CN114885830A - Method for cultivating pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding - Google Patents
Method for cultivating pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
- A01H6/822—Capsicum sp. [pepper]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to the technical field of plant breeding, and particularly relates to a cultivation method of pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding. The pepper germplasm carrying target excellent genes is taken as a parent for hybridization, microspores are separated from the full-bloom stage of the first filial generation plant, the microspores are cultivated to obtain a haploid plant, the chromosome doubling is carried out on the plant after the pepper single plant with excellent gene polymerization is obtained by screening, a plurality of excellent genes can be polymerized together in a short time to obtain a plant with gene homozygosis, and the excellent pepper germplasm with multi-gene polymerization is obtained. Meanwhile, the breeding time can be obviously shortened and the breeding efficiency can be improved by adopting the breeding method, and compared with the traditional breeding time (more than 5 years), the breeding time is greatly shortened as the gene homozygous plant obtained by the method only needs 2.5 years.
Description
Technical Field
The invention belongs to the technical field of plant breeding, and particularly relates to a cultivation method of pepper polygene polymerization germplasm based on combination of molecular marker-assisted selection and ploidy breeding.
Background
Sweet pepper, capsicum of the solanaceae family, a subspecies of sweet berry, an annual or perennial herb, one of the peppers. The sweet pepper is evolved from the original pepper in the tropical region of central and south America in North America, and through long-term cultivation and artificial selection, the fruit has the characteristics of increased volume, thickened pulp, reduced pungency, increased heart skin and ovary cavity number and the like. Meanwhile, because the fruit pulp is thick, crisp and tender, the nutrient contents of raw food and cooked food are almost the same, and the fruit pulp is rich in carotenoid, is popular in the international market and has higher price.
In the breeding process of pepper, pepper varieties or inbred lines with multiple excellent traits are usually obtained, however, different excellent genes of pepper are usually located at different positions on different chromosomes, such as root knot nematode resistant genes and non-pungent genes. Therefore, it is not easy to create pepper germplasm including a plurality of excellent genes, and it takes a long time to produce pepper germplasm by a conventional breeding method, and it is difficult to solve the technical problem of obtaining excellent pepper varieties in a short time.
Furthermore, breeding of pepper, especially yellow sweet pepper, in China just starts, bred varieties are few, for example, yellow sweet pepper (non-spicy) breeding materials (strains) for resisting root-knot nematode are rare, the requirement of the market on resisting root-knot nematode yellow sweet pepper varieties cannot be met, and a large amount of root-knot nematode-resisting yellow sweet pepper seeds need to be introduced to abroad every year to relieve the production seeds of the root-knot nematode-resisting yellow sweet pepper. But the imported seeds are expensive, which increases the production cost; imported seeds are produced by people, so that the phenomenon of neck clamping is easily caused, and the healthy development of the sweet pepper industry is influenced; there is also a risk of unknown pest input. Therefore, it is highly desirable to enhance the creation of excellent breeding materials (lines) to meet the needs of society.
Disclosure of Invention
The invention aims to provide a cultivation method of pepper polygenic polymerization germplasm based on the combination of molecular marker-assisted selection and ploidy breeding.
The invention provides a cultivation method of pepper polygene polymerization germplasm based on the combination of molecular marker-assisted selection and ploidy breeding, which comprises the following steps:
1) hybridizing pepper parents comprising excellent genes pairwise to obtain first-filial generation seeds;
2) sowing the first-filial generation seeds to obtain first-filial generation plants;
3) microspores are separated from the first-filial generation plant in the full-bloom stage, microspore cultivation is carried out to obtain a pepper haploid plant, and a pepper single plant with excellent gene polymerization is obtained through screening;
4) carrying out chromosome doubling on the excellent gene polymerized pepper single plant to obtain an excellent gene polymerized pepper diploid plant;
5) performing rooting culture and planting management on the excellent gene polymerized pepper diploid plant to obtain pepper polygene polymerized germplasm;
the pepper parents in the step 1) at least comprise one excellent gene.
Preferably, the pepper parents in the step 1) are all pepper inbred line varieties.
Preferably, when two pepper parents including an excellent gene are hybridized in the step 1), the two pepper parents are the root knot nematode-resistant yellow pepper spicy pepper and the red pepper non-spicy pepper, respectively.
