CN110129481B - Method for simultaneously breeding hot pepper male sterile line and homozygous restoring gene line by restoring gene linked markers - Google Patents
Method for simultaneously breeding hot pepper male sterile line and homozygous restoring gene line by restoring gene linked markers Download PDFInfo
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Abstract
The invention provides a method for simultaneously breeding a pepper male sterile line and a pepper homozygous restoring gene strain by using a restoring gene linked marker. The invention provides a method for identifying individuals with homozygous restorer genes by constructing a male sterile pool and a fertile pool in an F2 generation population. By using the Capsicum-R-82CAPS marker, whether pepper has homozygous restoring gene strains or not can be distinguished, cytoplasmic male sterile single plants can be identified on the DNA level, and the breeding workload increased by sterility caused by environmental factors can be reduced. The original pepper breeding material required by the invention is easy to obtain, can reduce breeding algebra, shorten breeding time and quickly enrich the pepper male sterile three-line material storage of breeders.
Description
Technical Field
The invention belongs to the technical field of breeding, and particularly relates to a method for simultaneously breeding a pepper male sterile line and a homozygous restoring gene line by using a restoring gene linked marker.
Background
The capsicum belongs to the plant of the capsicum genus of the solanaceae family, and is popular with people because of the abundant capsanthin, capsaicin and other substances. At present, the demand for capsorubin is increasing, and the capsorubin is widely applied to the industries of feed additives, cosmetics and medicines. The pigment capsicum is a main raw material for extracting capsicum red pigment, and has been widely popularized in Xinjiang, Gansu, inner Mongolia and the like. However, in production, the special pigment pepper has few varieties and uneven quality, so that the breeding of the first-generation excellent hybrid with high capsorubin content is very important for the development of the pepper industry.
The hot pepper belongs to the common cross pollination crop, has obvious heterosis, and has the advantages of simplifying seed production process, reducing seed reproduction cost, improving hybrid purity and the like by utilizing a male sterile three-line matched breeding technology. In a male sterile three-line matched breeding system, the breeding of a sterile line and a restorer line is very important. Regarding the breeding of the female sterile line of the pepper, besides ensuring 100 percent of male sterility, the breeding method also needs to select materials with a plurality of excellent properties such as good fruiting performance, large fruit number, high capsorubin content, strong resistance and the like. The recovery line material with good quality in the pigment pepper is less, and the breeding of the material with homozygous recovery gene and strong recovery can reduce the popularization risk of the first generation hybrid. Therefore, the conservation of pepper breeding resources is enriched, and the breeding of sterile lines with various excellent properties and a restoring line with homozygous restoring genes and excellent quality becomes the central importance in the matched breeding work of the pepper male sterility three lines.
At present, the breeding of sterile lines in three-line mating hybrid breeding in pepper breeding is mainly completed by multi-generation backcross, selfing or material screening through special marked characters. One of the breeding procedures is: the sterile line is bred by a method of test crossing and backcross of a male sterile source and a plurality of pepper materials, but the breeding period of the method is long, generally more than 6 generations are needed, and the germplasm resources are easy to degrade. The other breeding process is as follows: the method for breeding the sterile line by backcrossing the leaf yellow and the bud yellow as the marker characters with the maintainer line needs special materials and is not suitable for most materials in breeding.
The method for breeding the pepper restorer line mainly comprises a conventional breeding method and molecular marker-assisted screening. One method is as follows: and performing excellent character aggregation by a multi-generation backcross or multi-generation selfing method. However, the method not only needs a large amount of manpower and material resources, but also is very easy to cause the degradation of plants and lose the commodity value. The other method is as follows: hybridizing a cytoplasmic male sterile line of the yellow bud pepper with a selfing line pepper male parent, and screening a restorer line according to a leaf color mark; this method requires a material with yellow shoots and is not suitable for all breeding units. In addition, the restoring line is cultivated after pepper anther culture and doubling, and the method has high operation difficulty and strict requirements on a technical system and experimental conditions. With the development of molecular biology, molecular marker assisted breeding has been widely applied to rice, cabbage and pepper. In the pepper, a restoring gene linked marker can be used for assisting in screening a pepper restoring line. The method has high screening efficiency, but the research only screens molecular markers which can be used for auxiliary screening of the restorer line, and whether the individual plant with homozygous restorer genes is identified is not described. The present invention uses the first generation of hybrid meeting the breeding target as basic material, and can breed plant line with homozygous restoring gene and male sterile line simultaneously to widen the material reserve of breeder.
