Molecular marker assisted breeding method for olives

Abstract

The invention relates to the technical field of economic crop planting, in particular to an olive molecular marker assisted breeding method which comprises the steps of land selection and preparation, germplasm resource collection, molecular marker screening, molecular marker assisted selection, field test and evaluation, variety breeding and popularization and the like. According to the method, efficient and accurate genetic diversity analysis, germplasm resource identification and genetic variation detection are realized in the olive breeding process by utilizing an advanced molecular marker technology, so that the breeding process is accelerated, and new varieties with target characters are better screened. The molecular marker-assisted breeding method for the olives has a wide application prospect and important value in actual production and research. By improving the breeding accuracy, performing variety genetic evaluation and other applications, the new olive variety with excellent characters can be more efficiently cultivated, and powerful support is provided for the development of the olive industry.

Description

Technical Field

[0001] The invention relates to the technical field of economic crop planting, and in particular to an olive molecular marker-assisted breeding method. Background Art

[0002] Olive, also known as yellow olive, green fruit, mountain olive, white olive, green seed, admonition fruit, loyal fruit, etc., is a tree plant of the genus Olive in the family Oleaceae. Olives are mainly produced in Fujian, Sichuan, Guangdong, Yunnan, Guangxi and other places in China, as well as Vietnam, Laos, Cambodia, Thailand, Myanmar, India and Malaysia. In China, olives are an economic fruit tree planted in some southern regions, such as Chaozhou, Yunfu, Maoming, Meizhou and other places in Guangdong, where they are cultivated on a large scale.

[0003] Olives are rich in 18 kinds of amino acids and nutrients and trace elements such as calcium, iron, ascorbic acid, etc. that are needed by the human body. Its kernel contains 58.1% oil, which has the effects of lowering blood lipids, aiding digestion, promoting body fluid and detoxification, strengthening the stomach, and promoting children's bone development. It has an incomparable effect on throat diseases. Chinese medicine believes that olives are sweet, astringent, sour, and flat, and enter the lung and stomach meridians. They have the effects of clearing heat, relieving sore throat, promoting body fluid, and detoxification. They can be used to treat symptoms such as sore throat, cough, thirst, fish and crab poisoning, etc. They can also be used to lower blood pressure, blood sugar, and blood lipids, and have a certain effect on softening blood vessels.

[0004] However, traditional olive breeding methods have problems such as long cycles and low efficiency, which lead to delayed benefits for growers. Therefore, the efficiency and accuracy of olive breeding can be significantly improved through molecular marker-assisted breeding technology. Molecular marker technology is a method of genetic analysis by detecting DNA sequence polymorphism in organisms. In recent years, with the development of high-throughput sequencing technology, a variety of molecular marker technologies have been widely used in olive breeding research, including RFLP, RAPD, AFLP, SSR, ISSR, SRAP, etc.

[0005] Among them, ISSR (Inter-Simple Sequence Repeat) molecular marker technology has the advantages of good repeatability, high sensitivity, strong ability to detect polymorphism, etc., and is easy to operate and does not require prior knowledge of sequence information, which greatly saves time and cost. In addition, SNP (Single Nucleotide Polymorphism) molecular marker technology has begun to be applied to the construction of high-density genetic maps of olive germplasm resources and the identification of sites related to important agronomic traits due to its advantages of rich sites, wide distribution, high sensitivity and accuracy.

[0006] In the existing technology, there are still problems such as low amplification effect and accuracy in olive breeding technology. To this end, we use advanced molecular marker technology to achieve efficient and accurate genetic diversity analysis, germplasm resource identification and genetic variation detection in the olive breeding process, thereby accelerating the breeding process and better screening new varieties with target traits. Summary of the invention

[0007] The main purpose of the present invention is to provide an olive molecular marker-assisted breeding method, which utilizes advanced molecular marker technology to achieve efficient and accurate genetic diversity analysis, germplasm resource identification and genetic variation detection in the olive breeding process, thereby accelerating the breeding process and better screening new varieties with target traits.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A molecular marker-assisted breeding method for olives, the method comprising the following steps: site selection and preparation, germplasm resource collection, molecular marker screening, molecular marker-assisted selection, field trials and evaluation, variety breeding and promotion.

[0010] Preferably, the site selection and land preparation specifically include the following steps: selecting a sloping land with good ventilation and light transmission, good drainage, good light, soil pH of 6-8 and high calcium content for planting, and performing deep plowing, fertilization and land preparation.

[0011] Preferably, the germplasm resource collection is specifically the following steps: collecting olive germplasm resources of different varieties and different geographical origins to ensure genetic diversity.