Preferably, the hybridization is reciprocal crossing.
Preferably, the screening in step 4) comprises molecular marker assisted screening; the molecular markers of the excellent genes comprise the molecular markers of a root-knot nematode resistant Me gene, a peppery-taste-free at3 gene and a yellow fruit ccs gene.
Preferably, the detection primer of the molecular marker of the root-knot nematode resistant Me gene is Me-1, the sequence of the forward primer of the Me-1 is shown as SEQ ID No.1, and the sequence of the reverse primer of the Me-1 is shown as SEQ ID No. 2;
the detection primer of the molecular marker of the spicy-taste-free at3 gene is at3-2, the sequence of the forward primer of at3-2 is shown as SEQ ID No.3, and the sequence of the reverse primer of at3-2 is shown as SEQ ID No. 4;
the detection primer of the yellow fruit ccs gene molecular marker is ccs-3, the forward primer sequence of the ccs-3 is shown as SEQ ID No.5, and the reverse primer sequence of the ccs-3 is shown as SEQ ID No. 6.
Preferably, the multi-gene polymeric germplasm of capsicum obtained in the step 5) is a root-knot nematode-resistant yellow fruit pepper without peppery taste.
Preferably, the microspores in step 3) are mononuclear and border-stage microspores.
Preferably, the culture medium for culturing the microspores in the step 3) takes NTh as a basic culture medium, and 0.5mg/L KT, 0.25 mg/L2, 4-D and 4mg/L AgNO are added 3 0.2 wt.% AC, 3 wt.% sucrose, 0.7 wt.% agar, pH 5.8.
Preferably, the method for doubling chromosomes in the step 4) comprises the following steps: wrapping the stem tip of the single pepper plant for 30h by colchicine with the mass concentration of 0.45% to obtain the excellent gene polymerized pepper diploid plant.
Has the advantages that:
the invention provides a method for cultivating hot pepper polygene polymerization germplasm based on the combination of molecular marker-assisted selection and ploidy breeding, which is characterized in that hot pepper germplasm carrying excellent target genes is used as a parent, microspore culture is utilized to obtain haploid plants, chromosome doubling is carried out on the plants after screening, a plurality of excellent genes can be polymerized together in a short time to obtain plants with homozygous genes, and the excellent hot pepper germplasm with polygene polymerization is obtained.
Meanwhile, the breeding time can be obviously shortened and the breeding efficiency can be improved by adopting the breeding method, and compared with the traditional breeding time (more than 5 years), the gene homozygous plant obtained by the method is at least 2.5 years, so that the breeding time is greatly shortened.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 shows the results of the amplification of the primers for root-knot nematode (Me) resistant No-spicy (at3) and yellow fruit (ccs) resistant in example 1 on root-knot nematode, spicy and red fruit-trait Capsicum annuum, respectively.
Detailed Description
The invention provides a cultivation method of pepper polygene polymerization germplasm based on the combination of molecular marker-assisted selection and ploidy breeding, which comprises the following steps:
1) hybridizing pepper parents comprising excellent genes pairwise to obtain first-filial generation seeds;
2) sowing the first-filial generation seeds to obtain first-filial generation plants;
3) microspores are separated from the first-filial generation plant in the full-bloom stage, microspore cultivation is carried out to obtain a pepper haploid plant, and a pepper single plant with excellent gene polymerization is obtained through screening;
4) carrying out chromosome doubling on the excellent gene polymerized pepper single plant to obtain an excellent gene polymerized pepper diploid plant;
5) performing rooting culture and planting management on the excellent gene polymerized pepper diploid plant to obtain pepper polygene polymerized germplasm;
the pepper parents in the step 1) at least comprise one excellent gene.
The pepper parents are preferably pepper inbred line varieties, and the pepper inbred line varieties are more homozygous and have more stable characters. The hybridization according to the invention is preferably a reciprocal cross. The variety of the pepper parent is not specially limited, and the pepper parent only needs to carry corresponding excellent genes according to breeding targets.