Disclosure of Invention
The invention aims to provide a method for simultaneously breeding a pepper male sterile line and a pepper homozygous restoring gene strain by using a restoring gene linked marker.
In order to realize the purpose of the invention, the invention is realized by the following technical scheme:
the invention provides a restoring gene linked marker Capsicum-R-82CAPS for simultaneously breeding a hot pepper male sterile line and a homozygous restoring gene strain, wherein the sequence of the linked marker Capsicum-R-82CAPS is as follows:
the invention also provides a method for simultaneously breeding a pepper male sterile line and a pepper homozygous restorer gene line by using the restorer gene linked marker Capsicum-R-82CAPS, and the breeding method comprises the following steps:
(1) constructing a pepper F2 generation population;
(2) identifying the field fertility of the single plant of the pepper F2 generation;
(3) identifying the individual plants with homozygous restorer genes in a pepper F2 population by using the restorer gene linked markers;
(4) when the strain with homozygous restoring gene is bred, a new pepper sterile line is created.
Further: the construction process of the step (1) comprises the following steps:
selecting three-line mating hybrid seeds of hot pepper cytoplasmic male sterility which meet the breeding target of a breeder; breeding the hybrid F1, and planting the hybrid F1 in the isolated net room; selfing is carried out in the flowering phase, isolation is well carried out, and seeds are collected after fruits are ripe, namely F2 generation seeds; f2 seeds are cultivated, isolated and individually planted, and the number of each individual plant is marked; a pepper F2 generation population was established.
Further: the field fertility identification process of the step (2) comprises the following steps:
1) f2 Single plant planting
F2 single plants are subjected to plug seedling in a sunlight greenhouse, and single plant field planting is carried out in an isolation room at the 6-leaf stage;
2) f2 population fertility field survey
In the flowering period, performing fertility investigation on an F2 population, recording the fertility of each plant, and marking a single plant in the field by a tag; wherein, fertility identification is carried out according to the amount of pollen in the flowering phase; it is divided into fertile and sterile.
Further: the fertility is identified as: the pollen grains on the surface of the anther are dense, and the fertility is fertile; otherwise, no pollen grain on the anther is sterile.
Further: the identification process of the step (3) comprises the following steps:
1) in the flowering period of F2 colony, randomly taking DNA of 10 fertile individuals to establish fertile pools and DNA of 10 sterile individuals to establish sterile pools according to the identification result of field fertility; extracting DNA from the F2 single plant, the fertile tank and the sterile tank by a CTAB method;
2) polymorphism screening is carried out in a fertile pool and a sterile pool by utilizing the linkage marker Capsicum-R-82 CAPS; if only one band with the molecular weight of 82bp exists, selecting the single plant with homozygous restoring genes; while the single plants of other belt types are eliminated;
3) and (3) pulling out the heterozygous single plant of the restoring gene locus in the flowering phase by utilizing the result of the identification of the linkage marker, removing the opened flower, selfing and establishing a plant line with the homozygous restoring gene.
Further: the number of the F2 population single plants is in the range of 100-150 strains.
Further: the creation process of the step (4) comprises the following steps:
1) in F2 colony individual plants, the individual plant which is only provided with one band with the molecular weight of 108bp is cytoplasmic male sterile individual plant in the flowering survey and molecular marker identification, and is subjected to label hanging numbering, and test crossing with the existing pepper maintainer line of the same type of a breeder in the flowering period;
2) carrying out seedling raising and field planting on the seeds subjected to test crossing, and carrying out fertility identification in a full-bloom stage;
3) if the hybrid progeny is 100% male sterile in the flowering phase, the hybrid progeny is a created sterile line;
if the sterile rate is 51-99%, continuous backcross is needed until the progeny reaches the sterile rate of 100%, the newly created sterile line is obtained;
if the sterility rate is lower than 50%, the corresponding strains are eliminated.