[0012] Preferably, the molecular marker screening is specifically carried out in the following steps: using ISSR marker technology to perform genetic diversity analysis on the collected germplasm resources, and screening out germplasm resources with excellent traits and rich genetic variations.

[0013] Preferably, the molecular marker-assisted selection is specifically the following steps: using molecular marker technology to perform early genetic identification and screen out individuals with target traits.

[0014] Preferably, the field test and evaluation specifically include the following steps: conducting field tests on the selected excellent individuals to evaluate their yield, quality and stress resistance traits.

[0015] Preferably, the variety breeding and promotion specifically include the following steps: based on the field test evaluation results, excellent varieties are selected and promoted for planting.

[0016] Preferably, the molecular marker screening is to use ISSR molecular marker technology to perform genetic diversity analysis and genetic variation detection.

[0017] Preferably, the molecular marker-assisted selection is to optimize the PCR amplification reaction system and determine the optimal reaction parameters. In the PCR amplification reaction system, the optimal Taq enzyme amount, Mg2+ concentration, template DNA content, NTPs concentration and primer concentration parameters are determined. The specific reaction system is as follows:

[0018] Taq enzyme: 1.5U;

[0019] Mg 2+ :2.5mmol / L;

[0020] Template DNA: 60 ng;

[0021] dNTPs: 0.2mmol / L;

[0022] Primer: 0.15 μmol / L.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. In the present invention: the olive molecular marker-assisted breeding method, by utilizing advanced molecular marker technology, realizes efficient and accurate genetic diversity analysis, germplasm resource identification and genetic variation detection in the olive breeding process, thereby accelerating the breeding process and better screening new varieties with target traits.

[0025] 2. In the present invention: the olive molecular marker-assisted breeding method significantly shortens the breeding cycle, can screen out individuals with excellent traits in a shorter time, and accelerate the breeding process. Compared with traditional breeding methods, the present invention has higher accuracy; molecular marker technology can directly detect DNA sequence variations, thereby revealing the genetic differences and similarities between breeding materials. This DNA sequence-based detection method is more accurate and reliable than traditional phenotypic identification, which helps us more accurately evaluate the genetic value and breeding potential of breeding materials. DETAILED DESCRIPTION

[0026] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0027] Example

[0028] The present invention provides a technical solution:

[0029] A molecular marker-assisted breeding method for olives, the method comprising the following steps: site selection and preparation, germplasm resource collection, molecular marker screening, molecular marker-assisted selection, field trials and evaluation, variety breeding and promotion.

[0030] Furthermore, the site selection and land preparation specifically include the following steps: selecting a sloping land with good ventilation and light transmission, good drainage, good light, soil pH of 6-8 and high calcium content for planting, and performing deep plowing, fertilization and land preparation.

[0031] Furthermore, the germplasm resource collection is specifically carried out in the following steps: collecting olive germplasm resources of different varieties and different geographical origins to ensure genetic diversity.

[0032] Furthermore, the molecular marker screening specifically comprises the following steps: using ISSR marker technology to perform genetic diversity analysis on the collected germplasm resources, and screening out germplasm resources with excellent traits and rich genetic variations.

[0033] Furthermore, the molecular marker-assisted selection specifically comprises the following steps: using molecular marker technology to conduct early genetic identification and screen out individuals with target traits.

[0034] Furthermore, the field test and evaluation specifically include the following steps: conducting field tests on the selected excellent individuals to evaluate their yield, quality and stress resistance traits.

[0035] Furthermore, the variety breeding and promotion specifically include the following steps: based on the field test evaluation results, excellent varieties are selected and promoted for planting.

[0036] Furthermore, the molecular marker screening is to use ISSR molecular marker technology to perform genetic diversity analysis and genetic variation detection.

[0037] Furthermore, the molecular marker-assisted selection is to optimize the PCR amplification reaction system and determine the optimal reaction parameters. In the PCR amplification reaction system, the optimal Taq enzyme amount, Mg2+ concentration, template DNA content, NTPs concentration and primer concentration parameters are determined. The specific reaction system is as follows:

[0038] Taq enzyme: 1.5U;

[0039] Mg 2+ :2.5mmol / L;

[0040] Template DNA: 60 ng;

[0041] dNTPs: 0.2mmol / L;

[0042] Primer: 0.15 μmol / L.

[0043] Example 1

[0044] The present invention uses a series of ISSR primers to analyze the genetic diversity of olive germplasm resources. ISSR primers can produce high polymorphic bands and are suitable for germplasm resource identification and genetic background analysis; SNP primers can accurately detect single nucleotide polymorphisms and are suitable for constructing high-density genetic maps and identifying sites related to important agronomic traits.

