CN115462314A

CN115462314A - Rapid pepper cultivation method using molecular marker

Info

Publication number: CN115462314A
Application number: CN202211240606.3A
Authority: CN
Inventors: 李明; 吴新荣; 黄晓川
Original assignee: Zhongke Xiliang Functional Agriculture Research Co ltd
Current assignee: Zhongke Xiliang Functional Agriculture Research Co ltd
Priority date: 2022-10-11
Filing date: 2022-10-11
Publication date: 2022-12-13

Abstract

The invention relates to a rapid pepper cultivation method by using molecular markers, which relates to a related series of molecular markers of a pepper specific inbred line, and applies various molecular biological technologies, including but not limited to related sequence deletion, addition and point mutation; or chemically synthesizing corresponding primers, and establishing an analysis model on the basis, wherein the method is characterized in that different inbred lines can be effectively distinguished at low cost to determine the purity of the inbred lines; the method is characterized in that a pepper immature embryo sterile separation and culture technical system is established, and immature embryos are generally from immature fruits pollinated for 12-20 days, so that the method can be applied to any plant, and the purification time after hybridization can be effectively shortened; by developing a series of molecular markers of specific pepper inbred lines and establishing an analysis model, different pepper inbred lines can be effectively identified and distinguished at low cost, purification of the inbred lines after specific excellent character transformation and hybridization of the peppers is quickly realized by combining a pepper young embryo culture technology and an all-weather greenhouse culture system, and the time required by 4-5 generations of backcross purification is controlled within 12-15 months.

Description

Rapid pepper cultivation method using molecular marker

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a rapid pepper cultivation method by using molecular markers.

Background

Molecular markers (molecular markers), genetic markers based on nucleotide sequence variations in the genetic material between individuals, are a direct reflection of genetic polymorphisms at the DNA level. Compared with other genetic markers, namely morphological markers, biochemical markers and cytological markers, the DNA molecular marker has the following advantages: most molecular markers are co-dominant, and selection of recessive characters is very convenient; the genome variation is extremely abundant, and the number of molecular markers is almost unlimited; at different stages of biological development, DNA from different tissues can be used for marker analysis; molecular markers reveal changes from DNA

Iso; the expression is neutral, the expression of target characters is not influenced, and no linkage exists between the target characters and undesirable characters; the detection means is simple and rapid. With the development of molecular biology technology, there are dozens of DNA molecular marker technologies, and the DNA molecular marker technologies are widely applied to aspects such as genetic breeding, genome mapping, gene localization, species genetic relationship identification, gene library construction, gene cloning and the like. The ideal molecular marker must meet several requirements: the method has high polymorphism; the method comprises the following steps of (1) co-dominant inheritance, namely, heterozygous and homozygous genotypes in the diploid can be identified by utilizing a molecular marker; the allele can be clearly distinguished; fourthly, the whole genome is distributed; fifthly, requiring that molecular markers are uniformly distributed in the whole genome except for markers of special sites; sixthly, selecting neutrality (namely nongenic pleiotropic); the detection method is simple and quick (the method is easy to automate like an experiment program); development cost and use cost are as low as possible; the self-checking feature is highly reproducible both in and between laboratories (facilitating data exchange). However, any of the molecular markers found separate the different biological DNA molecules by electrophoresis, which are then hybridized with labeled specific DNA probes to reveal DNA polymorphisms by autoradiography or non-isotopic visualization techniques.

Molecular marker technology has been developed rapidly and is widely used in genetic research of animals and plants. In the molecular marker, a plurality of application researches are carried out in breeding and production of animals and plants such as corn, soybean, chicken, pig and the like, the application researches are mainly focused on the aspects of gene positioning, auxiliary breeding, disease treatment and the like, and a plurality of application achievements are obtained. The development of molecular labeling technology is a hot spot in the field of molecular biology. With the rapid development of molecular biology theory and technology, a molecular marking technology with higher analysis speed, lower cost and larger information content must be developed. The combination of molecular marking technology, extraction programming, electrophoresis film analysis automation, information (data) processing and computerization will speed up the construction of genetic map, gene location, gene cloning, species genetic relationship identification and the diagnosis and analysis of human related pathogenic gene.

