CN113308565A - Molecular marker primer combination for rapidly identifying morphological characters of waxberry leaves and application thereof - Google Patents
Molecular marker primer combination for rapidly identifying morphological characters of waxberry leaves and application thereof Download PDFInfo
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Abstract
The invention discloses a group of molecular marker primer combinations for rapidly identifying the morphological characters of waxberry leaves and application thereof, which are expected to be applied to the auxiliary selection of the molecular markers of the morphological characters of the waxberry leaves. The invention provides a group of molecular marker primer combinations capable of rapidly identifying the morphological characters of the leaves of the waxberries, which are Y9766732, are suitable for high-throughput typing detection platforms such as a fluorescent quantitative PCR instrument and the like, have high sensitivity, and can be used for the early rapid identification of the morphological characters of the leaves of the waxberries. The method for assisting in screening the excellent waxberry germplasm resources based on the developed molecular marker primer combination has important guiding significance on the predication of the morphological character phenotype of the waxberry leaves, and has the advantages of simplicity in operation, high efficiency, rapidness, low cost and the like.
Description
Technical Field
The invention relates to a group of molecular marker primer combinations for rapidly identifying morphological characters of waxberry leaves and application thereof, belonging to the field of molecular biology.
Background
Fruit tree leaves are the main places for photosynthesis, the shape of the leaves is closely related to the yield and the quality of fruits, and the leaves play an important role in resisting stress response of the environmental protection. Generally, the establishment of morphological characteristics of plant leaves is a very complex physiological and biochemical process, and the same plant may have great differences. Different blade shapes have different efficiencies of light energy utilization. Therefore, the research on the plant leaf morphogenetic molecular mechanism will be further deepened by applying the bioinformatics, modern molecular biology, genomics and the like, and a new way for cultivating new high-quality and stress-resistant fruit tree varieties is expected to be developed.
Waxberry (Myrica rubra Sieb. and Zucc.) is a special fruit tree produced in south China, and the fruit is rich in anthocyanin, has high nutritional value and is suitable for fresh eating and processing. The red bayberry leaves can be divided into three types, namely, inverted lanceolate, inverted oval and spoon shapes according to the shape of the red bayberry leaves. The study of the class question group at the earlier stage discovers that the leaf shape of the waxberry is closely related to the development period and the quality of the fruits, wherein the leaf shape of the waxberry variety is mostly in a shape of a reversed cape needle, and the quality is good; the waxberry varieties with the shape of the spoon leaves are the early-maturing waxberry varieties. In addition, the improvement of the leaf form has important significance on the photosynthesis and transpiration of the waxberries and the improvement of the quality of the waxberries, and a breeding way for creating an ideal plant type of the waxberries through the leaf shape transformation is feasible. Therefore, the method combines the leaf shape of the waxberry with the physiology and combines the molecular design theory with the traditional breeding practice, namely the transformation of the leaf shape with the high-light-efficiency breeding, improves the photosynthetic efficiency, and straightens the library and the source relationship, and simultaneously adopts the method combining the molecular biology technical theory with the breeding practice to realize the overall improvement of the plant type of the waxberry or the directional transformation of a certain character, thereby being an effective way for the directional breeding of the waxberry in the future. However, the waxberry has a long childhood period (more than 8 years after the seedling propagation and permanent planting), the seedling is in a seedling stage, the leaf shape of the seedling is not fixed, and other characteristics can clearly show the leaf shape types in the late development stage and the adult stage and delay the leaf shape directional breeding process of the waxberry. The development of modern biotechnology provides an effective method for the early prediction and identification of the leaf morphology of the waxberry, namely a DNA molecular marker, and the leaf morphology of the waxberry breeding material can be identified early in a short time by utilizing the specific DNA molecular marker related to the leaf morphology, so that the breeding process can be greatly accelerated. Therefore, establishing an effective molecular marker combination and a detection method thereof, and realizing high-throughput typing detection-assisted screening become one of the problems to be solved by researchers in the field. The bayberry leaf morphological character specific site is obtained by DNA re-sequencing results of current local main cultivars and excellent plant populations (more than 100 parts) of bayberries and developing Genome wide association analysis (GWAS), and the identification accuracy is reliable after the group verification of large-sample bayberry varieties.