[0045] Amplification reaction system:

[0046] In the PCR amplification reaction, the present invention optimizes the reaction system and determines the optimal Taq enzyme amount, Mg2+ concentration, template DNA content, dNTPs concentration, primer concentration and other parameters to improve the amplification effect and accuracy. The specific reaction system is as follows:

[0047] Taq enzyme: 1.5U

[0048] Mg 2+ :2.5mmol / L

[0049] Template DNA: 60ng

[0050] dNTPs: 0.2mmol / L

[0051] Primer: 0.15 μmol / L

[0052] Furthermore, in terms of optimizing the PCR amplification reaction system to improve the amplification effect and accuracy, the present invention adopts a number of specific measures for the ISSR-PCR reaction system of olives. The following is a detailed description of these optimization measures:

[0053] Optimization of primer concentration:

[0054] Determine the optimal primer concentration: Through repeated experiments, the present invention determined that the optimal concentration of ISSR primers is 0.25 μmol / L. At this concentration, the primers can ensure sufficient binding sites and avoid excessive competition and ineffective amplification, thereby improving the amplification effect and accuracy.

[0055] Optimization of template DNA concentration:

[0056] Selecting the appropriate amount of template DNA: In PCR amplification, the concentration of template DNA directly affects the yield and specificity of amplification. The present invention has been optimized to determine that 40ng of template DNA is the best amount to ensure that there is enough template for amplification while avoiding competitive inhibition caused by too high a concentration of template.

[0057] Optimization of dNTPs concentration:

[0058] Balanced dNTPs concentration: dNTPs are the raw materials for providing nucleotides in PCR amplification. Too high a concentration will increase the base mismatch rate, while too low a concentration will affect the amplification efficiency. The present invention determines that the optimal concentration of dNTPs is 0.2mmol / L, which can both ensure the progress of the amplification reaction and reduce the risk of base mismatch.

[0059] Optimization of Taq polymerase concentration:

[0060] Ensure sufficient enzyme activity: Taq polymerase is a key enzyme in PCR amplification, and its activity directly affects the amplification effect and accuracy. In the optimized reaction system of the present invention, the concentration of Taq polymerase is 1U / 20μL reaction system, which is sufficient to support efficient amplification reaction. Optimization of renaturation temperature:

[0061] Improve specificity: The annealing temperature is a key factor affecting the specificity of PCR amplification. Through optimization experiments, the present invention has determined that 51.6°C-53°C is the best annealing temperature range, which can specifically bind to the target sequence at this temperature and improve the accuracy of amplification.

[0062] Optimization of buffer and other reaction components:

[0063] Adjusting pH and ionic strength: The PCR amplification system contains a specific buffer, which can not only maintain the stability of enzyme activity, but also adjust the pH and ionic strength of the reaction system. The present invention selects 1× buffer (without Mg 2+ ), and on this basis, add appropriate concentration of Mg 2+ , providing a suitable environment for enzymatic reactions.

[0064] Other factors to consider:

[0065] Optimizing the amplification program: In addition to the optimization of the above components, the present invention also optimizes the amplification program, including the time and temperature of pre-denaturation, denaturation, annealing and extension, to improve the amplification efficiency and accuracy.

[0066] In summary, the present invention establishes an efficient, accurate and stable reaction system suitable for olive ISSR-PCR amplification by accurately adjusting the primer concentration, template DNA concentration, dNTPs concentration, Taq polymerase concentration, renaturation temperature, buffer and other reaction components, and optimizing the amplification program.

[0067] Example 2

[0068] The olive molecular marker-assisted breeding method of the present invention is first applied to the construction of a genetic map. Using high-density molecular markers (such as SNPs) to scan the whole genome of olive germplasm resources can reveal a large number of genetic variations and linkage disequilibrium regions. These variations and regions provide a basis for subsequent genetic analysis and breeding work. By constructing a genetic map, we can have a deeper understanding of the genetic structure and genomic characteristics of olives, and provide strong support for subsequent genetic improvement and breeding practices. On the basis of the genetic map, we further use molecular marker technology to identify sites that are closely linked to important olive traits. These important traits include but are not limited to yield, quality, stress resistance, etc. Through methods such as association analysis or gene positioning, we can identify molecular markers that are significantly correlated with these traits, and then determine their corresponding gene regions. This not only helps to reveal the genetic mechanism of important olive traits, but also provides a basis for subsequent molecular marker-assisted selection.