In recent years, the development of high-throughput genotyping technology and the increase in computational power have made it possible to perform association studies on genome-wide sets of genetic variations, a method known as genome-wide association studies (GWAS), which has greatly changed the localization of quantitative traits. While low resolution and time consuming methods (such as linkage mapping) are commonly used to map parental populations, the advent of GWAS provides the opportunity to find genes or regions associated with a given trait in a relatively high resolution and unbiased manner in a wide and diverse population. GWAS may also reveal the global pattern of a trait, i.e., its genetic structure, which term is used to describe the genetic basis of a trait based on information about the number of disease-causing genes or alleles, their interactions, and their distribution and pattern of influence. In the plant breeding practice, the method of artificially controlling self-pollination is adopted, bad plant rows are continuously eliminated through a plurality of generations (generally 5-8 generations, which need 3-5 years), and single plants with better agronomic characters are selected for selfing, so that the obtained agronomic characters are more orderly

Lines with consistent and relatively simple genetic basis are called inbred lines. The hybrid progeny with strong viability and high yield generated by the cross between the excellent inbred lines is called the hybrid between the inbred lines. The agronomic character is superior to the variety or the hybrid between the varieties, and the yield is increased by 20 to 30 percent usually. Based on the fact that the application is more in corn and partial vegetable crops such as pepper, the applicant hopes to provide a method utilizing molecular markers so as to solve the problems of long purification process, low purification efficiency and high cost of plants in the hybridization or selfing process.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a rapid pepper cultivation method by utilizing molecular markers, which is used for solving the problems of long purification process, low purification efficiency and high cost of pepper plants in the prior art in the hybridization or selfing process in the background art.

The technical purpose of the invention is realized by the following technical scheme:

a rapid pepper cultivation method using molecular markers is characterized by comprising the following two steps:

the method comprises the following steps: establishing an analysis model by developing a series of molecular markers of a special pepper inbred line;

step two: and (3) combining a pepper immature embryo culture technology and an all-weather greenhouse culture system, and quickly purifying the inbred line after the specific excellent character transformation and hybridization of the pepper.

Preferably, one of the steps comprises the following steps:

the first step is as follows: selecting different pepper core inbred line seeds, adopting a conventional cultivation and planting technology, adopting a completely random block design in a field experiment, and repeating for 2-3 times;

the second step: acquiring important agronomic character data;

the third step: sampling at different growth periods, and determining related biochemical and physiological indexes;

the fourth step: sampling (leaves) before the ridge sealing period, extracting DNA, sequencing and obtaining SNP data;

the fifth step: analyzing genetic variation and population structure, discovering high-quality SNP obviously associated with important agronomic and biochemical traits according to the principle of linkage disequilibrium, and developing corresponding molecular markers;

and a sixth step: SNP and molecular markers are used for constructing a data analysis model, and different pepper inbred lines can be distinguished through DNA analysis.

Preferably, the agronomic trait data to be obtained in the second step include growth period, plant height, disease resistance, yield.

Preferably, the related biochemical and physiological indicators measured in the third step include the inhibition activity indicators of the extracts of different pepper lines on alpha-glucosidase.

Preferably, there are no more than 25 immature embryos on the plate of solid medium in step seven.

Preferably, the second step comprises the following steps:

the first step is as follows: planting pepper inbred line seeds in a greenhouse in a flowerpot containing a soilless substrate, and planting by adopting a conventional cultivation and planting technology;

the second step is that: selecting young pepper fruits, and washing away dust on the surfaces of the fruits;

the third step: putting young fruits into a beaker in an ultraclean workbench, pouring 75% alcohol for disinfection for 30 seconds, removing the alcohol, pouring 0.5% sodium hypochlorite solution for disinfection for 5-10 minutes, and washing with sterile water for 3 times;

the fourth step: using a sterile scalpel to longitudinally take the fruit, and using sterile forceps to take out sterile young embryos in the fruit;

the fifth step: immersing the separated immature embryos in 2.0 ml microcentrifuge tubes in 1 ml of liquid culture medium until the immature embryo separation process is completed;

and a sixth step: centrifuging briefly with a bench centrifuge, and removing the liquid culture medium;

the seventh step: transferring the young embryo to a solid culture medium by using a forceps, sealing a culture dish by using a sealing film, and planting by using a conventional planting technology;

eighth step: sampling pepper plantlets at a height of 5-10 cm for DNA analysis, screening plants meeting the requirements, and transferring the plants to a greenhouse for cultivation;

the ninth step: repeating the steps one to eight until obtaining homozygous plants which meet the target.

Preferably, the conventional cultivation and planting techniques described in the first step and in the seventh step use: 16/8 (day/night) light cycle, average temperature 27 ℃ in the day and 21 ℃ in the night.