Compared with the prior art, the invention has the advantages and the technical effects that:
1. the invention establishes a method for rapidly and simultaneously breeding the pigment type pepper restoring gene locus homozygous restoring line and creating the male sterile line, and can reduce the problems of degeneration caused by multi-generation backcross and multi-generation selfing and large hybridization workload in breeding.
2. The invention develops 1 linkage marker which can be used for rapidly identifying whether the strain has homozygous restorer gene or not by using the pigment pepper: (1) Capsicum-R-82CAPS (5 '-3', F: TTCTCATCATAGCATTGCTGTGCAAACT, R: CATCAGGCTTCGGTTAGTCA).
3. The invention provides a method for identifying individuals with homozygous restorer genes in an F2 generation population by constructing a sterile pool and a fertile pool. When the electrophoresis band type of the linked molecular marker in the sterile pool and the fertile pool is that two bands are arranged in the fertile pool, one band with the molecular weight different from that in the sterile pool and the other band with the molecular weight same as that in the sterile pool are the markers for identifying whether the marker has the homozygous restoring gene. Then, the single plant in the fertile pond is identified, and only one band with the molecular weight of 82bp is a single plant with homozygous restoring genes.
4. The marker screened by the method of the invention can not only distinguish whether the pepper has homozygous restoring gene strains, but also identify the single plant of the male sterile line at the DNA level, thereby reducing the influence of environmental factors.
5. The original pepper breeding material required by the invention is easy to obtain, the number of self-breeding generations can be reduced, the breeding time is shortened, and the pepper three-line matched breeding material storage of a breeder is rapidly enriched.
Drawings
FIG. 1 shows the results of screening for polymorphisms of restorer gene linked markers; f represents a fertile pool, and S represents a sterile pool;
FIG. 2 is the result of electrophoresis in the F2 population using the linked molecular marker, Capsicum-R-82 CAPS.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and specific embodiments.
Example 1
The invention takes pepper male sterile three-line hybrid as a base material, and an F2 generation segregating population is constructed in a 40-mesh isolation net room. And (3) constructing a sterile pool and a fertile pool according to the result of the florescence field fertility investigation, and performing polymorphism screening by using the pepper restorer gene linked marker. And (3) as a result of electrophoresis in the fertile pond, when the band type is that two bands are arranged at the position with the molecular weight of 82bp, one band has a molecular weight different from the size in the sterile pond, and the other band has the same molecular weight, the restoring gene linked marker is utilized to identify the restoring gene locus homozygous single plant in the F2 population. And then, screening all fertile individuals in an F2 population by using molecular markers, and selecting the individuals with only one strip and different molecular weights from the individuals in the sterile pool, namely the individuals homozygous for the pepper restoring gene locus. Finally, selecting individual plants for selfing, wherein the progeny is a pepper recovery line with homozygous recovery genes. Meanwhile, the molecular marker and the flowering phenotype are used for identifying and screening sterile single plants, the self-owned maintainer line with excellent properties of the same type of pepper is taken as a male parent, and a new pepper sterile line is bred by adopting a test cross and backcross method. The hybrid line is a homozygous restoring gene line and a new male sterile line which are reserved for pepper male sterile three-line matched hybrid breeding.
The embodiment specifically comprises the following steps:
1. the pepper F2 generation group required for breeding pepper sterile line and strain with homozygous restoring gene is constructed
(1) Selecting the pepper male sterile three-line mating hybrid which meets the breeding target, such as yield, fruit shape, capsorubin content and the like.
(2) The hybrid seeds are cultured in a seedling culture shed, and the hybrid seeds F1 are planted in a 40-mesh isolation net room. Selfing is carried out in the flowering phase, isolation is well carried out, and seeds are collected after fruits are ripe, namely F2 generation seeds.
(3) F2 generation seeds are cultivated in a seedling cultivation shed, single plant field planting is carried out in a 40-mesh isolation net room, and the single plant number is marked by a tag.
(4) And (5) making a record, and preparing fertility identification at the flowering stage.
