Method for rapidly identifying far-source hybrid progeny of fruit tree

Abstract

The invention relates to the field of remote source identification of fruit trees, in particular to a method for rapidly identifying remote source hybrid progenies of fruit trees, which comprises a morphological feature identification module, a physiological feature identification module and a molecular marker identification module, a morphological feature identification module, a physiological feature identification module and a molecular marker identification module are arranged to perform PCR amplification on different fruit tree far-source hybrid offspring samples, clear and stable strips are observed, it is indicated that the selected molecular marker is suitable for identification of the fruit tree far-source hybrid offspring, genotypes of male parents, female parents and hybrid offspring are compared, and the identification accuracy is high. The genotype of the far-source hybrid progeny of the fruit tree can be accurately judged, and a breeder can screen the far-source hybrid progeny with excellent characters in an early stage through rapid identification, so that the breeding efficiency is improved, the genetic background of the hybrid progeny can be known, and powerful support is provided for subsequent genetic improvement and variety breeding; and the rapid identification method can reduce the breeding cost and shorten the breeding period.

Description

Technical Field

[0001] The invention relates to the technical field of distant origin identification of fruit trees, in particular to a method for rapidly identifying distant origin hybrid offspring of fruit trees. Background Art

[0002] The offspring of distant hybrids of fruit trees refer to the offspring produced by hybridization between different varieties or species, and have the genetic characteristics of both parents. This type of offspring is of great significance in the genetic improvement and variety innovation of fruit trees, which can enrich the genetic resources of fruit trees and improve the quality, yield and stress resistance of fruits. However, at present, the identification of distant hybrid offspring of fruit trees often relies on a single identification method, and the identification time is long. The traditional method usually takes several years or even more than ten years to obtain the identification results, which is not suitable for early screening and has low identification accuracy. The traditional method is affected by multiple factors such as the environment and growth conditions, resulting in certain errors in the identification results, which cannot fully reflect the genetic diversity of hybrid offspring. The traditional method can often only reveal some genetic characteristics, and it is difficult to fully evaluate the genetic background of hybrid offspring. The labor intensity is high: the traditional method requires a lot of manual operations, which consumes a lot of time and energy. Therefore, it is necessary to propose a method for rapid identification of distant hybrid offspring of fruit trees. Summary of the invention

[0003] In view of the problems in the prior art, the present invention provides a method for rapidly identifying distant hybrid offspring of fruit trees.

[0004] The technical solution adopted by the present invention to solve the technical problem is: a method for rapidly identifying distant hybrid offspring of fruit trees, comprising a morphological feature identification module, a physiological feature identification module and a molecular marker identification module.

[0005] The morphological feature identification module performs morphological scanning on the fruit tree through a scanning camera. By observing the external morphology of the fruit tree, its genetic characteristics can be preliminarily determined. The identification of the distant hybrid offspring of the fruit tree is mainly carried out by observing its morphological characteristics, which specifically includes three aspects: fruit morphological characteristics, tree morphological characteristics and growth habit identification. The identification of fruit morphological characteristics is one of the important methods for judging the distant hybrid offspring of the fruit tree. The fruit size, shape, color, leather grain, etc. of different varieties of fruit trees have obvious differences. By observing and comparing the fruits of the hybrid offspring, its parental source can be preliminarily determined. For example, each morphological feature of the fruit can be compared with the known parent, and the similarity can be calculated to infer the genetic background of the hybrid offspring. Secondly, the identification of tree morphological characteristics is also an important means to judge the offspring of distant hybrids of fruit trees. The morphological characteristics of trees include tree shape, leaf shape, flower color, etc. These characteristics are largely affected by genetic factors. By observing and comparing the tree morphological characteristics of hybrid offspring, its genetic characteristics can be further verified. For example, it can be observed whether the tree shape of the hybrid offspring is between the parents, whether the leaf shape shows the characteristics of the parents, whether the flower color has changed, etc. Finally, the identification of growth habits also has a certain reference value for judging the offspring of distant hybrids of fruit trees. Growth habits include growth rate, adaptability, disease resistance, etc. These characteristics are often affected by the genetic characteristics of the parents. By observing and comparing the growth habits of hybrid offspring, its genetic characteristics can be indirectly understood. For example, the similarity between the hybrid offspring and the parents in growth rate, adaptability, etc. can be compared to infer its genetic background, so as to preliminarily infer the genetic characteristics of the offspring of distant hybrids of fruit trees.