Disclosure of Invention
The invention aims to provide a group of molecular marker primer combinations for rapidly identifying the morphological characters of waxberry leaves.
In order to achieve the purpose, the invention adopts the technical scheme that: a group of molecular marker primer combinations for rapidly identifying the morphological traits of waxberry leaves comprises Y9766732-F and Y9766732-R, and the specific sequences are as follows:
Y9766732-F：5’-GGCACGTGTCACGTATTAATTGGTGGTGACCTTC-3’，
Y9766732-R：5’-ATAGATCCCTCTATCCACTCACCGTCAT-3’。
the invention also discloses application of the molecular marker primer combination in rapid identification of the morphological characters of the leaves of the waxberry.
The method comprises the following steps:
(1) primer synthesis: synthesizing primers Y9766732-F and Y9766732-R according to claim 1;
(2) extraction of DNA: extracting genome DNA of waxberry leaves;
(3) and (3) PCR amplification: performing PCR amplification by using the primers Y9766732-F and Y9766732-R synthesized in the step (1) based on the genome DNA of the leaves of the waxberry, and adding at least two sample DNAs with determined leaf shapes as internal references in advance;
(4) detection and analysis of amplification products: and (3) carrying out genotype detection analysis on the PCR products, wherein if the PCR amplification products appear and are gathered with the internal reference sample into a group, the PCR products can be regarded as the same leaf form type, and if no PCR amplification product appears, the PCR products can be regarded as other leaf form types.
The detailed steps are as follows:
(1) primer synthesis: the biotechnology company was entrusted with the synthesis of primer Y9766732;
(2) extraction of DNA:
A. grinding about 0.2g of young leaves of the waxberries into powder by using liquid nitrogen, and putting the powder into a 2mL centrifuge tube;
B. extracting the sample genome DNA by using an Ezup Column type Plant genome DNA extraction Kit (EZ-10Spin Column Plant Genomic DNA Purification Kit, Shanghai Biotech engineering Co., Ltd.), and performing specific operation steps according to the instruction;
C. detecting 1-2 mul on 1.0% agarose gel, and diluting the DNA stock solution into working solution with the concentration of 20 ng/mul for later use;
(3) the PCR amplification reaction system is as follows: sample genomic DNA template 2.0. mu.L (20 ng/. mu.L), 2 XSG Fast qPCR Master Mix (Low Rox, Shanghai Biotech engineering Co., Ltd.) 5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M) 0.5. mu.L, ddH2Make up to 10 μ L of O.
(4) And (3) detecting and analyzing PCR amplification products: PCR amplification is a two-step procedure, i.e., 95 ℃ for 3min, (95 ℃ for 3s, 60 ℃ for 30s) for 25 cycles; after the PCR reaction is finished, the morphological characters of the leaves of the sample can be known directly by the automatic analysis result (Allelic diagnosis Plot) of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform Genotyping functional module without other processes; wherein, if PCR amplification products appear (2 waxberry germplasm samples with determined leaf shape as spoon shape are respectively added as internal reference) and are gathered with the internal reference sample into a group, the waxberry germplasm samples can be regarded as the same leaf shape character type; in addition, the absence of any PCR amplification products is considered to be another leaf morphology type.
Compared with the prior art, the invention has the beneficial effects that:
the fragment of the amplified product of the molecular marker primer Y9766732 is only 91bp, and the method is particularly suitable for high-throughput typing detection platforms such as a fluorescence quantitative PCR instrument, and the PCR amplification in the application process is short in time consumption and only has 25 cycle numbers; only 1.5 hours are needed from the extraction of the DNA of the waxberry leaf sample to the final identification result; the whole process from PCR amplification to the acquisition of the final typing result data is closed tube operation, subsequent gel electrophoresis detection is not needed, and zero pollution is realized; meanwhile, the amount of the sample genome DNA template can only need 40ng to obtain a typing detection result, and the method has the characteristics of high speed, high flux, high sensitivity and high accuracy.