2. Identification of field fertility of single plant of pepper F2 generation
2.1F2 Individual planting
And (4) carrying out plug seedling on the substrate in the seedling raising shed, and carrying out single plant field planting in a 40-mesh isolation net room in a 6-leaf stage. Wherein the number of F2 population strains is within the range of 100-150, and the following application examples are 123F 2 generation strains.
2.2F2 population fertility field survey
At the flowering stage, fertility investigation was performed on the F2 population, and fertility was recorded for each plant and field individual plant tagging was performed. Wherein fertility identification is performed according to the amount of pollen in the flowering phase. The pollen grains on the surface of the anther are dense, and the fertility is fertile; otherwise, no pollen grain on the anther is sterile.
TABLE 1 Pepper F2 generation population individual plant fertility questionnaire
3. Individual identification of homozygous restorer genes in pepper F2 population using molecular markers
3.1 constructing sterile pool and fertile pool of F2 colony according to the field fertility investigation result of single plant and extracting DNA
And F2, randomly selecting DNA of 10 fertile single plants to establish fertile pools and DNA of 10 sterile single plants to establish sterile pools according to field fertility investigation and fertility identification results. And extracting DNA from the F2 single plant, the fertile tank and the sterile tank by a CTAB method.
3.2 adaptive detection of restorer Gene-linked markers in fertile and sterile pools
Polymorphism screening is carried out on the restorer gene linked markers in the fertile pool and the sterile pool, and the result shows that the Capsicum-R-82CAPS markers have polymorphism (figure 1) and can be used for auxiliary screening of restorer lines, and the two markers belong to co-dominant markers, so that whether the restorer gene sites are homozygous can be distinguished, the restorer lines with homozygous restorer gene sites can be screened more effectively in an auxiliary mode, and the workload of distinguishing whether the restorer gene sites are homozygous is reduced.
TABLE 2 restorer Gene-linked marker information
The fertile pool and sterile pool marked in the pepper F2 colony can be used only by the co-dominant polymorphism, and the single plant homozygous restoring gene identification and screening of the next step is carried out, namely: if in the marker screening, a band with the same molecular weight can be found in both the sterile pool and the fertile pool like the marker AFRF3CAPS (FIG. 1-1), the band pattern has no polymorphism between materials and is not suitable for being applied to the pepper material population to be identified. Neither marker like the marker AFRF1CAPS (FIGS. 1-2) amplified a banded marker nor was it applicable. Markers such as 3336-last2-SCAR (FIGS. 1-3) in which only one band was present in the fertile pool and no target band was present in the sterile pool were not able to be used, although polymorphisms were present between the two pools, but it was not possible to identify whether they had homozygous restorer genes. The marker Capsicum-R-82CAPS (figures 1-4) can have two bands in the fertile pool of the F2 population, one band with molecular weight different from that in the sterile pool and the other band with molecular weight the same as that in the sterile pool, and can distinguish whether the individual plants constructing the fertile pool are the individual plants with homozygous restorer genes or not, so that the marker of the banding pattern can identify the marker with the homozygous restorer genes.
3.3 identification of individuals having homozygous restorer Gene selected from individuals of Capsicum annuum F2 Generation by marker
The results of the detection in the population of generations F2 using the marker, Capsicum-R-82CAPS, are shown in FIG. 2 and Table 3. The comprehensive analysis of the fertility identification result shows that the marker can be used for screening the auxiliary restorer line. Thus, individuals having only 82bp bands were selected as individuals having homozygous restorer genes. Wherein, the numbers of 3, 16, 27, 30, 31, 32, 36, 37, 39, 49, 60, 64, 67, 70, 72, 81, 88, 97, 105, 110, 112, 113, 116, 119, 120 and 123 in the F2 generation population total 26 individuals with homozygous restoring gene loci, and the individuals with other banding patterns are eliminated.
If the applicability of the marker needs to be checked again in a population constructed by other pepper materials, namely after the banding pattern of figures 1-4 is determined, the identification of the homozygous individual plant at the restoring gene locus is carried out, and the identification workload is reduced.