Preferably, the young fruits of the peppers in the second step are olives obtained 12 to 20 days after pollination of the peppers.

Preferably, the pepper plants used to select the olives are pepper plants obtained by crossing or selfing.

Preferably, there are no more than 25 immature embryos on the plate of solid medium in the seventh step.

Preferably, the liquid culture medium is beef extract peptone liquid culture medium, the solid culture medium is liquid culture medium, about 1.5-2.0% of agar is added into the liquid culture medium, the mixture is heated to 100 ℃ for dissolution, and the solid culture medium is obtained after cooling and solidification at 40 ℃ and becomes a solid state.

The invention has the beneficial effects that:

1. the invention relates to a pepper specific inbred line related series molecular marker, which applies a plurality of molecular biology techniques, including but not limited to related sequence deletion, addition and point mutation; or chemically synthesizing corresponding primer, and establishing analysis model, and it features that different selfing lines can be effectively distinguished at low cost to determine their purity.

2. The invention establishes a capsicum young embryo sterile separation and culture technical system, and the young embryo is generally from young fruit pollinated for 12-20 days, so the invention can be applied to any plant and can effectively shorten the purification time after hybridization.

3. By developing a series of molecular markers of the special pepper inbred lines and establishing an analysis model, different pepper inbred lines can be effectively identified and distinguished at low cost, and purification of the inbred lines after specific excellent character transfer hybridization of the peppers is quickly realized by combining a pepper young embryo culture technology and an all-weather greenhouse culture system. The method is characterized in that the time required by the 4-5 generation backcross purification can be controlled within 12-15 months.

Drawings

FIG. 1 is a block flow diagram of the present invention.

FIG. 2 is a block diagram of the steps of developing SNP molecular markers and analytical models of a specific excellent inbred line of capsicum annuum in the invention.

FIG. 3 is a block diagram of the procedure for harvesting young pepper fruits and culturing young pepper embryos according to the present invention.

FIG. 4 is a brief introduction of SNPs localized on the first chromosome in the isolated population of Capsicum annuum according to the present invention.

Detailed Description

To further clarify the contents, features and effects of the present invention, the following examples are given by way of illustration and the following detailed description.

The system for rapidly cultivating the pepper by utilizing the molecular marker comprises the following technical steps:

(1) Developing a molecular marker and an analysis model of the special excellent pepper inbred line, and effectively distinguishing the special inbred line.

(2) An all-weather greenhouse pepper cultivation system is established, indoor annual pepper planting is realized, and the influence of external climate factors is avoided.

(3) And (3) establishing a sterile separation and cultivation system for pepper immature embryos, wherein the pepper immature embryos are generally from immature fruits 12-20 days after pollination.

The method comprises the following steps: experimental procedures without specific conditions noted in the examples below, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or the conditions as suggested in the literature.

Harvesting young pepper fruits and culturing young embryos

1. Planting the selfing line seeds of the hot pepper in a greenhouse in a flowerpot containing a soilless substrate, and adopting a conventional cultivation and planting technology. A16/8 (day/night) photoperiod was used with an average temperature of 27 ℃ during the day and 21 ℃ at night.

2. Picking green fruits obtained 12-20 days after pollination of hot peppers (hybridization or selfing), and washing off dust on the surfaces of the fruits.

3. And (4) putting the young fruits into a beaker in an ultra-clean workbench, and pouring 75% alcohol for disinfection for 30 seconds. Removing alcohol, adding 0.5% sodium hypochlorite solution for sterilization for 5-10 min, and washing with sterile water for 3 times.

4. The fruit is vertically cut by a sterile scalpel, and the sterile young embryo in the fruit is taken out by a sterile forceps.

5. The separated embryos are immersed in 2.0 ml microcentrifuge tubes in 1 ml of medium until the embryo separation process is complete.

6. The liquid medium was removed by brief centrifugation with a bench centrifuge.

7. Transfer the immature embryos to solid medium with tweezers and seal the petri dish with a sealing membrane. A16/8 (day/night) photoperiod was used with an average temperature of 27 ℃ during the day and 21 ℃ at night. A maximum of 25 embryos can be placed on one plate.

8. And (3) sampling the pepper plantlets at a height of 5-10 cm for DNA analysis, screening plants meeting the requirements, and transferring the plants to a greenhouse for cultivation.

9. Repeating the steps 1-8 until a homozygous plant meeting the target is obtained.

SNP molecular marker for developing special pepper excellent inbred line and analysis model

1. Selecting different pepper core inbred line seeds, and adopting a conventional cultivation and planting technology. The field experiment adopts a completely random block design and is repeated for 2-3 times.

