CN110499384B - Molecular markers for identifying interspecific hybrids of cabbage mustard and red cabbage moss and tracking the segregation of A08 and C08 chromosomes in progeny materials thereof - Google Patents
Molecular markers for identifying interspecific hybrids of cabbage mustard and red cabbage moss and tracking the segregation of A08 and C08 chromosomes in progeny materials thereof Download PDFInfo
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Abstract
The invention provides a molecular marker for identifying the segregation condition of interspecific hybrids of cabbage mustard and red cabbage moss and progeny materials A08 and C08 chromosomes of the interspecific hybrids, and belongs to the field of plant genetic breeding. The molecular marker for identifying the interspecific hybrid of cabbage mustard and red cabbage moss and the separation condition of the chromosomes of the progeny materials A08 and C08 thereof is a pair of co-dominant molecular markers, and can be used for identifying the interspecific hybrid true hybrid of the cabbage mustard and the red cabbage moss and also can be used for identifying the selfing progeny, the backcross progeny, the chromosome additional line, the recombinant segregation population and the plant of the distant hybrid.
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 kale belongs to cabbage vegetables originated from China, and has the advantages of good resistance, excellent quality and quick harvesting. Is an important bolting vegetable in south China. The red-vegetable moss belongs to a variety 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 Yangtze river basin. 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 C08 chromosomes of cabbage mustard and the a08 chromosomes of red laver 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 C08 of cabbage mustard and A08 chromosome of red cabbage moss in filial generation can obtain new agronomic character, and improve the disease resistance, stress resistance and nutritional quality of vegetables.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a molecular marker and an identification method for identifying the segregation condition of chromosomes of interspecific hybrids of cabbage mustard and red cabbage moss and progeny materials A08 and C08 thereof so as to quickly and accurately identify the interspecific hybrids of cabbage mustard and red cabbage moss and the progeny materials thereof.
The technical scheme of the invention is as follows:
an SSR molecular marker for identifying interspecific hybrids of cabbage mustard and red cabbage moss and tracking the segregation condition of progeny materials A08 and C08 chromosomes, wherein the SSR molecular marker is obtained by amplifying a primer C08a, and the forward primer sequence of the primer C08a is C08a-F: 5'-TTGGTCGCCTTCTTCGTAGT-3' (SEQ ID No. 1); the backward primer sequence of primer C08a was C08a-R: 5'-AGCGAACCTCTCTTGTCCTG-3' (SEQ ID No. 2).
Molecular marker PCR fragment expected length: 270-290 bp. Indeed, the expected length of the amplified fragment in the genome of red cabbage moss (A) is 277bp, and the expected length of the amplified fragment in the genome of cabbage mustard (C) is 283 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 on the chromosomes of the cabbage mustard and the red laver: located at Chromosome 8, 2.76Mb in the genome of Brassica oleracea (according to Chiifu cabbage V1.0 genome; download address: http:// branched. org/branched/dates/pub/Brassicaceae genome/Brassicaceae _ rapa/V1.0/), and located at Chromosome 8, 0.46Mb in the genome of Brassica juncea (C) (according to cabbage _ Chromosome _ V1.1 genome; download address: http:// branched. org/branched/dates/pub/Brassicaceae genome/Brassica _ Chronoma/Bol _ 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 C08a for identifying the interspecific hybrid of cabbage mustard and red cabbage moss and tracking the segregation condition of progeny materials A08 and C08 chromosome is characterized in that the forward primer sequence of the primer C08a is shown as SEQ ID No.1, and the backward primer sequence of the primer C08a is shown as SEQ ID No. 2.
The SSR molecular marker or primer C08a is used in identifying interspecific hybrid between cabbage mustard and red cabbage moss and tracking the chromosome separation of progeny materials A08 and C08.
Specifically, a primer C08a is used as a primer for PCR amplification, a plant to be identified, cabbage mustard and red laver parent DNA are used as templates for PCR amplification, and a 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 F1When growing plants, if F1The plants show the cabbage mustard zone type and the red cabbage moss zone type together, so that the plants are true hybrids; if detected F1If the plant only shows the cabbage mustard zone type or only shows the red cabbage moss zone type, the plant is a false hybrid.