[0006] The physiological special diagnosis and identification module records the growth of plants through recording instruments, stores plant physiological data, and further determines the genetic characteristics by measuring some physiological indicators of fruit trees. The identification of distant hybrid offspring of fruit trees requires not only morphological and molecular markers, but also needs to be further verified by physiological characteristics identification. The determination and analysis of physiological indicators, fruit quality identification and stress resistance identification are three main aspects. First, the determination and analysis of physiological indicators is an important method for evaluating the physiological state of distant hybrid offspring of fruit trees. By measuring growth indicators such as tree height, ground diameter, leaf area, etc., the growth status of hybrid offspring can be understood. In addition, physiological parameters such as photosynthetic rate and respiration rate can also be measured to evaluate their physiological functions. The determination and analysis of these indicators help to understand the physiological adaptability of hybrid offspring, thereby providing a basis for their cultivation management and reproduction strategies; Secondly, fruit quality identification is the key to evaluating the economic value of distant hybrid offspring of fruit trees. Fruit quality can be evaluated by measuring appearance indicators such as fruit size, shape, color, and internal quality indicators such as soluble solids, vitamin C content, and acidity. In addition, the sensory characteristics of the fruit such as taste and flavor can also be evaluated. Through fruit quality identification, hybrid offspring with excellent traits can be screened out, providing high-quality resources for the development of the fruit industry. Finally, stress resistance identification is an important aspect of evaluating the adaptability of distant hybrid offspring of fruit trees. Stress resistance includes cold resistance, drought resistance, disease resistance, etc. It can be evaluated by artificially simulating adverse conditions, such as low temperature and drought, and observing the growth conditions and physiological responses of hybrid offspring. In addition, molecular marker technology can be used to detect the expression of stress resistance-related genes in hybrid offspring to gain a deeper understanding of their stress resistance mechanism. Stress resistance identification helps to screen out hybrid offspring with strong adaptability and improve the stability of the fruit industry. Through the measurement and analysis of physiological indicators, fruit quality identification and stress resistance identification, the physiological characteristics of distant hybrid offspring of fruit trees can be comprehensively evaluated.

[0007] The molecular marker identification module includes a PCR molecular marker module and a DNA identification module, which respectively perform DNA extraction, molecular markers and electrophoresis analysis on the fruit trees to fully identify the fruit trees; the PCR molecular marker module amplifies the fruit tree DNA in a short time, thereby using in vitro amplification of specific DNA sequences for identification. By designing specific primers, the PCR technology can amplify a large number of target DNA fragments in a short time, thereby facilitating subsequent detection and analysis. In the identification of distant hybrid offspring of fruit trees, the PCR technology can be used to amplify specific DNA marker sites, and the size of the amplified product can be analyzed by electrophoresis to determine the genetic background of the hybrid offspring; The DNA identification module identifies fruit trees by sequencing the genomic DNA of an organism and analyzing its sequence information to identify genetic characteristics. The identification method based on DNA sequencing is to identify genetic characteristics by sequencing the genomic DNA of an organism and analyzing its sequence information. With the development of high-throughput sequencing technology, the identification method based on DNA sequencing has been widely used in the identification of distant hybrid offspring of fruit trees. By sequencing and analyzing the genome of the hybrid offspring, its genetic background can be accurately determined, including the genetic contribution of the father and the mother. The morphological feature identification module, the physiological feature identification module and the molecular marker identification module increase the overall identification efficiency of distant fruit trees by quickly mixing the morphological data, physiological data and molecular data of the fruit trees.