The invention crosses pepper parents comprising excellent genes pairwise to obtain first-filial generation seeds. The pepper parents of the present invention comprise at least one excellent gene, and more preferably the excellent genes contained in the pepper parents are different from each other. The specific type of the excellent gene and the number of the excellent genes carried by each pepper parent are not particularly limited, and the excellent genes are selected according to breeding targets. The number of pepper parents is not limited in the invention, and two-parent hybridization or multiple-parent hybridization belong to the protection scope of the invention. The number of rounds of hybridization is not particularly limited, and one round of hybridization or multiple rounds of hybridization are all within the scope of the present invention. When the present invention preferably comprises a plurality of pepper parents, the present invention preferably further comprises pairwise crossing the plurality of pepper parents, and continuing pairwise crossing the resulting filial generation until two pepper material remain; and hybridizing the remaining two pepper materials serving as parents to obtain first-filial generation seeds.
When the pepper parents of the present invention are preferably two pepper parents that comprise excellent genes, the two pepper parents are preferably root knot nematode resistant yellow pepper peppery and red pepper pepperless peppery. The root-knot nematode-resistant yellow-fruit peppery pepper and the red-fruit peppery-free pepper are hybridized to obtain the first-filial generation seeds.
The root-knot nematode resistant yellow-fruit spicy pepper disclosed by the invention preferably carries a root-knot nematode resistant gene Me, a normal capsaicin synthase gene AT3 and a mutated capsorubin synthase gene ccs. The normal capsaicin synthetase gene AT3 has the accession number AY819026 in NCBI; the mutant capsorubin synthase gene CCS is preferably a deletion of 240 bases at the 3' end of a normal capsorubin synthase gene CCS having an accession number of KM037687 in NCBI; the marker of the root knot nematode resistant gene Me is an SCAR molecular marker. The chilli fruit non-spicy chilli fruits of the invention preferably carry a mutated capsaicin synthetase gene at3 and a normal capsaicin synthetase gene CCS. The accession number of the mutant capsaicin synthetase gene at3 in NCBI is FJ 871985.
After the first filial generation seeds are obtained, the first filial generation seeds are sown to obtain first filial generation plants. The sowing mode is not particularly limited, and the conventional sowing mode in the field can be adopted.
After the first-filial generation plant is obtained, microspores are separated from the first-filial generation plant in the full-bloom stage and are cultured to obtain a pepper haploid plant. The microspores of the present invention are preferably those in the unikaryon penultimate stage. The microspores are placed on a culture medium to culture the microspores, the culture medium for culturing the microspores preferably takes NTh as a basic culture medium and also comprises 0.5mg/L KT, 0.25 mg/L2, 4-D and 4mg/L AgNO 3 0.2 wt.% AC, 3 wt.% sucrose, 0.7 wt.% agar, pH 5.8, the percentages of AC, sucrose and agar preferably being mass percentages of the culture medium of microspore cultivation. The preferable conditions for culturing the microspores comprise dark culture, light-dark alternate culture until embryoid is formed, and illumination culture until the pepper haploid plant is obtained. The temperature of the dark culture is preferably 35 ℃, and the duration of the dark culture is preferably 7 days. The light-dark alternate culture period is preferably performed alternately in normal day and night, the temperature in the day is preferably 25-27 ℃, and the temperature in the night is preferably 20-22 ℃. The illumination intensity of the illumination culture is preferably 2000lux, and the temperature of the illumination culture is preferably 25-27 ℃. The length of the pepper haploid plant obtained by culturing the microspore is preferably selectedIs 3-4 cm.