If the detected self-bred progeny plants of the distant hybrids show the cabbage mustard zone type and the red laver zone type together, the plants are heterozygous for the whole or part of the cabbage mustard C08 chromosome and the red laver A08 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage type, the plant does not contain the whole or part of the chromosome of the red laver A08; if the detected self-bred progeny plant of the distant hybrid only shows the red vegetable moss type, the plant does not contain the whole or part of the cabbage C08 chromosome.
When backcross progeny taking cabbage as backcross parents are detected, if the detected plant shows a cabbage belt type and a red cabbage moss belt type together, the plant contains the whole or part of a red cabbage moss A08 chromosome; if only the cabbage type is shown, the plant does not contain the whole or part of the chromosome of the red-laver A08.
When backcross progeny taking the red cabbage moss as backcross parents are detected, if the detected plant shows the cabbage mustard zone type and the red cabbage moss zone type together, the plant contains the whole or part of the cabbage mustard C08 chromosome; if only the red laver type is shown, the plant does not contain the whole or part of the cabbage C08 chromosome.
The PCR amplification parameters were:
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 C08a-F, 0.5. mu.M primer C08a-R, 1U Taq enzyme.
Reaction conditions are as follows: 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 high-throughput sequencing detection.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a codominant molecular marker and an identification method for identifying interspecific hybrids of cabbage mustard and red cabbage moss and progeny materials thereof, and the method can quickly and accurately identify the interspecific hybrids of the cabbage mustard and the red cabbage moss and the progeny materials thereof. The method identifies distant hybridization of cabbage mustard and red cabbage moss and backcross progeny thereof, can expand genetic resources of cabbage mustard and red cabbage moss, transfer new characters, enrich vegetable types, and enrich daily dietary nutrition of people. The method identifies distant hybridization of cabbage mustard and red cabbage moss and recombination and separation of part of homologous (horologous) chromosomes of backcross offspring A08 and C08 of the cabbage mustard and red cabbage moss, can expand germplasm genetic resources of the cabbage mustard and the red cabbage moss, 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.
Drawings
FIG. 1 is a distant hybrid F of cabbage mustard and red bolts as provided in example 1 of the present invention1A 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. C08 is the C08 chromosome characteristic band of cabbage mustard, and A08 is the A08 chromosome characteristic band of red cabbage moss.
FIG. 2 is the distant hybrid F of cabbage mustard and red bolts of example 11Phenotype comparison of plants and their parents.
FIG. 3 is the distant hybrid F of cabbage mustard and red bolts of example 11And (4) tabletting the chromosome of the root tip of the plant.
FIG. 4 shows the backcross BC between the cabbage mustard and the red cabbage moss as the distant hybrid of the cabbage mustard and the red cabbage moss provided in example 2 of the present invention1A polypropylene gel electrophoresis chart of the identification result of the plant generation; lane a is the red moss lane; lane C is cabbage mustard; lane H is cabbage mustard X red moss F1Hybrid; lanes 16, 17, 18 are (cabbage mustard. times. Red bolts). times.BC of Red bolts1And (5) plant growing. C08 is the C08 chromosome characteristic band of cabbage mustard and A08 is the A08 chromosome characteristic band of red cabbage moss.
FIG. 5 is a high throughput sequencing chromosome overlay in example 2.