TABLE 3 Individual identification results in the Capsicum F2 population using the marker Capsicum-R-82CAPS
Note: RR represents that the material has homozygous restorer gene; rr represents cytoplasmic male sterile plant material;
rr represents that the material has a heterozygous restorer gene; representative of no amplified bands in the material
3.4 selfing to establish lines with homozygous restorer genes
In the isolated net room, by utilizing the result of molecular marker identification, pulling out the single plant heterozygous at the restoring gene locus in the flowering phase, removing the opened flower, selfing and establishing a plant line with the homozygous restoring gene. Then according to the breeding target and the quality economic character, the excellent recovery strain with homozygous recovery gene is bred.
4. Creating new male sterile line of hot pepper while breeding strain with homozygous restoring gene
4.1 in F2 colony single plant, the flowering phase investigation phenotype is no pollen and the molecular marker identification shows that the single plant with only one molecular weight 108bp strip is cytoplasmic male sterile single plant, by combining the molecular marker identification result and the field identification result, the sterile plant in the F2 colony can be identified more accurately, and the phenomenon of inaccurate field fertility identification caused by plant sterility due to environmental change is prevented. Wherein the individuals in the F2 population are marked with the numbers of 8, 9, 14, 17, 20, 23, 24, 33, 44, 48, 52, 61, 62, 68, 69, 76, 82, 83, 86, 89, 92, 98, 99, 102, 103, 104, 108, 111 and 121 and are male sterile individuals. And (5) carrying out plate-hanging numbering, carrying out test crossing with the existing pepper maintainers of the same type in the flowering phase and recording.
4.2, carrying out seedling raising and planting on the hybridized seeds, and carrying out fertility identification in the full-bloom stage.
4.2.1 if the 100% male sterility of the filial generation in the flowering phase is established, the new sterile line is established;
4.2.2 if the male sterility rate is 51-99%, continuous backcross is needed until the progeny reaches the sterility rate of 100%, and the new created sterile line is obtained;
4.2.3 if the male sterility rate is lower than 50%, the corresponding strains are eliminated.
4.3 using newly created male sterile line of hot pepper as female parent and the existing recovery line of hot pepper of the same type as male parent to prepare hybrid combination, and comparing and evaluating the combining ability of the female parent sterile line.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao agricultural university
<120> method for simultaneously breeding hot pepper male sterile line and homozygous restorer gene line by utilizing restorer gene linked markers
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<213> Artificial Sequence (Artificial Sequence)
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ttctcatcat agcattgctg tgcaaact 28
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<213> Artificial Sequence (Artificial Sequence)
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ccatcaggct tcggttagtc a 21
Claims (8)
1. A restorer gene linked marker Capsicum-R-82CAPS for simultaneously breeding a hot pepper male sterile line and a homozygous restorer gene line is characterized in that: the sequence of the linkage marker, Capsicum-R-82CAPS, is:
F: 5'-TTCTCATCATAGCATTGCTGTGCAAACT-3';
R: 5'-CCATCAGGCTTCGGTTAGTCA-3';
polymorphism screening is carried out in a fertile pool and a sterile pool by utilizing the linkage marker Capsicum-R-82 CAPS; if only one band with the molecular weight of 82bp exists, selecting the single plant with homozygous restoring genes; while the single plants of other belt types are eliminated;
in F2 colony individual plants, the florescence survey shows that the individual plant with male sterility and molecular marker identification only has one band with molecular weight of 108bp is cytoplasmic male sterile individual plant, and the individual plants are subjected to plate hanging and numbering.
2. The method for simultaneously breeding a male sterile line and a homozygous restorer gene line of pepper by using the restorer gene linked marker Capsicum-R-82CAPS of claim 1, wherein the breeding method comprises the steps of:
(1) constructing a pepper F2 generation population;
(2) identifying the field fertility of the single plant of the pepper F2 generation;
(3) identifying the individual plants with homozygous restorer genes in a pepper F2 population by using the restorer gene linked markers;
(4) when the strain with homozygous restoring gene is bred, a new pepper sterile line is created.
3. The breeding method according to claim 2, characterized in that: the construction process of the step (1) comprises the following steps:
selecting three-line mating hybrid seeds of hot pepper cytoplasmic male sterility which meet the breeding target of a breeder; breeding the hybrid F1, and planting the hybrid F1 in the isolated net room; selfing is carried out in the flowering phase, isolation is well carried out, and seeds are collected after fruits are ripe, namely F2 generation seeds; f2 seeds are cultivated, isolated and individually planted, and the number of each individual plant is marked; a pepper F2 generation population was established.