After the pepper haploid plant is obtained, the pepper haploid plant is screened to obtain a pepper single plant with excellent gene polymerization. The screening according to the invention is preferably a molecular marker assisted screening. The invention preferably extracts the genome DNA of the capsicum single-plant, respectively adopts the molecular markers of the root-knot nematode resistant Me gene, the non-pungent at3 gene and the yellow fruit ccs gene to carry out PCR amplification, and obtains the capsicum single-plant with excellent gene polymerization according to the amplification result. The method for extracting the genomic DNA is not particularly limited, and a conventional genomic DNA extraction method in the field can be adopted. The detection primer of the root-knot nematode resistant Me gene molecular marker is preferably Me-1, and the forward primer sequence of the Me-1 is shown as SEQ ID No.1, and specifically comprises the following steps: 5 '-GAAGCTTATGTGGTAMCC-3', the reverse primer sequence of ME-1 is shown in SEQ ID NO.2, and specifically comprises: 5'-GCAAAGTAATTATATGCAAGAGT-3' are provided. The detection primer of the spicy-taste-free AT3 gene molecular marker is AT3-2, and the sequence of the forward primer of the AT3-2 is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-TGGCAGTTTCCCTTCTCTC-3', the reverse primer sequence of the AT3-2 is shown as SEQ ID NO.4, and specifically comprises: 5'-GGGAATAGCCATCAGTGTATGCTTTTCG-3', respectively; the non-spicy character is generated by mutation of a normal capsaicin synthetase gene AT3, and one primer is in a mutation region when the primer is designed, so that the normal capsaicin synthetase gene can be amplified to a strip, the pepper character is represented, and the mutation of the AT3 gene can be detected without description, and the non-spicy character is represented. The detection primer of the yellow fruit CCS gene molecular marker is CCS-3, the sequence of the forward primer of the CCS-3 is shown as SEQ ID NO.5, and the detection primer specifically comprises the following components: 5'-CCTTTTCCATCTCCTTTACTTTCCATT-3', the reverse primer sequence of CCS-3 is shown in SEQ ID NO.6, which specifically comprises: 5'-AAGGCTCTCTATTGCTAGATTGCCCAG-3' are provided. The yellow fruit character of the invention is generated by the mutation of the normal capsorubin synthetase gene CCS, and one primer is arranged in a mutation region when the primer is designed, so that the normal capsorubin synthetase gene can be amplified to a strip, and the yellow fruit character is represented by the red fruit character, and the detection fails to indicate that the CCS gene is mutated. The system and procedure for PCR amplification in the present invention is not particularly limited, and any conventional system and procedure in the art may be used.
After the PCR amplification is completed, the present invention preferably further comprises subjecting the obtained PCR amplification product to electrophoresis to confirm the screening of the individual Capsicum annuum containing the above excellent gene. The invention has no special limitation on the specific flow of electrophoresis, and the conventional electrophoresis flow in the field can be adopted.
After obtaining the pepper individual plant with excellent gene polymerization, the invention performs chromosome doubling on the pepper individual plant with excellent gene polymerization to obtain a pepper diploid plant with excellent gene polymerization. The method for chromosome doubling according to the invention preferably comprises: wrapping the stem tip of the single pepper plant for 30h by colchicine with the mass concentration of 0.45% to obtain the excellent gene polymerized pepper diploid plant. According to the invention, the stem tip of the single pepper plant is wrapped after the cotton is soaked in colchicine.
After obtaining the excellent gene polymerized pepper diploid plant, the invention carries out rooting culture on the excellent gene polymerized pepper diploid plant to obtain pepper plantlets. The culture medium for rooting culture preferably takes 1/2MS culture medium as a basic culture medium, and also comprises 0.1mg/LNAA, and the pH value is 5.8. The rooting culture is preferably light-dark alternate culture, more preferably day-night alternate culture, the day culture temperature is preferably 25-27 ℃, and the night culture temperature is preferably 20-22 ℃. The illumination intensity of the rooting culture is preferably 2000lux, and the illumination culture temperature is preferably 25-27 ℃.
After the pepper plantlets are obtained, the method carries out planting management on the plantlets to obtain the pepper polygene polymerization germplasm. The planting management of the invention preferably comprises seedling exercising, transplanting, bagging in flowering phase and harvesting seeds according to single plants. The specific steps of the planting management are not particularly limited, and the conventional planting management steps in the field can be adopted. The multi-gene polymeric seed substance of the capsicum obtained by the invention is preferably yellow fruit peppery pepper with root-knot nematode resistance.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the examples and the accompanying drawings, but they should not be construed as limiting the scope of the present invention.
Example 1
The breeding method of the new spicy pepper strain of yellow fruit capable of resisting root-knot nematode comprises the following steps:
materials: the pepper inbred line '8731' (obtained from pepper germplasm resource library of garden gardening academy of agriculture and horticulture of Yunnan university) carries a root knot nematode resistant gene (Me), a normal capsaicin synthase gene (AT3, AY819026) and a mutated capsorubin synthase gene (ccs) (240 bases are deleted AT the end of KM037687), and shows root knot nematode resistance, yellow fruit resistance and peppery taste; the capsicum inbred line '9917' carries a mutated capsaicin synthetase gene (at3) (FJ871985) and a normal capsorubin synthetase gene (CCS, KM037687), turns into red fruit and has no spicy taste.