Detailed Description
The molecular markers and methods for identifying the segregation of the chromosomes of the interspecific hybrids of cabbage mustard and red cabbage moss and the progeny material A08 and C08 of the present invention 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 an identification method for identifying the interspecific hybrid of cabbage mustard and red cabbage moss and the segregation condition of A08 and C08 chromosomes of progeny materials thereof, wherein the identification method comprises the following steps:
extracting genome DNA of a plant to be detected and a parent thereof;
synthesizing a primer:
C08a-F:5’-TTGGTCGCCTTCTTCGTAGT-3’(SEQ ID No.1)；
C08a-R:5’-AGCGAACCTCTCTTGTCCTG-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 C08a-F, 0.5. mu.M primer C08a-R, 1U Taq enzyme. And (3) PCR reaction conditions: 94-95 deg.C for 0.5-3 min; 30S at 94-95 ℃, 30S at 57-60 ℃, 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,
band statistics and genotype analysis: if F1The plants show the cabbage mustard zone type and the red cabbage moss zone type together, so that the plants are true hybrids; if detected F1If the plant only shows the cabbage mustard zone type or only shows the red cabbage moss zone type, the plant is a false hybrid. If the detected self-bred progeny plants of the distant hybrids show the cabbage mustard zone type and the red laver zone type together, the plants are heterozygous for the whole or part of the cabbage mustard C08 chromosome and the red laver A08 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage type, the plant does not contain the whole or part of the chromosome of the red laver A08; if the detected self-bred progeny plant of the distant hybrid only shows the red vegetable moss type, the plant does not contain the whole or part of the cabbage C08 chromosome. When backcross progeny taking cabbage as backcross parents are detected, if the detected plant shows a cabbage belt type and a red cabbage moss belt type together, the plant contains the whole or part of a red cabbage moss A08 chromosome; if only the cabbage type is shown, the plant does not contain the whole or part of the chromosome of the red-laver A08. When backcross progeny taking the red cabbage moss as the backcross parents are detected, if the detected plants show the cabbage mustard typeAnd a red cabbage lace type, the plant contains the whole or part of the cabbage mustard C08 chromosome; if only the red laver type is shown, the plant does not contain the whole or part of the cabbage C08 chromosome.
In addition to polyacrylamide gel electrophoresis, PCR amplification products can be detected by capillary electrophoresis or high throughput sequencing. The genotype determination method is the same.
The features and properties of the present invention are described in further detail below with reference to examples.
EXAMPLE 1 this example identifies interspecific hybrid F of cabbage mustard and red cabbage moss1Plant, method for producing the same and plant
1.1 extraction of F to be detected1Genomic DNA of the plant and its parent.
1.2 primer synthesis:
C08a-F:5’-TTGGTCGCCTTCTTCGTAGT-3’(SEQ ID No.1)；
C08a-R:5’-AGCGAACCTCTCTTGTCCTG-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 C08a-F, 0.5. mu.M primer C08a-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 94 ℃; 30S at 94 ℃, 30S at 57.8 ℃, 30S at 72 ℃ and 35 cycles; 5min 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. 1.
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 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.
1.8 karyotyping
The F1The chromosome pressing of the root tip of the plant is shown in FIG. 3, and the plant is a true hybrid according to the chromosome morphology and number
Therefore, the molecular marker of the invention can identify the interspecific hybrid F of cabbage mustard and red cabbage moss1And (5) plant growing.
Example 2 this example identifies the progeny (BC) of interspecific hybrid backcross between cabbage mustard and red cabbage moss1) Material
1.1 extracting the genome DNA of the plant to be detected and the parent thereof.
Synthesizing a primer:
C08a-F:5’-TTGGTCGCCTTCTTCGTAGT-3’(SEQ ID No.1)；
C08a-R:5’-AGCGAACCTCTCTTGTCCTG-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)+) 1ng template DNA, 0.2mM dNTPs, 0.5. mu.M primer C08a-F, 0.5. mu.M primer C08a-R, 1U Taq enzyme. And (3) PCR reaction conditions: 3min at 95 ℃; 30S at 95 ℃, 30S at 57.8 ℃ 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. 4.
1.6 band statistics and genotyping: detecting backcross progeny taking the red cabbage moss as the backcross parents, wherein the detected 16 th and 17 th plants show the cabbage mustard zone type and the red cabbage moss zone type together, which indicates that the plants contain the whole or part of the cabbage C08 chromosome; the 18 th plants tested exhibited only the red laver lace pattern, indicating that these plants did not contain the whole or part of the cabbage C08 chromosome.
1.7 sequencing identification
Identifying positive 16 th plants by molecular markers, and carrying out illumina sequencing, wherein the sequencing depth is 10X; comparing the reads with the reference genome of A and C, and judging whether the target chromosome exists in the plant according to the coverage degree.