[0069] Based on the results of the identification of important trait loci, we screened out molecular markers that are closely linked to the target traits and are easy to detect. These markers have high application value in actual production and can be used to quickly and accurately identify the genotype of breeding materials. By selecting these markers, we can ensure that only individuals with excellent traits are retained during the breeding process, thereby improving the efficiency and accuracy of breeding.

[0070] In the breeding process, we use molecular marker technology to screen breeding materials. By detecting molecular markers that are closely linked to important traits, we can quickly and accurately identify individuals with excellent traits, thereby avoiding selection errors caused by inaccurate phenotypic identification in traditional breeding. This method not only improves the efficiency of breeding, but also ensures the genetic stability and consistency of breeding materials.

[0071] During the breeding process, we also need to continuously test the target traits. Using molecular marker technology, we can conduct regular genotyping tests on breeding materials to monitor their genetic variation and trait changes. This helps us identify problems in a timely manner and take appropriate measures to make adjustments, thereby ensuring the smooth progress of breeding work.

[0072] The olive molecular marker-assisted breeding method of the present invention significantly shortens the breeding cycle. Traditional breeding methods require a lot of time and effort for phenotypic identification and genetic analysis, while molecular marker technology can quickly reveal the genetic characteristics and variations of breeding materials. This enables us to screen out individuals with excellent traits in a shorter period of time and accelerate the breeding process. Compared with traditional breeding methods, the olive molecular marker-assisted breeding method of the present invention has higher accuracy. Molecular marker technology can directly detect variations in DNA sequences, thereby revealing genetic differences and similarities between breeding materials. This DNA sequence-based detection method is more accurate and reliable than traditional phenotypic identification, which helps us more accurately evaluate the genetic value and breeding potential of breeding materials.

[0073] Finally, the olive molecular marker-assisted breeding method of the present invention is also applied to the genetic evaluation of varieties. By performing whole genome scanning and molecular marker detection on new varieties, we can comprehensively evaluate their performance in terms of genetic characteristics, genetic stability and adaptability. This helps us to more accurately understand the genetic background and genetic value of new varieties, and provide a scientific basis for their application and promotion in actual production.

[0074] In summary, the olive molecular marker-assisted breeding method of the present invention has broad application prospects and important value in actual production and research. Through the application of constructing genetic maps, identifying important trait loci, selecting molecular markers, screening breeding materials, detecting target traits, shortening breeding cycles, improving breeding accuracy, and performing variety genetic evaluation, we can more efficiently cultivate new olive varieties with excellent traits, providing strong support for the development of the olive industry.

[0075] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A molecular marker-assisted breeding method for olives, characterized in that: The method comprises the following steps: site selection and preparation, germplasm resource collection, molecular marker screening, molecular marker assisted selection, field trial and evaluation, variety breeding and promotion.

2. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The site selection and land preparation specifically include the following steps: selecting a sloping land with good ventilation, light transmission, good drainage, good light, soil pH of 6-8 and high calcium content for planting, and performing deep plowing, fertilizing and land preparation.

3. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The germplasm resource collection specifically includes the following steps: collecting olive germplasm resources of different varieties and different geographical origins to ensure genetic diversity.

4. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The molecular marker screening specifically includes the following steps: using ISSR marker technology to perform genetic diversity analysis on the collected germplasm resources, and screening out germplasm resources with excellent traits and rich genetic variations.

5. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The molecular marker-assisted selection specifically comprises the following steps: using molecular marker technology to perform early genetic identification and screen out individuals with target traits.

6. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The field test and evaluation specifically include the following steps: conducting field tests on the selected excellent individuals to evaluate their yield, quality and stress resistance traits.

7. The method for olive molecular marker-assisted breeding according to claim 6, characterized in that: The variety breeding and promotion specifically includes the following steps: according to the field test evaluation results, excellent varieties are selected and promoted for planting.

8. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The molecular marker screening is to use ISSR molecular marker technology to perform genetic diversity analysis and genetic variation detection.

9. The method for olive molecular marker-assisted breeding according to claim 1, characterized in that: The molecular marker-assisted selection is to optimize the PCR amplification reaction system and determine the optimal reaction parameters. In the PCR amplification reaction system, the optimal Taq enzyme amount, Mg 2+ The parameters of concentration, template DNA content, NTPs concentration and primer concentration are as follows: Taq enzyme: 1.5U; Mg 2+ :2.5mmol / L; Template DNA: 60 ng; dNTPs: 0.2mmol / L; Primer: 0.15 μmol / L.