[0008] A method for using a method for rapidly identifying distant hybrid offspring of fruit trees comprises the following steps: S1: When it is necessary to perform remote identification of external fruit trees, the scanning camera in the morphological feature identification module is used to perform morphological scanning of the fruit trees, observe the external morphology of the fruit trees, such as leaves, fruits, and flowers, and record the observed data; S2: Use the physiological special diagnosis and identification module with recording instruments to record plant growth and store plant physiological data. By measuring some physiological indicators of fruit trees, such as photosynthetic rate, respiratory rate, and nutrient content, further determine their genetic characteristics and assist in data preparation in the later stage. S3: Prepare experimental plant materials at this time, select representative samples of distant hybrid offspring of fruit trees, including male and female parents and hybrid offspring, and ensure that the samples are fresh and free of pests and diseases; then prepare experimental reagents, including DNA extraction kit, PCR amplification kit, DNA molecular weight standard, agarose; experimental instruments, including PCR amplification instrument, gel imaging system, high-speed centrifuge, DNA electrophoresis instrument; S4: According to the instructions of the DNA extraction kit, genomic DNA was extracted from the prepared sample fruit tree leaves, and the concentration and purity of the extracted DNA were detected by ultraviolet spectrophotometer. Fruit tree-specific DNA fragments were selected, primers were designed, and PCR amplification was performed. The reaction system was: 1 μL DNA template, 1 μL upstream and downstream primers each, 25 μL DNA polymerase mixture, and ddH2O was added to 50 μL. The reaction conditions were pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 35 cycles; extension at 72°C for 10 minutes. After the above operations were completed, DNA molecular labeling was performed, and the PCR amplification products were subjected to DNA molecular labeling analysis. The results were recorded using a gel imaging system and used for identification based on morphological, physiological and molecular data.

[0009] Beneficial effects of the present invention: The method for rapidly identifying distant hybrid offspring of fruit trees disclosed by the present invention can observe clear and stable bands by PCR amplification of distant hybrid offspring samples of different fruit trees by setting a morphological feature identification module, a physiological feature identification module and a molecular marker identification module, indicating that the selected molecular markers are suitable for the identification of distant hybrid offspring of fruit trees. By comparing the genotypes of the paternal parent, the maternal parent and the hybrid offspring, the genotypes of the distant hybrid offspring of fruit trees can be accurately determined. Through rapid identification, breeders can screen out distant hybrid offspring with excellent traits at an early stage, improve breeding efficiency, help understand the genetic background of hybrid offspring, and provide strong support for subsequent genetic improvement and variety selection. In addition, the rapid identification method can reduce breeding costs and shorten the breeding cycle. DETAILED DESCRIPTION

[0010] 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.

[0011] The method for rapidly identifying the offspring of distant hybrids of fruit trees disclosed in the present invention comprises a morphological feature identification module, a physiological feature identification module and a molecular marker identification module; the morphological feature identification module performs morphological scanning on the fruit tree by means of a scanning camera, and by observing the external morphology of the fruit tree, its genetic characteristics can be preliminarily determined, and the identification of the offspring of distant hybrids of fruit trees is mainly carried out by observing their morphological features, which specifically include three aspects: fruit morphological features, tree body morphological features and growth habit identification; the identification of fruit morphological features is one of the important methods for determining the offspring of distant hybrids of fruit trees, and different varieties of fruit trees have obvious differences in fruit size, shape, color, skin texture, etc., and by observing and comparing the fruits of the hybrid offspring, a preliminarily determined genetic characteristics can be determined. The first step is to determine the origin of its parents. For example, the various morphological characteristics of the fruit can be compared with the known parents, and their similarities can be calculated to infer the genetic background of the hybrid offspring. Secondly, the identification of tree morphological characteristics is also an important means to determine the offspring of distant hybrids of fruit trees. The morphological characteristics of the tree include tree shape, leaf shape, flower color, etc. These characteristics are largely affected by genetic factors. By observing and comparing the tree morphological characteristics of the hybrid offspring, its genetic characteristics can be further verified. For example, it can be observed whether the tree shape of the hybrid offspring is between the parents, whether the leaf shape shows the characteristics of the parents, whether the flower color has changed, etc. Finally, the identification of growth habits also has a certain reference value for judging the offspring of distant hybrids of fruit trees. Growth habits include growth rate, adaptability, disease resistance, etc. These characteristics are often affected by the genetic characteristics of the parents. By observing and comparing the growth habits of hybrid offspring, we can indirectly understand their genetic characteristics. For example, we can compare the similarities between hybrid offspring and their parents in growth rate, adaptability, etc., and thus infer their genetic background. From this, we can preliminarily infer the genetic characteristics of distant hybrid offspring of fruit trees more accurately as a basis for preliminary judgment.