Taking a pepper inbred line '8731' and a pepper inbred line '9917' as parents (obtained from a pepper germplasm resource library of garden gardening academy of Yunnan agricultural university), and carrying out cross breeding on the pepper inbred line and the pepper inbred line in 2017 for 5 months to obtain cross-breeding first-generation seeds. Sowing in 12 months in 2017, culturing the plant to full-bloom stage, respectively taking pollen in positive and negative cross first generation mononuclear edgewise stage in 3 months in 2018, and placing the pollen in NTh + KT 0.5mg/L +2, 4-D0.25 mg/L + AgNO 3 4mg/L + AC 0.2 wt.%, sucrose 3 wt.%, agar 0.7 wt.%, and on a culture medium with pH of 5.8, performing dark treatment at 35 ℃ for 7 days, then culturing at 26 +/-1 ℃ in the daytime and 21 +/-1 ℃ at night, and after an embryoid is formed, moving to 2000lux light for continuous culture to obtain a haploid plant with the length of 3-4 cm.
Extracting haploid plant DNA in 2018 and 9 months, carrying out PCR amplification screening by using molecular markers closely linked with root-knot nematode (Me) (forward primer, SEQ ID No.1, reverse primer, SEQ ID No.2), non-peppery (at3) (forward primer, SEQ ID No.3, reverse primer, SEQ ID No.4), yellow fruit (ccs) (forward primer, SEQ ID No.5, reverse primer and SEQ ID No.6), and screening pepper haploid single plants with root-knot nematode (Me), non-peppery (at3) and yellow fruit (ccs) resistance polymerized by 3 genes
Wherein, the primer (forward primer, SEQ ID No.1, reverse primer and SEQ ID No.2) for resisting root-knot nematode (Me) can be amplified to obtain a specific product of 250bp, and the pepper haploid single strain which has no pepper (at3) and no amplification band of yellow fruit (ccs) is polymerized by 3 genes of resisting root-knot nematode (Me), no pepper (at3) and yellow fruit (ccs).
The design rationality of the primers for identifying the genes of the non-spicy (at3) and the yellow fruit (ccs) in the invention is proved by respectively amplifying primers of the non-spicy (at3) (a forward primer, SEQ ID No.3, a reverse primer, SEQ ID No.4) and the yellow fruit (ccs) (a forward primer, SEQ ID No.5, a reverse primer and SEQ ID No.6) on a pepper plant with spicy taste and red fruit, amplifying a primer of the non-spicy (at3) to obtain a product of 949bp and amplifying a primer of the yellow fruit (ccs) to obtain a product of 1476bp (as shown in figure 1).
Dipping 0.45 percent of colchicine in cotton containing the 3 gene polymerized haploid plants in 2018 and 10 months, and doubling chromosomes when wrapping stem tips for 30 hours to obtain diploid plants; placing the diploid plant in 1/2MS + NAA 0.1mg/L, pH 5.8, culturing at 26 +/-1 ℃ in the day and 21 +/-1 ℃ in the evening under 2000lux for rooting, hardening and transplanting the plantlet, bagging in the flowering phase, harvesting seeds according to the single plant in 2019 for 6 months, and obtaining 16 single plants with 3 excellent character polymerization (the method for screening the gene polymerization pepper haploid single plant is the same and is not repeated) through gene detection to obtain the pepper with the non-pungent taste of the root-knot nematode yellow fruit, wherein the whole culturing process is 2.5 years in total.
The obtained root-knot nematode-resistant yellow fruit pepper-free pepper germplasm is subjected to a planting test, and the method specifically comprises the following steps:
in 2020 and 2021, planting is carried out on plots with root-knot nematodes such as Kunming, Wenshan and Dehong, and the root-knot nematode resistance, fruit color and pungency (capsaicin content) are mainly evaluated. The average data of 3 different test points in 2 years are counted.