2. Obtaining important agronomic character data including growth period, plant height, disease resistance, yield and the like.

3. Sampling (leaves or fruits) in different growth periods, and measuring related biochemical and physiological indexes, such as the inhibition activity of extracts of different pepper lines on alpha-glucosidase and the like.

4. Sampling (leaves) before the ridge sealing period, extracting DNA, sequencing and obtaining SNP data.

5. Analyzing genetic variation and population structure, discovering high-quality SNP (shown in figure 4) obviously associated with important agronomic and biochemical traits according to the principle of linkage disequilibrium, and developing corresponding molecular markers.

And constructing a data analysis model by using the SNP and the molecular marker, and distinguishing different pepper inbred lines (genotypes) through DNA analysis.

Different genotype materials can be effectively distinguished at low cost by developing a series of molecular markers and analytical models of specific inbred lines; by combining the young pepper embryo culture technology and an all-weather greenhouse culture system, the purification of the inbred line or the culture of a new inbred line after the specific excellent character transformation and hybridization of the peppers can be quickly realized. The method is characterized in that the time required by the backcross purification of 4-5 generations can be controlled to be completed within 12-15 months.

The invention has the beneficial effects that:

1. the invention relates to a pepper specific inbred line related series molecular marker, which applies a plurality of molecular biology techniques, including but not limited to related sequence deletion, addition and point mutation; or chemically synthesizing corresponding primers, and establishing an analysis model on the basis of the primers. It features that different inbred lines can be effectively distinguished at low cost to determine their purity.

2. The invention establishes a technological system for sterile separation and culture of pepper immature embryos, wherein the immature embryos are generally from immature fruits pollinated for 12-20 days. Therefore, the invention can be applied to any plants, and can effectively shorten the purification time after hybridization.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for rapidly cultivating hot pepper by using molecular markers is characterized by comprising the following two steps:

step two: the purification of the inbred line after the transformation and hybridization of the specific excellent characters of the hot pepper is quickly realized by combining the hot pepper immature embryo culture technology and an all-weather greenhouse culture system.

2. The method for rapidly cultivating pepper using molecular markers as claimed in claim 1, wherein the step one comprises the steps of:

the first step is as follows: selecting seeds of different pepper core inbred lines, adopting a conventional cultivation and planting technology, adopting a completely random block design in a field experiment, and repeating for 2-3 times;

the second step: acquiring important agronomic character data;

the fifth step: analyzing genetic variation and population structure, discovering high-quality SNP obviously associated with important agronomic and biochemical traits according to the linkage disequilibrium principle, and developing corresponding molecular markers;

and a sixth step: the SNP and the molecular marker are used for constructing a data analysis model, and different pepper inbred lines can be distinguished through DNA analysis.

3. The method as claimed in claim 2, wherein the agronomic trait data required to be obtained in the second step include growth period, plant height, disease resistance, yield.

4. The method as claimed in claim 2, wherein the related biochemical and physiological indicators measured in the third step include the α -glucosidase inhibitory activity of extracts of different capsicum strains.

5. The method as claimed in claim 1, wherein the second step comprises the following steps:

the second step is that: selecting young pepper fruits, and washing off dust on the surfaces of the fruits;

the third step: putting young fruits into a beaker in an ultra-clean workbench, pouring 75% alcohol for disinfection for 30 seconds, removing the alcohol, pouring 0.5% sodium hypochlorite solution for disinfection treatment for 5-10 minutes, and washing with sterile water for 3 times;

6. The method for rapidly cultivating capsicum using molecular marker as claimed in claim 5, wherein said conventional cultivation and planting technique in the first step and in the seventh step uses: 16/8 (day/night) light cycle, average temperature 27 ℃ in the day and 21 ℃ in the night.

7. The method as claimed in claim 5, wherein the young pepper is olive obtained 12-20 days after pollination of pepper in the second step.

8. The method as claimed in claim 7, wherein the pepper plants used for picking the olive are hybrid pepper plants or selfed pepper plants.

9. The rapid pepper cultivation method using molecular markers as claimed in claim 5, wherein there are no more than 25 immature embryos on the solid medium plate in the seventh step.

10. The method as claimed in claim 5, wherein the liquid medium is beef extract peptone liquid medium, and the solid medium is liquid medium added with about 1.5% to 2.0% agar, heated to 100 ℃ to dissolve, cooled at 40 ℃ and solidified to become solid medium.