[0012] The physiological special diagnosis and identification module records the growth of plants through recording instruments, stores the physiological data of plants, and further determines the genetic characteristics by measuring some physiological indicators of fruit trees. The identification of distant hybrid offspring of fruit trees requires not only morphological and molecular markers, but also further verification through physiological characteristics identification. The determination and analysis of physiological indicators, fruit quality identification and stress resistance identification are three main aspects. First of all, the determination and analysis of physiological indicators is an important method to evaluate the physiological state of distant hybrid offspring of fruit trees. By measuring growth indicators such as tree height, ground diameter, leaf area, etc., the growth status of hybrid offspring can be understood. In addition, physiological parameters such as photosynthetic rate and respiratory rate can also be measured to evaluate their physiological functions. The measurement and analysis of these indicators help to understand the physiological adaptability of hybrid offspring, thereby providing a basis for their cultivation management and breeding strategies; secondly, fruit quality identification is the key to evaluating the economic value of distant hybrid offspring of fruit trees. Fruit quality can be evaluated by measuring appearance indicators such as fruit size, shape, and color, as well as intrinsic quality indicators such as soluble solids, vitamin C content, and acidity. In addition, sensory characteristics such as the taste and flavor of the fruit can also be evaluated. Through fruit quality identification, hybrid offspring with excellent traits can be screened out, providing support for the development of the fruit industry. Provide high-quality resources; finally, stress resistance identification is an important aspect of evaluating the adaptability of distant hybrid offspring of fruit trees. Stress resistance includes cold resistance, drought resistance, disease resistance and other abilities. It can be evaluated by artificially simulating adverse conditions, such as low temperature and drought, and observing the growth conditions and physiological responses of hybrid offspring. In addition, molecular marker technology can be used to detect the expression of stress resistance-related genes in hybrid offspring to gain a deeper understanding of their stress resistance mechanism. Stress resistance identification helps to screen out hybrid offspring with strong adaptability and improve the stability of the fruit industry. Through the measurement and analysis of physiological indicators, fruit quality identification and stress resistance identification, the physiological characteristics of distant hybrid offspring of fruit trees can be comprehensively evaluated.

[0013] The molecular marker identification module includes the PCR molecular marker module and the DNA identification module, which respectively perform DNA extraction, molecular markers and electrophoresis analysis on the fruit trees to fully identify the fruit trees; the PCR molecular marker module amplifies the fruit tree DNA in a short time, thereby using the in vitro amplification of specific DNA sequences for identification. By designing specific primers, the PCR technology can amplify a large number of target DNA fragments in a short time, thereby facilitating subsequent detection and analysis. In the identification of distant hybrid offspring of fruit trees, the PCR technology can be used to amplify specific DNA marker sites, and the size of the amplified products can be analyzed by electrophoresis to determine the genetic background of the hybrid offspring; the DNA identification module amplifies the organism Fruit tree identification is carried out by sequencing the genomic DNA and analyzing its sequence information to identify genetic characteristics. The identification method based on DNA sequencing is to identify genetic characteristics by sequencing the genomic DNA of the organism and analyzing its sequence information. With the development of high-throughput sequencing technology, the identification method based on DNA sequencing has been widely used in the identification of distant hybrid offspring of fruit trees. By sequencing and analyzing the genome of the hybrid offspring, its genetic background can be accurately determined, including the genetic contribution of the father and the mother. The morphological characteristic identification module, the physiological characteristic identification module and the molecular marker identification module increase the overall identification efficiency of distant fruit trees by quickly mixing the morphological data, physiological data and molecular data of the fruit trees.

[0014] Specific use: When it is necessary to conduct distant source identification of external fruit trees, the scanning camera in the morphological feature identification module is used to scan the morphology of the fruit trees, and the external morphology of the fruit trees, such as leaves, fruits, and flowers, is observed, and the observed data is recorded; the growth of the plants is recorded through the physiological special diagnosis identification module in conjunction with the recording instrument, and the physiological data of the plants is stored. By measuring some physiological indicators of the fruit trees, such as photosynthetic rate, respiratory rate, and nutrient content, its genetic characteristics are further determined to assist in data preparation in the later stage; at this time, prepare experimental plant materials, select representative samples of distant hybrid offspring of fruit trees, including paternal, maternal and hybrid offspring, and ensure that the samples are fresh and free of diseases and pests; then prepare experimental reagents, including DNA extraction kits, PCR amplification kits, DNA molecular weight standards, and agarose; experimental instruments, including PCR amplifiers, gel imaging systems, and high-speed centrifuges machine, DNA electrophoresis instrument; operate according to the instructions of the DNA extraction kit, extract genomic DNA from the prepared sample fruit tree leaves, use an ultraviolet spectrophotometer to detect the concentration and purity of the extracted DNA, select fruit tree-specific DNA fragments, design primers, and perform PCR amplification. The reaction system is: 1 μL DNA template, 1 μL upstream and downstream primers each, 25 μL DNA polymerase mixture, add ddH2O to 50 μL, and the reaction conditions are 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 35 cycles; 72°C extension for 10 minutes. After the above operations are completed, DNA molecular labeling is performed, and the PCR amplification products are subjected to DNA molecular labeling analysis. The results are recorded using a gel imaging system, and the morphological data, physiological data, and molecular data are used for identification and use, so as to quickly identify the fruit trees and determine the distant source species of the fruit trees.