Drawings
FIG. 1 is a classification chart of the shapes of waxberry leaves (from the left, they are respectively in the shape of a reversed lanceolate, a reversed oval, and a spoon).
FIG. 2 shows the typing effect of the Y9766732 primer on the fluorescent quantitative PCR instrument platform for the red bayberry germplasm resource DNA sample.
Detailed Description
The present invention will be described more specifically with reference to the following examples and drawings. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
Example 1
A group of molecular marker primer combinations for identifying the morphological characters of waxberry leaves and the application thereof, the detailed steps are as follows:
(1) primer synthesis: according to the sequence information in Table 1, primer Y9766732 was synthesized by Competition Biotechnology engineering (Shanghai) Ltd;
table 1: sequence information of the Y9766732 primer
(2) Extracting DNA of leaves of a waxberry germplasm resource sample to be detected:
A. grinding about 0.2g of young leaves of the waxberries into powder by using liquid nitrogen, and putting the powder into a 2mL centrifuge tube;
B. extracting the sample genome DNA by using an Ezup Column type Plant genome DNA extraction Kit (EZ-10Spin Column Plant Genomic DNA Purification Kit, Shanghai Biotech engineering Co., Ltd.), and performing specific operation steps according to the instruction;
C. detecting 1-2 mul on 1.0% agarose gel, and diluting the DNA stock solution into working solution with the concentration of 10 ng/mul for later use;
(3) the PCR amplification reaction system is as follows: sample genomic DNA template 2.0. mu.L (20 ng/. mu.L), 2 XSG Fast qPCR Master Mix (Low Rox, Shanghai Biotech engineering Co., Ltd.) 5. mu.L, forward primer (10. mu.M) 0.5. mu.L, reverse primer (10. mu.M) 0.5. mu.L, ddH2Make up to 10 μ L of O.
(4) And (3) detecting and analyzing PCR amplification products: PCR amplification is a two-step procedure, i.e., 95 ℃ for 3min, (95 ℃ for 3s, 60 ℃ for 30s) for 25 cycles; after the PCR reaction is finished, the morphological characters of the leaves of the sample can be known directly by the automatic analysis result (Allelic diagnosis Plot) of the ABI Q6 Flex real-time fluorescent quantitative PCR system platform Genotyping functional module without other processes; wherein, if PCR amplification products appear (2 waxberry germplasm sample DNAs with determined leaf shape as spoon shape are added in advance as internal reference) and are gathered with the internal reference sample into a group, the waxberry germplasm sample can be regarded as the shape type of the spoon-shaped leaf, and the blade is positive and marked with a plus; other genotypes or those without any PCR amplification product were considered as other leaf morphology types, negative, marked with a "-". Finally, all the leaf shape identification results of the waxberry germplasm samples are recorded by using a table (table 2), the real leaf shape characters of the waxberry germplasm samples are determined by observation in a result period, and only 9 waxberry germplasm leaf shape identification errors exist, so that the final waxberry leaf shape character identification accuracy rate is about 91%, and the specific typing effect is shown in fig. 1. Meanwhile, the primer can be prepared into a kit or a biological preparation for early auxiliary identification of the leaf morphology of the waxberry.
In the example, the 96-hole heating module of the fluorescence quantitative platform can be replaced by 384 holes, a fluid chip and the like as required so as to meet the requirement of higher-flux typing detection.
TABLE 2 leaf shape and character identification results of 96 waxberry germplasm resources used in the examples of the present invention
Note: the typing result graph is shown as "+" which is similar to the spoon-shaped internal reference sample of the waxberry leaves, and the rest samples are shown as "-".