4. The breeding method according to claim 2, characterized in that: the field fertility identification process of the step (2) comprises the following steps:
1) f2 Single plant planting
F2 single plants are subjected to plug seedling in a sunlight greenhouse, and single plant field planting is carried out in an isolation room at the 6-leaf stage;
2) f2 population fertility field survey
In the flowering period, performing fertility investigation on an F2 population, recording the fertility of each plant, and marking a single plant in the field by a tag; wherein, fertility identification is carried out according to the amount of pollen in the flowering phase; it is divided into fertile and sterile.
5. The breeding method according to claim 4, characterized in that: the fertility is identified as: the pollen grains on the surface of the anther are dense, and the fertility is fertile; otherwise, no pollen grain on the anther is sterile.
6. The breeding method according to claim 2 or 4, characterized in that: the identification process of the step (3) comprises the following steps:
1) in the flowering period of F2 colony, randomly taking DNA of 10 fertile individuals to establish fertile pools and DNA of 10 sterile individuals to establish sterile pools according to the identification result of field fertility; extracting DNA from the F2 single plant, the fertile tank and the sterile tank by a CTAB method;
2) polymorphism screening is carried out in a fertile pool and a sterile pool by utilizing the linkage marker Capsicum-R-82 CAPS; if only one band with the molecular weight of 82bp exists, selecting the single plant with homozygous restoring genes; while the single plants of other belt types are eliminated;
3) and (3) pulling out the heterozygous single plant of the restoring gene locus in the flowering phase by utilizing the result of the identification of the linkage marker, removing the opened flower, selfing and establishing a plant line with the homozygous restoring gene.
7. The breeding method according to claim 4, characterized in that: the number of the F2 population single plants is in the range of 100-150 strains.
8. The breeding method according to claim 4, characterized in that: the creation process of the step (4) comprises the following steps:
1) in F2 colony individual plants, the individual plant which is only provided with one band with the molecular weight of 108bp is cytoplasmic male sterile individual plant in the flowering survey and molecular marker identification, and is subjected to label hanging numbering, and test crossing with the existing pepper maintainer line of the same type of a breeder in the flowering period;
2) carrying out seedling raising and field planting on the seeds subjected to test crossing, and carrying out fertility identification in a full-bloom stage;
3) if the hybrid progeny is 100% male sterile in the flowering phase, the hybrid progeny is a created sterile line;
if the sterile rate is 51% -99%, continuous backcross is needed until the sterile rate of the progeny reaches 100%, the new created sterile line is obtained;
if the sterility rate is lower than 50%, the corresponding strains are eliminated.
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CN104561297A (en) * | 2014-12-29 | 2015-04-29 | 浙江省农业科学院 | Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker |
CN107312870A (en) * | 2017-09-04 | 2017-11-03 | 河南省农业科学院园艺研究所 | With molecular labeling, method and the application of capsicum sterile restoring gene close linkage |
CN108300800A (en) * | 2018-04-19 | 2018-07-20 | 河南省农业科学院园艺研究所 | Molecular labeling, primer and the application of hot pepper male sterile restoring gene close linkage |
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CN103598083A (en) * | 2013-09-11 | 2014-02-26 | 山西省农业科学院棉花研究所 | Method of synchronously breeding cotton restorer line and sterile line |
CN104561297A (en) * | 2014-12-29 | 2015-04-29 | 浙江省农业科学院 | Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker |
CN107312870A (en) * | 2017-09-04 | 2017-11-03 | 河南省农业科学院园艺研究所 | With molecular labeling, method and the application of capsicum sterile restoring gene close linkage |
CN108300800A (en) * | 2018-04-19 | 2018-07-20 | 河南省农业科学院园艺研究所 | Molecular labeling, primer and the application of hot pepper male sterile restoring gene close linkage |
CN108330208A (en) * | 2018-05-09 | 2018-07-27 | 绿亨科技股份有限公司 | A kind of molecular labeling of detection capsicum cytoplasmic male sterility restoring gene |
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