if the detected distant hybrid selfing progeny plant shows both the A genome banding pattern and the C genome banding pattern, the plant is a whole or partial heterozygous plant of the A genome vegetable A06 chromosome and the C05 chromosome of the C genome plant; if the detected distant hybrid selfing progeny plant only shows the A genome banding pattern, the plant does not contain the whole or part of the C05 chromosome of the C genome plant; if the detected distant hybrid self-bred progeny plant only shows the C genome banding pattern, the plant does not contain the whole or part of the A genome vegetable A06 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 A genome vegetable as a backcross parent, if the detected plant shows both the A genome banding pattern and the C genome banding pattern, the plant contains the whole or part of the C05 chromosome of the C genome plant; if only the A genotype is shown, the plant does not contain the whole or part of C05 chromosome of the C genome plant.
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 a C or BC genome plant as a backcross parent, if the detected plant shows a C genome banding pattern and an A genome banding pattern together, the plant contains the whole or part of the A genome vegetable A06 chromosome; if only the C genome banding pattern is shown, the plant does not contain the whole or part of the A genome vegetable A06 chromosome.
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 μ M forward primer shown in SEQ ID No.1, 0.5 μ M backward primer shown in SEQ ID No.2, 1U Taq enzyme; reaction procedure for PCR amplification: 94-95 deg.C for 0.5-3 min; 94-95 ℃ for 30s30S at 55-60 ℃, 30S at 72 ℃ and 35 cycles, and 5-10min at 72 ℃.
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