[0015] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand 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. The scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for rapidly identifying distant hybrid offspring of fruit trees, characterized in that: The following steps are involved: Step 1: When it is necessary to conduct remote identification of external fruit trees, the scanning camera in the morphological feature identification module is used to scan the morphology of the fruit trees, observe the external morphology of the fruit trees, such as leaves, fruits, and flowers, and record the observed data; Step 2: Use the physiological special diagnosis and identification module with recording instruments to record plant growth and store plant physiological data. By measuring some physiological indicators of fruit trees, such as photosynthetic rate, respiratory rate, and nutrient content, further determine their genetic characteristics and assist in data preparation in the later stage. Step 3: Prepare experimental plant materials at this time, select representative samples of distant hybrid offspring of fruit trees, including male and female parents and hybrid offspring, and ensure that the samples are fresh and free of pests and diseases; then prepare experimental reagents, including DNA extraction kit, PCR amplification kit, DNA molecular weight standard, agarose; experimental instruments, including PCR amplification instrument, gel imaging system, high-speed centrifuge, DNA electrophoresis instrument; Step 4: According to the instructions of the DNA extraction kit, genomic DNA was extracted from the prepared sample fruit tree leaves, and the concentration and purity of the extracted DNA were detected by ultraviolet spectrophotometer. Fruit tree-specific DNA fragments were selected, primers were designed, and PCR amplification was performed. The reaction system was: 1 μL DNA template, 1 μL upstream and downstream primers, 25 μL DNA polymerase mixture, and ddH2O was added to 50 μL. The reaction conditions were pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 35 cycles; extension at 72°C for 10 minutes. After the above operations were completed, DNA molecular labeling was performed, and the PCR amplification products were subjected to DNA molecular labeling analysis. The results were recorded using a gel imaging system and used for identification based on morphological, physiological and molecular data.

2. A method for rapidly identifying distant hybrid offspring of fruit trees, characterized in that: It includes a morphological feature identification module, a physiological feature identification module and a molecular marker identification module.

3. The method for rapidly identifying distant hybrid offspring of fruit trees according to claim 1, characterized in that: The morphological feature identification module performs a morphological scan on the fruit tree through a scanning camera, and can preliminarily determine its genetic characteristics by observing the external morphology of the fruit tree.

4. A method for rapidly identifying distant hybrid offspring of fruit trees according to claim 1, characterized in that: The physiological special diagnosis and identification module records the growth of the plant through a recording instrument, stores the physiological data of the plant, and further determines its genetic characteristics by measuring some physiological indicators of the fruit tree.

5. The method for rapidly identifying distant hybrid offspring of fruit trees according to claim 1, characterized in that: The molecular marker identification module includes a PCR molecular marker module and a DNA identification module, which respectively perform DNA extraction, molecular markers and electrophoresis analysis on the fruit trees to conduct a complete identification of the fruit trees.

6. A method for rapidly identifying distant hybrid offspring of fruit trees according to claim 4, characterized in that: The PCR molecular marker module amplifies the fruit tree DNA in a short time, thereby utilizing in vitro amplification of specific DNA sequences for identification.

7. A method for rapidly identifying distant hybrid offspring of fruit trees according to claim 4, characterized in that: The DNA identification module sequences the genomic DNA of the organism and analyzes the sequence information to identify the genetic characteristics for fruit tree identification.

8. The method for rapidly identifying distant hybrid offspring of fruit trees according to claim 1, characterized in that: The morphological feature identification module, the physiological feature identification module and the molecular marker identification module increase the overall identification efficiency of the distant sources of the fruit trees by quickly mixing the morphological data, physiological data and molecular data of the fruit trees.