Claims (5)
1. A group of molecular marker primer combinations for rapidly identifying the morphological traits of waxberry leaves is characterized by comprising Y9766732-F and Y9766732-R, and the specific sequences are as follows:
Y9766732-F：5’-GGCACGTGTCACGTATTAATTGGTGGTGACCTTC-3’，
Y9766732-R：5’-ATAGATCCCTCTATCCACTCACCGTCAT-3’。
2. the application of the molecular marker primer combination of claim 1 in rapidly identifying the morphological traits of the leaves of the waxberry.
3. The use of claim 2, the steps comprising:
(1) primer synthesis: synthesizing primers Y9766732-F and Y9766732-R according to claim 1;
(2) extraction of DNA: extracting genome DNA of waxberry leaves;
(3) and (3) PCR amplification: based on the genome DNA of the waxberry leaves, carrying out PCR amplification by using primers Y9766732-F and Y9766732-R synthesized in the step (1), and adding two waxberry sample DNAs with the leaf shape determined as a spoon shape in advance as internal references;
(4) detection and analysis of amplification products: carrying out genotype detection and analysis on the PCR product, and if the PCR amplification product appears and is gathered with the internal reference sample into a group, determining that the PCR amplification product and the internal reference sample are in the same leaf form, namely the spoon-shaped type; otherwise, if no PCR amplification product appears, the leaf morphology type is considered as other leaf morphology types.
4. Use according to claim 3, characterized in that: the PCR amplification reaction system in the step (3) is as follows: 2.0. mu.L of sample genomic DNA template at 20 ng/. mu.L, 2 XSG Fast qPCR Master Mix 5. mu.L, 0.5. mu.L of 10. mu.M forward primer, 0.5. mu.L of 10. mu.M reverse primer, ddH2Supplementing O to 10 μ L; PCR amplification was a two-step procedure, with 95 ℃ for 3min, followed by 25 cycles at 95 ℃ for 3s and 60 ℃ for 30 s.
5. Use according to claim 3, characterized in that: and (4) adopting an ABI Q6 Flex real-time fluorescent quantitative PCR system platform Genotyping functional module to automatically analyze the result.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110778765.8A CN113308565B (en) | 2021-07-09 | 2021-07-09 | Molecular marker primer combination for rapidly identifying morphological characters of waxberry leaves and application thereof |
| ZA2022/00803A ZA202200803B (en) | 2021-07-09 | 2022-01-18 | Molecular marker primer combinations for rapidly identifying myrica rubra leaf shape characters and application thereof |
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| CN202110778765.8A CN113308565B (en) | 2021-07-09 | 2021-07-09 | Molecular marker primer combination for rapidly identifying morphological characters of waxberry leaves and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113621734A (en) * | 2021-09-14 | 2021-11-09 | 宁波市农业科学研究院 | Molecular marker primer combination for rapidly identifying super-large fruit type characters of waxberries and application thereof |
| CN114107548A (en) * | 2021-12-07 | 2022-03-01 | 浙江省农业科学院 | KASP molecular marker for detecting color of waxberry fruits and application thereof |
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- 2021-07-09 CN CN202110778765.8A patent/CN113308565B/en active Active
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- 2022-01-18 ZA ZA2022/00803A patent/ZA202200803B/en unknown
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| CN108085405A (en) * | 2017-04-06 | 2018-05-29 | 浙江大学 | A kind of molecular labeling, primer pair and its application for being used to differentiate candleberry male and female |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113621734A (en) * | 2021-09-14 | 2021-11-09 | 宁波市农业科学研究院 | Molecular marker primer combination for rapidly identifying super-large fruit type characters of waxberries and application thereof |
| CN113621734B (en) * | 2021-09-14 | 2022-04-05 | 宁波市农业科学研究院 | Molecular marker primer combination for rapidly identifying super-large fruit type characters of waxberries and application thereof |
| CN114107548A (en) * | 2021-12-07 | 2022-03-01 | 浙江省农业科学院 | KASP molecular marker for detecting color of waxberry fruits and application thereof |
| CN114107548B (en) * | 2021-12-07 | 2023-11-28 | 浙江省农业科学院 | KASP molecular marker for detecting color of waxberry fruits and application thereof |
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