The results are shown in FIG. 5, which shows that the A08 and C08 chromosomes are densely and uniformly covered, and the average coverage depth is more than 8 times; the 2 chromosomes are shown in the detected plant, and the molecular marker identification result is proved to be reliable.
1.8 assay of glucosinolate content As shown in Table 1, BC 16 introduced into C08 chromosome1The total glucosinolate content of the plant is 3-4 times of that of the parent. Glucosinolates are an important anticancer component in cruciferous vegetables; the method is proved to be successful in improving the quality of the vegetables.
TABLE 1 glucosinolate composition and content
In summary, the molecular marker and the identification method for identifying the interspecific hybrid of cabbage mustard and red cabbage moss and the progeny material thereof provided by the embodiment of the invention can identify the interspecific hybrid true hybrid of cabbage mustard and red cabbage moss through a pair of codominant SSR molecular markers, can also be used for identifying the cabbage mustard C08 chromosome and the red cabbage moss A08 chromosome additional line or introgression line of the backcross progeny of distant hybrids, and effectively improve the quality of vegetables.
Sequence listing
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> molecular markers for identifying interspecies hybrids of cabbage mustard and red laver and for tracking the segregation of chromosomes of progeny materials A08 and C08
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttggtcgcct tcttcgtagt 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agcgaacctc tcttgtcctg 20
Claims (7)
1. The primer C08a for identifying the interspecific hybrid of cabbage mustard and red cabbage moss and tracking the segregation condition of progeny materials A08 and C08 chromosome is characterized in that the forward primer sequence of the primer C08a is shown as SEQ ID No.1, and the backward primer sequence of the primer C08a is shown as SEQ ID No. 2.
2. The use of the primer C08a in identifying interspecific hybrids of cabbage mustard and red cabbage moss and tracking the segregation of its progeny A08 and C08 chromosomes according to claim 1, wherein the primer C08a is used as a PCR amplification primer, the DNA of the plant to be identified and the parent DNA of cabbage mustard and red cabbage moss are used as templates for PCR amplification, the PCR amplification products are detected for band statistics and genotype analysis, the band type of cabbage mustard is an amplified fragment of 283bp, and the red cabbage moss is an amplified fragment of 277 bp.
3. Use according to claim 2, characterized in that the method of band statistics and genotyping is: if F1The plants show the cabbage mustard zone type and the red cabbage moss zone type together, so that the plants are true hybrids; if detected F1If the plant only shows the cabbage mustard zone type or only shows the red cabbage moss zone type, the plant is a false hybrid.
4. The use according to claim 2, wherein the method of band statistics and genotyping is: if the detected self-bred progeny plants of the distant hybrids show the cabbage mustard zone type and the red laver zone type together, the plants are heterozygous for the whole or part of the cabbage mustard C08 chromosome and the red laver A08 chromosome; if the detected self-bred progeny plant of the distant hybrid only shows the cabbage type, the plant does not contain the whole or part of the chromosome of the red laver A08; if the detected self-bred progeny plant of the distant hybrid only shows the red vegetable moss type, the plant does not contain the whole or part of the cabbage C08 chromosome.
5. The use according to claim 2, wherein the method of band statistics and genotyping is: when backcross progeny taking cabbage as backcross parents are detected, if the detected plant shows a cabbage belt type and a red cabbage moss belt type together, the plant contains the whole or part of a red cabbage moss A08 chromosome; if only the cabbage type is shown, the plant does not contain the whole or part of the chromosome of the red-laver A08.
6. The use according to claim 2, wherein the method of band statistics and genotyping is: when backcross progeny taking the red cabbage moss as backcross parents are detected, if the detected plant shows the cabbage mustard zone type and the red cabbage moss zone type together, the plant contains the whole or part of the cabbage mustard C08 chromosome; if only the red laver type is shown, the plant does not contain the whole or part of the cabbage C08 chromosome.
7. Use according to any one of claims 2 to 6, wherein the PCR amplification parameters are:
the reaction system is as follows: 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 conditions are as follows: 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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