Wherein, the root knot nematode resistance is determined according to NY/T2060.1-2011, the disease resistance identification technical specification of the pepper, part 5: identifying the technical specification of identifying the southern root knot nematode resistance of the pepper;
whether the peppery taste exists or not is detected according to the methods of measuring capsaicin substances and expressing the peppery degree in the GB/T21266-2007 pepper and pepper products, if one of the capsaicin substances and the dihydrocapsaicin substances can be detected, the peppery taste exists, and if the two monomers can not be detected, the peppery taste does not exist. The results are shown in Table 1, where the capsaicin content is the total amount of capsaicin and dihydrocapsaicin in Table 1.
TABLE 1 quality and yield index results for root-knot nematode resistant yellow fruit pepperless pepper germplasm
The embodiments can show that the breeding method provided by the invention can aggregate a plurality of excellent genes together in a short time to obtain a plant with homozygous genes, obtain excellent pepper germplasm with polymerized multiple genes, and shorten the breeding time by more than 2 years compared with the breeding time of the traditional breeding method.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
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Claims (10)
1. A cultivation method of pepper polygene polymerization germplasm based on combination of molecular marker assisted selection and ploidy breeding is characterized by comprising the following steps:
1) hybridizing pepper parents comprising excellent genes pairwise to obtain first-filial generation seeds;
2) sowing the first-filial generation seeds to obtain first-filial generation plants;
3) microspores are separated from the first-filial generation plant in the full-bloom stage, microspore cultivation is carried out to obtain a pepper haploid plant, and a pepper single plant with excellent gene polymerization is obtained through screening;
4) carrying out chromosome doubling on the excellent gene polymerized pepper single plant to obtain an excellent gene polymerized pepper diploid plant;
5) performing rooting culture and planting management on the excellent gene polymerized pepper diploid plant to obtain pepper polygene polymerized germplasm;
the pepper parents in the step 1) at least comprise one excellent gene.
2. The cultivation method as claimed in claim 1, wherein the pepper parents in step 1) are all pepper inbred line varieties.
3. The breeding method as claimed in claim 1, wherein when two pepper parents including excellent genes are crossed in the step 1), the two pepper parents are the root knot nematode resistant yellow pepper and the red pepper non-peppery pepper, respectively.
4. A method of cultivation as claimed in claim 3, wherein the cross is a reciprocal cross.
5. The method of claim 3, wherein the screening of step 4) comprises molecular marker assisted screening; the molecular markers of the excellent genes comprise the molecular markers of a root-knot nematode resistant Me gene, a peppery-taste-free at3 gene and a yellow fruit ccs gene.
6. The cultivation method as claimed in claim 5, wherein the detection primer of the molecular marker of the root-knot nematode resistant Me gene is Me-1, the sequence of the forward primer of Me-1 is shown in SEQ ID No.1, and the sequence of the reverse primer of Me-1 is shown in SEQ ID No. 2;
the detection primer of the molecular marker of the spicy-taste-free at3 gene is at3-2, the sequence of the forward primer of at3-2 is shown as SEQ ID No.3, and the sequence of the reverse primer of at3-2 is shown as SEQ ID No. 4;
the detection primer of the yellow fruit ccs gene molecular marker is ccs-3, the forward primer sequence of the ccs-3 is shown as SEQ ID No.5, and the reverse primer sequence of the ccs-3 is shown as SEQ ID No. 6.
7. The cultivation method as claimed in any one of claims 1 to 6, wherein the polygenic polymeric germplasm of capsicum obtained in step 5) is root-knot nematode-resistant yellow fruit pepper with no peppery taste.
8. A culture method according to any one of claims 1 to 6, wherein the microspores in step 3) are unicellular marginal-stage microspores.
9. The culture method according to any one of claims 1 to 6, wherein the culture medium for microspore culture in step 3) is NTh basal medium supplemented with 0.5mg/LKT, 0.25 mg/L2, 4-D, 4mg/LAgNO 3 0.2 wt.% AC, 3 wt.% sucrose, 0.7 wt.% agar, pH 5.8.
10. A breeding method according to any one of claims 1 to 6, wherein the chromosome doubling in step 4) comprises: wrapping the stem tip of the single pepper plant for 30h by colchicine with the mass concentration of 0.45% to obtain the excellent gene polymerized pepper diploid plant.
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